NO781404L - PROCEDURE FOR THE PREPARATION OF THYMOSIN ALFA-1 AND AN ANALOG COMPOSITION - Google Patents
PROCEDURE FOR THE PREPARATION OF THYMOSIN ALFA-1 AND AN ANALOG COMPOSITIONInfo
- Publication number
- NO781404L NO781404L NO781404A NO781404A NO781404L NO 781404 L NO781404 L NO 781404L NO 781404 A NO781404 A NO 781404A NO 781404 A NO781404 A NO 781404A NO 781404 L NO781404 L NO 781404L
- Authority
- NO
- Norway
- Prior art keywords
- glu
- obzl
- bzl
- ala
- lys
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims description 26
- 108010046075 Thymosin Proteins 0.000 title claims description 18
- 102000007501 Thymosin Human genes 0.000 title claims description 18
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 title claims description 18
- 239000000203 mixture Substances 0.000 title description 39
- 238000002360 preparation method Methods 0.000 title description 3
- 125000006239 protecting group Chemical group 0.000 claims description 27
- 150000003839 salts Chemical class 0.000 claims description 24
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical class OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 23
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical class OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 18
- 150000001875 compounds Chemical class 0.000 claims description 15
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 14
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 11
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims description 10
- 238000009833 condensation Methods 0.000 claims description 9
- 230000005494 condensation Effects 0.000 claims description 9
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 claims description 9
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 claims description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 3
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 2
- 239000004473 Threonine Substances 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 229910000040 hydrogen fluoride Inorganic materials 0.000 claims description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- 125000003354 benzotriazolyl group Chemical group N1N=NC2=C1C=CC=C2* 0.000 claims 1
- SLRMQYXOBQWXCR-UHFFFAOYSA-N 2154-56-5 Chemical compound [CH2]C1=CC=CC=C1 SLRMQYXOBQWXCR-UHFFFAOYSA-N 0.000 description 176
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 160
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 108
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 94
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 81
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 65
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 63
- 239000000047 product Substances 0.000 description 43
- 235000019439 ethyl acetate Nutrition 0.000 description 36
- 239000007787 solid Substances 0.000 description 36
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 34
- 238000006243 chemical reaction Methods 0.000 description 29
- -1 tert.-butyl Chemical group 0.000 description 29
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 24
- 239000003921 oil Substances 0.000 description 24
- 239000003208 petroleum Substances 0.000 description 24
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 22
- 229910004373 HOAc Inorganic materials 0.000 description 22
- 239000000706 filtrate Substances 0.000 description 18
- 239000011734 sodium Substances 0.000 description 18
- 239000002253 acid Substances 0.000 description 16
- 239000000463 material Substances 0.000 description 16
- 230000015572 biosynthetic process Effects 0.000 description 15
- 238000003786 synthesis reaction Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 13
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical class C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 12
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 12
- 238000001704 evaporation Methods 0.000 description 12
- 230000008020 evaporation Effects 0.000 description 12
- 239000002904 solvent Substances 0.000 description 12
- 108010016626 Dipeptides Proteins 0.000 description 11
- 239000012043 crude product Substances 0.000 description 11
- 150000002148 esters Chemical class 0.000 description 11
- 239000000843 powder Substances 0.000 description 11
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 10
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 10
- 239000011541 reaction mixture Substances 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 8
- 238000004809 thin layer chromatography Methods 0.000 description 8
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 7
- 239000006227 byproduct Substances 0.000 description 7
- 239000012044 organic layer Substances 0.000 description 7
- 239000006188 syrup Substances 0.000 description 7
- 235000020357 syrup Nutrition 0.000 description 7
- BGGHCRNCRWQABU-JTQLQIEISA-N (2s)-2-amino-5-oxo-5-phenylmethoxypentanoic acid Chemical compound OC(=O)[C@@H](N)CCC(=O)OCC1=CC=CC=C1 BGGHCRNCRWQABU-JTQLQIEISA-N 0.000 description 6
- FHOAKXBXYSJBGX-YFKPBYRVSA-N (2s)-3-hydroxy-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](CO)C(O)=O FHOAKXBXYSJBGX-YFKPBYRVSA-N 0.000 description 6
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- SNEHQGZSMYSVFT-DYVFJYSZSA-N (2,5-dioxopyrrolidin-1-yl) (2s,3r)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylmethoxybutanoate Chemical compound O([C@H](C)[C@H](NC(=O)OC(C)(C)C)C(=O)ON1C(CCC1=O)=O)CC1=CC=CC=C1 SNEHQGZSMYSVFT-DYVFJYSZSA-N 0.000 description 5
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 description 5
- KEULITJLZYZYPU-AWEZNQCLSA-N 4-o-benzyl 1-o-(2,5-dioxopyrrolidin-1-yl) (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanedioate Chemical compound C([C@H](NC(=O)OC(C)(C)C)C(=O)ON1C(CCC1=O)=O)C(=O)OCC1=CC=CC=C1 KEULITJLZYZYPU-AWEZNQCLSA-N 0.000 description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 5
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 5
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical class BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000002198 insoluble material Substances 0.000 description 5
- 238000005342 ion exchange Methods 0.000 description 5
- 229960004592 isopropanol Drugs 0.000 description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 5
- NTFYOALQRQBBDG-AWEZNQCLSA-N (2,5-dioxopyrrolidin-1-yl) (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylmethoxypropanoate Chemical compound C([C@H](NC(=O)OC(C)(C)C)C(=O)ON1C(CCC1=O)=O)OCC1=CC=CC=C1 NTFYOALQRQBBDG-AWEZNQCLSA-N 0.000 description 4
- BZNDDHWTEVCBAD-BQBZGAKWSA-N (2s)-2-[[(2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoyl]amino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)OC(C)(C)C BZNDDHWTEVCBAD-BQBZGAKWSA-N 0.000 description 4
- FYYSQDHBALBGHX-YFKPBYRVSA-N N(alpha)-t-butoxycarbonyl-L-asparagine Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC(N)=O FYYSQDHBALBGHX-YFKPBYRVSA-N 0.000 description 4
- 125000000217 alkyl group Chemical group 0.000 description 4
- 229960003116 amyl nitrite Drugs 0.000 description 4
- NDKBVBUGCNGSJJ-UHFFFAOYSA-M benzyltrimethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)CC1=CC=CC=C1 NDKBVBUGCNGSJJ-UHFFFAOYSA-M 0.000 description 4
- 239000013058 crude material Substances 0.000 description 4
- PAFZNILMFXTMIY-UHFFFAOYSA-N cyclohexylamine Chemical compound NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- POBDBYGSGKMZPH-NSHDSACASA-N (2,5-dioxopyrrolidin-1-yl) (2s)-3-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanoate Chemical compound CC(C)(C)OC(=O)N[C@@H](C(C)C)C(=O)ON1C(=O)CCC1=O POBDBYGSGKMZPH-NSHDSACASA-N 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- CKGCFBNYQJDIGS-LBPRGKRZSA-N (2s)-2-azaniumyl-6-(phenylmethoxycarbonylamino)hexanoate Chemical compound [O-]C(=O)[C@@H]([NH3+])CCCCNC(=O)OCC1=CC=CC=C1 CKGCFBNYQJDIGS-LBPRGKRZSA-N 0.000 description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- DEFJQIDDEAULHB-IMJSIDKUSA-N L-alanyl-L-alanine Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(O)=O DEFJQIDDEAULHB-IMJSIDKUSA-N 0.000 description 3
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- 125000003118 aryl group Chemical group 0.000 description 3
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- 210000004899 c-terminal region Anatomy 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- VFRSADQPWYCXDG-LEUCUCNGSA-N ethyl (2s,5s)-5-methylpyrrolidine-2-carboxylate;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.CCOC(=O)[C@@H]1CC[C@H](C)N1 VFRSADQPWYCXDG-LEUCUCNGSA-N 0.000 description 3
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- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 3
- YWLICOCXPNQJPC-KRWDZBQOSA-N (2,5-dioxopyrrolidin-1-yl) (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-6-(phenylmethoxycarbonylamino)hexanoate Chemical compound C([C@H](NC(=O)OC(C)(C)C)C(=O)ON1C(CCC1=O)=O)CCCNC(=O)OCC1=CC=CC=C1 YWLICOCXPNQJPC-KRWDZBQOSA-N 0.000 description 2
- COMUWNFVTWKSDT-ZETCQYMHSA-N (2,5-dioxopyrrolidin-1-yl) (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CC(C)(C)OC(=O)N[C@@H](C)C(=O)ON1C(=O)CCC1=O COMUWNFVTWKSDT-ZETCQYMHSA-N 0.000 description 2
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- IBABAURSJXMCQJ-QWRGUYRKSA-N (2s)-3-methyl-2-[[(2s)-3-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanoyl]amino]butanoic acid Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)OC(C)(C)C IBABAURSJXMCQJ-QWRGUYRKSA-N 0.000 description 1
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 1
- IQHSSYROJYPFDV-UHFFFAOYSA-N 2-bromo-1,3-dichloro-5-(trifluoromethyl)benzene Chemical group FC(F)(F)C1=CC(Cl)=C(Br)C(Cl)=C1 IQHSSYROJYPFDV-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- VGALFAWDSNRXJK-VIFPVBQESA-N L-aspartic acid beta-benzyl ester Chemical group OC(=O)[C@@H](N)CC(=O)OCC1=CC=CC=C1 VGALFAWDSNRXJK-VIFPVBQESA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- IDGQXGPQOGUGIX-VIFPVBQESA-N O-BENZYL-l-SERINE Chemical compound OC(=O)[C@@H](N)COCC1=CC=CC=C1 IDGQXGPQOGUGIX-VIFPVBQESA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- ZSPQUTWLWGWTPS-HJGDQZAQSA-N Thr-Lys-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZSPQUTWLWGWTPS-HJGDQZAQSA-N 0.000 description 1
- KRNYOVHEKOBTEF-YUMQZZPRSA-N Val-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(O)=O KRNYOVHEKOBTEF-YUMQZZPRSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 125000005076 adamantyloxycarbonyl group Chemical group C12(CC3CC(CC(C1)C3)C2)OC(=O)* 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 125000000746 allylic group Chemical group 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003710 aryl alkyl group Chemical group 0.000 description 1
- 229940072107 ascorbate Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052792 caesium Inorganic materials 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000002668 chloroacetyl group Chemical group ClCC(=O)* 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012230 colorless oil Substances 0.000 description 1
- 239000002178 crystalline material Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- ZZVUWRFHKOJYTH-UHFFFAOYSA-N diphenhydramine Chemical group C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1 ZZVUWRFHKOJYTH-UHFFFAOYSA-N 0.000 description 1
- 125000005982 diphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- QRMKTNANRJCRCY-UHFFFAOYSA-N ethylammonium acetate Chemical compound CC[NH3+].CC([O-])=O QRMKTNANRJCRCY-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 239000012213 gelatinous substance Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 238000006698 hydrazinolysis reaction Methods 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000002816 methylsulfanyl group Chemical group [H]C([H])([H])S[*] 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000000612 phthaloyl group Chemical group C(C=1C(C(=O)*)=CC=CC1)(=O)* 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000001226 reprecipitation Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 229920002994 synthetic fiber Polymers 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
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 108700016958 thymosin fraction 5 Proteins 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N urethane group Chemical group NC(=O)OCC JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06086—Dipeptides with the first amino acid being basic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- C07K14/57581—Thymosin; Related peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
- C07K5/06043—Leu-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Description
Fremgangsmåte ved fremstilling av thymosin a-^og enProcedure for the production of thymosin a-^and en
analog forbindelse.analog connection.
Thymosin a, er en varme stabil, sterk sur polypeptid' forbindelse med 28 aminosyre rester med følgende sekvens: Thymosin a, is a heat stable, strongly acidic polypeptide' compound with 28 amino acid residues with the following sequence:
Dette peptidet som er et sterkt immunopotensierende middel blir isolert fra thymosin-fraksjon 5 av Goldstein et al. ved en komb-inasjon av ione-bytte kromatorgrafi og gel-filtrering (Proe. Nati. Acad. Sei. USA 74, 725-729 (1977)). This peptide which is a strong immunopotentiating agent is isolated from thymosin fraction 5 by Goldstein et al. by a combination of ion-exchange chromatography and gel filtration (Proe. Nat. Acad. Sei. USA 74, 725-729 (1977)).
Foreliggen2de oppfinnelse vedrører en syntese av thymosin a, eller dets [Asn ]-analog og deres farmaseutisk fordraglige salter ved fjerning av beskyttelses grupper fra et beskyttet oktacosapeptid med sekvensen: The present invention relates to a synthesis of thymosin a, or its [Asn] analogue and their pharmaceutically acceptable salts by removing protective groups from a protected octacosa peptide with the sequence:
hvor X er Asn eller Asp(OR 2), where X is Asn or Asp(OR 2),
R er en vanlig beskyttelses gruppe for hydroksyl-gruppen i serinresten, R is a common protecting group for the hydroxyl group in the serine residue,
2 4 6 2 4 6
R ,R og R er konvensjonelle karboksylgruppe beskyttelsesgrupper, R , R and R are conventional carboxyl protecting groups,
3 3
R er en konvensjonell beskyttelsesgruppe for hydroksylgruppen i treonin og R is a conventional protecting group for the hydroxyl group in threonine and
R 5 er en konvensjonell beskyttelsesgruppe for R 5 is a conventional protection group for
-aminogruppen i lysinresten,-the amino group in the lysine residue,
bg om ønsket, overføre den erholdte forbindelsen i et farmaseutisk fordrage! i.g salt. bg if desired, transfer the obtained compound in a pharmaceutical treaty! i.g salt.
Eksempler på Rx er ben syl, acetyl, benzyl,tert.-butyl, trityl,4-brombenzyl,2,6-diklorbenzyl og benzyloksykarbonyl hvor benzyl er foretrukket. Eksempler på R 2 ,R 4 , R 6 er arylgrupper, spesielt fenyl eller fenyl substituert med en lavere alkyl, halogen, nitro, markapto eller substituert merkapto som metyltio,aralkylgrupper som benzylgrupper eller benzyl substituert med metoksy, halogen eller nitro, lavere alkylgrupper som.metyl, etyl,tert.-butyl.og tert.-amyl, substituerte lavere lakylgrupper som 2-halogenetyl, 3, (3-dimetylaminoetyl og cyanometyl, benshydryl og f enacylgrupper Examples of Rx are ben syl, acetyl, benzyl, tert.-butyl, trityl, 4-bromobenzyl, 2,6-dichlorobenzyl and benzyloxycarbonyl where benzyl is preferred. Examples of R 2 , R 4 , R 6 are aryl groups, especially phenyl or phenyl substituted with a lower alkyl, halogen, nitro, marcapto or substituted mercapto such as methylthio, aralkyl groups such as benzyl groups or benzyl substituted with methoxy, halogen or nitro, lower alkyl groups such as .methyl, ethyl, tert.-butyl.and tert.-amyl, substituted lower alkyl groups such as 2-haloethyl, 3, (3-dimethylaminoethyl and cyanomethyl, benzhydryl and phenacyl groups
3 hvor benzyl er foretrukket. Eksempler på R er benzyl, acetyl, benzoyl,•tert.-butyl, trityl,2,6-diklorbenzyl,4-brombenzyl og benzyloksykaronyl hvor benzyl er foretrukket. Eksem' pler på R<5>er benzyloksykarbonyl som eventult kan være substituert i den aromatiske ringen, som ved 4-klor, . 2-brom, 4-brom, 2,4-diklor, 4-nitro, 4-metoksy, 3,5-dimetoksy,4-metyl, 2,4,6-trimetyl, 4-fen-ylasy,4-(4-metoksyfenylso), 2-(N,N-dimetylkarbamido) og 2-nitro-4,5-dimetoksy, uretantype beskyttelsesgruppe som 4--toluensulfon yloksykarbonyl,9-fluorenymetyloksykarbonyl og beslektede base-avspaltbare grupper, 5t;benzisoxazolylmetylenoksykarbonyl ,metyltio-og metylsulf onuletuloksykarbonyl, isonikotinyloksykarbonyl, halo-, etyloksykarbonyl,diisopropylmetyloksykarbonyl, benzhydryloksy-karbonyl, isobornyloksykarbony1, dinitrodifenylmetyldksykårbonyl, tert.-butyloksykarbonyl, tert.-amyloskykarbony1, adamantyloksy-karbonyl,cycloheksyloksykarbonyl, metylcyclobutyloksykarbonyl, metylcycloheksyloksykarbonyl,2-arylisoprpyloskykarbonyl grupper som2-(p-bifenylyl)-isopropyloksykarbonyl, 2-(4-pyridyl)-isopropy-•loksykarbonyl og beslektede nitrogenholdige uretan-grupper, acyl-grupper som formyl, triflouracetyl,ftaloyl, benaensulfonyl,klor-acetyl,2-nitrobensoyl>4-toluensulfonyl, sulfenylgrupper som benz ensulfenyl,o-nitrofenylsulfenyl og beslektede sulfenylgrupper, og aryl lavere alkylgrupper som difenylmetyl og trifenylmetyl hvor benzyloksykarbonylgruppen er foretrukket. 3 where benzyl is preferred. Examples of R are benzyl, acetyl, benzoyl, tert-butyl, trityl, 2,6-dichlorobenzyl, 4-bromobenzyl and benzyloxykaronyl, where benzyl is preferred. Examples of R<5> are benzyloxycarbonyl which may possibly be substituted in the aromatic ring, as with 4-chloro, . 2-bromo, 4-bromo, 2,4-dichloro, 4-nitro, 4-methoxy, 3,5-dimethoxy,4-methyl, 2,4,6-trimethyl, 4-phenylacyl,4-(4 -methoxyphenylso), 2-(N,N-dimethylcarbamido) and 2-nitro-4,5-dimethoxy, urethane-type protecting group such as 4--toluenesulfon yloxycarbonyl,9-fluorenymethyloxycarbonyl and related base leaving groups, 5t;benzisoxazolylmethyleneoxycarbonyl ,methylthio- and methylsulfonuletuloxycarbonyl, isonicotinyloxycarbonyl, halo-, ethyloxycarbonyl, diisopropylmethyloxycarbonyl, benzhydryloxycarbonyl, isobornyloxycarbonyl, dinitrodiphenylmethyldoxycarbonyl, tert.-butyloxycarbonyl, tert.-amyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, methylcyclobutyloxycarbonyl, methylcyclohexyloxycarbonyl, 2-(2-yl)pyroxycarbonyl groups )-isopropyloxycarbonyl, 2-(4-pyridyl)-isopropyl-•loxycarbonyl and related nitrogen-containing urethane groups, acyl groups such as formyl, trifluoroacetyl, phthaloyl, benaenesulfonyl, chloroacetyl, 2-nitrobenzoyl>4-toluenesulfonyl, sulfenyl groups such as benz one sulfenyl, o-nitrophenylsulfenyl and related sulfenyl groups, and aryl lower alkyl groups such as diphenylmethyl and triphenylmethyl where the benzyloxycarbonyl group is preferred.
Fjerning av beskyttelsesgruppen fra det beskyttede aktakosapeptidet med sekvens I utføres lett ved i og for seg kjente metoder som f.eks. ved behandling med vann-fri syre som hydrogenflorid,for- Removal of the protection group from the protected actacosa peptide with sequence I is easily carried out by methods known per se such as e.g. by treatment with anhydrous acid such as hydrogen fluoride, for-
I IN
trinnsvis i nærvær av anisol. stepwise in the presence of anisole.
Ifølge foreliggende oppfinnelse kan det beskyttede oktakosapeptid i denne sekvens I syntetiseres ved kondensasjon av et beskyttet tetradecapeptid med sekvensen According to the present invention, the protected octacosa peptide in this sequence I can be synthesized by condensation of a protected tetradecapeptide with the sequence
med et beskyttet tetradecapeptid med sekvensen with a protected tetradecapeptide with the sequence
hvor X,,,,,og R^ er som ovenforangitt where X,,,,, and R^ are as stated above
7 7
og R er hydrogen eller en aktiverende gruppe.and R is hydrogen or an activating group.
Den forannevnte koblingsreaksjonen kan.utføres ved å anvende velkjente fremgangsmåter innenfor oppløsningsfase peptidsyntesen. Således kan f.eks. amino terminal tetradekapeptidet omsettes med en hydroksy benzotriazole (HOBT) og dicycloheksylkarbodiimid (DCC) for å gi den aktiverte esteren som så omsettes med karboksyl terminaltetradekapeptidet for å gi det ønskede oktacosapeptidet i beskyttet form. The aforementioned coupling reaction can be carried out by using well-known methods within solution phase peptide synthesis. Thus, e.g. the amino terminal tetradecapeptide is reacted with a hydroxy benzotriazole (HOBT) and dicyclohexylcarbodiimide (DCC) to give the activated ester which is then reacted with the carboxyl terminal tetradecapeptide to give the desired octacosapeptide in protected form.
De beskyttede tetradecapeptidene kan fremstilles ifølge velkjente metoder. The protected tetradecapeptides can be prepared according to well-known methods.
Systemet som ble anvendt i den kjemiske syntese av det beskyttede karboksyl terminalede tetradecapeptide III [X=Asp(OBzl)] The system used in the chemical synthesis of the protected carboxyl-terminated tetradecapeptide III [X=Asp(OBzl)]
var som følger: ■ were as follows: ■
H-Glu(OBzl)-0H ble først koblet med Boc-Ala-OSu hvilket gav det beskyttede dipeptid fragmentet Boc-Ala-Glu(OBzl)-0H som så ble kondensert med HCL • H-Asn-OBzl via DCC/HOSu prosedyren til Wiinsch and Drees, Chem. Ber. 9>9,110 (1966). H-Glu(OBzl)-OH was first coupled with Boc-Ala-OSu giving the protected dipeptide fragment Boc-Ala-Glu(OBzl)-OH which was then condensed with HCL • H-Asn-OBzl via the DCC/HOSu procedure to Winsch and Drees, Chem. Pray. 9>9,110 (1966).
Hydrokloridsaltet til aspargin benzylester ble fremstilt fra Boc-Åsn-OBzl som igjen ble suntetisert fra handelsvare Boc-Asn-OH i(Dg ' benzylbromider ved bruk av cesiumsaltet av aminosyren. Boc-beskyttelsesgruppen ble fjernet ved en 30's behandling méd 4N HC1 i tørt THF. The hydrochloride salt of aspargine benzyl ester was prepared from Boc-Åsn-OBzl which in turn was synthesized from commercial Boc-Asn-OH i(Dg' benzyl bromides using the cesium salt of the amino acid. The Boc protecting group was removed by a 30's treatment with 4N HCl in dry THF .
Reaksjonen mellom H-Glu(OBzl)-OH og Boc-Glu(OBzl)-OSu gav Boc-Glu (OBzl).-Glu (OBzl)-OH som en fargeløs klar olje. Den ble så The reaction between H-Glu(OBzl)-OH and Boc-Glu(OBzl)-OSu gave Boc-Glu(OBzl).-Glu(OBzl)-OH as a colorless clear oil. It became so
brukt i syntesen av det beskyttede pentapeptid Boc-Glu(OBzl)-Glu (OBzl)-Asn-OBzl i en DCC/HOSuindirekte fragment kondensasjon ved bruk av HC1 ' H-Ala-Glu(OBzl)-Asn-OBzl som stammet fra Boc-Ala-Glu(OBzl)-Asn-OBZl etter 4N HC1/THF behandling. - Det forannevnte beskyttede pentapeptid ble oppnådd i gode utbytte som krystalinsk rent materiale. used in the synthesis of the protected pentapeptide Boc-Glu(OBzl)-Glu (OBzl)-Asn-OBzl in a DCC/HOSuindirect fragment condensation using HC1 ' H-Ala-Glu(OBzl)-Asn-OBzl which originated from Boc -Ala-Glu(OBzl)-Asn-OBZl after 4N HC1/THF treatment. - The aforementioned protected pentapeptide was obtained in good yields as crystalline pure material.
For fremstillingen av det beskyttede octapeptidet Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl, ble det det nødvendige beskyttede tripeptidet Boc-Glu(OBzl)-Val-Val-OH først fremstilt. Boc-Val-OSu fikk reagere med fritt valin hvilket gav Boc-Val-Val- For the preparation of the protected octapeptide Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl, the required protected tripeptide Boc-Glu(OBzl)-Val-Val- OH first produced. Boc-Val-OSu was allowed to react with free valine which gave Boc-Val-Val-
OH som etter avblokering med 4N HG1 i THF fulgt av omsetning med Boc-Glu(OBzl)-OSu gav det ønskede tripeptid som ble krystalisert OH which after deblocking with 4N HG1 in THF followed by reaction with Boc-Glu(OBzl)-OSu gave the desired tripeptide which was crystallized
som cycloheksylamin salt Boc-Glu(OBzl)-Val-Val-OH • CHA. Sykloheksylaminsaltet ble overført i den frisyren og ble så kob- as cyclohexylamine salt Boc-Glu(OBzl)-Val-Val-OH • CHA. The cyclohexylamine salt was transferred in that hairstyle and was then cob-
let med DCC i nærvær av HOSu til HC1 • H-Glu-(OBzl)-Glu(OBzl)-Ala-Glu (OBzl) -Asn-OBzl som stammet fra Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu (OBzl) -Asn-OBzl ved behandling med HC1 i THF. Det beskyttede octapeptidet Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl erholdtes i renset form som et amorft faststoff. Beskyttelsesgruppen ble fjernet hydrogenolytisk etterfulg av behandling med • triflouredikksyre på vanlig måte som gav det frie octapeptidet Glu-Val-Val-Glu-Glu-Ala-Glu-Asn. Dette produktet ble renset ved ione-byttekolonne kromatorgrafi for å gi et tynnskikt-kromatograf-isk papirelektroforetisk homogent materiale. easily with DCC in the presence of HOSu to HC1 • H-Glu-(OBzl)-Glu(OBzl)-Ala-Glu (OBzl) -Asn-OBzl which originated from Boc-Glu(OBzl)-Glu(OBzl)-Ala- Glu(OBzl)-Asn-OBzl by treatment with HC1 in THF. The protected octapeptide Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl was obtained in purified form as an amorphous solid. The protecting group was removed hydrogenolytically followed by treatment with • trifluoroacetic acid in the usual way, which gave the free octapeptide Glu-Val-Val-Glu-Glu-Ala-Glu-Asn. This product was purified by ion exchange column chromatography to give a thin layer chromatography paper electrophoretic homogeneous material.
For syntesen av det beskyttede undecapeptidet Boc-Glu(OBzl)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(Obzl)-Ala-Glu(OBzl)-Asn-OBzl, ble det nødvendige tripeptid fragmentet syntetisert utfra Boc-Lys(Z)-OSu og H-Lys(Z)-OH. Dipeptidet Boe- Lys(Z)-Lys(Z)-OH som således erholdtes ble behandlet med 4N HC1 i THF og det dannede salt HCl' H-Lys(Z)-Lys(Z)-OH fikk så reagere med.Boc-Glu(OBzl)-OSu hvilket gav det ønskede tripeptid Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH. Tripeptidet ble så aktivert med DCC og HOSu ifølge Weygand et al., For the synthesis of the protected undecapeptide Boc-Glu(OBzl)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(Obzl)-Ala-Glu(OBzl)-Asn-OBzl, it was necessary tripeptide fragment synthesized from Boc-Lys(Z)-OSu and H-Lys(Z)-OH. The dipeptide Boe-Lys(Z)-Lys(Z)-OH thus obtained was treated with 4N HCl in THF and the formed salt HCl' H-Lys(Z)-Lys(Z)-OH was then allowed to react with Boc- Glu(OBzl)-OSu which gave the desired tripeptide Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH. The tripeptide was then activated with DCC and HOSu according to Weygand et al.,
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Z. Naturforsch. 21b, 426 (1966), og løsningen av den aktive tripeptidester Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSu fremstilt in situ kombinert med trifluoracetatetsaltet H-Glu(OBzl)-Val-Val-Glu(Obzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl som stammet fra det tilsvarende blokerte octapeptidet'ved en 30 min behandling med TFA. Etter tilsetning av en liten mengde av en base ble det ønskede beskyttede undecapeptidet Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu (OBzl ) -Glu (OBzl) -Ala-Glu (OBzl ) -Asn-OBzl således erholdt. Fjerning av beskyttelsesgruppen i det beskyttede undecapeptidet Boc-Glu (OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu (OBzl)-Asn-OBzl med vannfri fluss-syre gav det free undecapeptidet Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn som var homogent ved papir elektroforese etter ione-bytte kolonne-kromatografi. Z. Naturforsch. 21b, 426 (1966), and the solution of the active tripeptide ester Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSu prepared in situ combined with the trifluoroacetate salt H-Glu(OBzl)-Val-Val-Glu( Obzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl which originated from the corresponding blocked octapeptide' by a 30 min treatment with TFA. After addition of a small amount of a base, the desired protected undecapeptide Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu (OBzl ) -Glu (OBzl) - Ala-Glu (OBzl ) -Asn-OBzl thus obtained. Removal of the protecting group in the protected undecapeptide Boc-Glu (OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu (OBzl)- Asn-OBzl with anhydrous hydrofluoric acid gave the free undecapeptide Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn which was homogeneous by paper electrophoresis after ion-exchange column chromatography.
Syntesen av det beskyttede tetradecapeptide fulgt et lignende skjema. Boc-Leu-OSu ble koblet med H-Lys(Z)-OH hvilket gav Boc-Leu-Lys(Z)-OH .. Etter fjerning av Na<->Boc-gruppen med 4N HC1 i ThF og omsetning med Boc-Asp(OBzl)-OSu erholdtes detbeskyttede tripeptidet Boc-Asp(OBzl)-Leu-Lys(Z)-OH som et krystalinskrent fast stoff. Det ble overført i den aktive esteren Boc-Asp(OBzl)-Leu-Lys (Z) -OSu og kondenser med trifloracetatsaltet av H-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Asn-OBzl som erholdtes fra TFA-behand.ling av det tilsvarende blokerte undecapeptidet. Det ønskede produktet Boc-Asp(OBzl)-Leu^LYs(Z)-Glu (OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Asn-OBzl erholdtes i godt utbytte. Tynnskikt kromatofrafien indikerte at produktet var homogent. Det fri tetradecapeptide Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn erholdtes fra den beskyttelde forbindelsen ved behandling med vannfri fluss-syre og rensning på ione-bytter kolonne. The synthesis of the protected tetradecapeptide followed a similar scheme. Boc-Leu-OSu was coupled with H-Lys(Z)-OH which gave Boc-Leu-Lys(Z)-OH .. After removal of the Na<->Boc group with 4N HC1 in ThF and reaction with Boc- Asp(OBzl)-OSu, the protected tripeptide Boc-Asp(OBzl)-Leu-Lys(Z)-OH was obtained as a crystalline solid. It was transferred into the active ester Boc-Asp(OBzl)-Leu-Lys(Z)-OSu and condensed with the trifluoroacetate salt of H-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val -Val-Glu(OBzl)-Glu(OBzl)-Asn-OBzl which was obtained from TFA treatment of the corresponding blocked undecapeptide. The desired product Boc-Asp(OBzl)-Leu^LYs(Z)-Glu (OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl) -Asn-OBzl was obtained in good yield. Thin layer chromatography indicated that the product was homogeneous. The free tetradecapeptide Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn was obtained from the protected compound by treatment with anhydrous hydrofluoric acid and purification on an ion-exchange column.
På en lignende måte ble det beskyttede amino terminalet tetradecapeptidet H3C-Co-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH sammensatt fra et acetyl tetrapeptid-fragment, et heksapeptid, og et annet tetrapeptid ved å bruke veikjentefremgangsmåter innenfor peptidsyntese-området. For syntesen av det aminoterminale acetyltetrapeptidet, ble H3C-Co-Ser(Bzl)-OBzl fremstilt fra H3C-CO-Ser(Bzl)-OH og benzylbromidet via sesiumsaltet av aminosyren. Hydrasinalyse av H3C-CO-Ser(Bzl)-OBzl gav H3C-CO-Ser(Bzl)-HNNH som et rent kryst alinsk fast-stoff i godt utbytte. Deblokering av Boc-Ala-Ala-OH gav dipeptidhydrokloridsaltet HC1 • H-Ala-Ala-OH. Kobling av dette dipeptidet med Boc-Asp(OBzl)-OSu gav det beskyttede tripeptid Boc-Asp(OBzl)-Ala-Ala-OH som ble isolert som dicykloheksyl aminsaltet. Fjerning ,av aminobeskyttelses gruppen og kondensajon-en med H^C-CO-Ser(Bzl)-HNNH2 via asidmetoden til Honzl and Ruding-er, Collection Czech. Chem.Commun. 2_6 , 2333 (1961), gav det part-sielt beskyttede tetrapeptidet H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH som et reaksjon ved ekvivalente mengder hydrasin behandlet med DGC i nærvær av HOBT gav det ønskede mellomprodukt H3C-CO-Ser(Bzl)-.Asp(OBzl)-Ala-Ala-HNNH2 . In a similar manner, the protected amino terminal tetradecapeptide H3C-Co-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl-Ser(Bzl)-Glu(OBzl)-Ile -Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH composed of an acetyl tetrapeptide fragment, a hexapeptide, and another tetrapeptide using procedures known in the field of peptide synthesis. For the synthesis of the amino-terminal acetyltetrapeptide, H3C was -Co-Ser(Bzl)-OBzl prepared from H3C-CO-Ser(Bzl)-OH and the benzyl bromide via the cesium salt of the amino acid Hydrasinalysis of H3C-CO-Ser(Bzl)-OBzl gave H3C-CO-Ser(Bzl)- HNNH as a pure cross-linked allylic solid in good yield. Deblocking Boc-Ala-Ala-OH gave the dipeptide hydrochloride salt HC1 • H-Ala-Ala-OH. Coupling this dipeptide with Boc-Asp(OBzl)-OSu gave the protected tripeptide Boc-Asp(OBzl)-Ala-Ala-OH which was isolated as the dicyclohexyl amine salt Removal of the amino protecting group and condensation with H^C-CO-Ser(Bzl)-HNNH2 via the acid method of Honzl and Rudinger , Collection Czech.Chem.Commun. 2_6 , 2333 (1961), gave the partially protected tetrapeptide H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH as a reaction with equivalent amounts of hydrazine treated with DGC in the presence of HOBT gave the desired intermediate H3C-CO-Ser(Bzl)-.Asp(OBzl)-Ala-Ala-HNNH2 .
For syntesen av det beskyttede hektapeptidet Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH2, ble en fremgangsmåte som omfattet trinnvis forlengelse av peptid-kjeden fra den C-terminale ende anvendt. Således ble H-Glu(OBzl)-OH koplet med Boc-Ser(Bzl)-OSu for å gi det'beskyttede dipeptidet Boc-Ser(Bzl)-Glu (OBzl)-OH som ved fjerning av Boc-gruppen og vidre omsetningmed Boc-SEr (Bzl)-OSu gav tripeptidet Boc-Ser (Bzl)-Ser (Bzl)-Glu (OBzl.)-OH. Deproteksjon av en aminogruppe etterfulgt av kondensasjon av det reulterende tripeptidsaltet HCl.'H-Ser(Bzl)-Glu(OBzl)-OH For the synthesis of the protected hectapeptide Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH2, a procedure involving stepwise extension of the peptide chain from the C-terminal end used. Thus, H-Glu(OBzl)-OH was coupled with Boc-Ser(Bzl)-OSu to give the protected dipeptide Boc-Ser(Bzl)-Glu (OBzl)-OH which by removal of the Boc group and further reaction with Boc-SEr (Bzl)-OSu gave the tripeptide Boc-Ser (Bzl)-Ser (Bzl)-Glu (OBzl.)-OH. Deprotection of an amino group followed by condensation of the resulting tripeptide salt HCl.'H-Ser(Bzl)-Glu(OBzl)-OH
med Boc-Thr(Bzl)-OSu gav det beskyttede tetrapeptide Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH som ved behandling HC1 i THF fjernet Boc-gruppen og vidre omsetning med Boc-Asp(OBzl)-OSu resulterte i dannelsen av det beskyttede pentapeptide Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH.. Boc-gruppen i denne forbindelsen ble fjernet ved HCl-behandling og det dannede produktet HC1 • H-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH ble så koblet tilBoc-Val-OSu hvilket gav Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH som et krystalinsk rent materiale. with Boc-Thr(Bzl)-OSu gave the protected tetrapeptide Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH which on treatment with HC1 in THF removed the Boc group and further reaction with Boc-Asp(OBzl)-OSu resulted in the formation of the protected pentapeptide Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH.. The Boc group in this the compound was removed by HCl treatment and the product formed HC1 • H-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH was then coupled to Boc-Val-OSu which afforded Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH as a crystalline pure material.
a-karboksylgruppen på den terminale glutamin-syreresten i det forannevnte heksapeptidet ble så spesifikk overført til hydrasid-funksjonen ved omsetning med en ekvivalent mengde hydrasin ved bruk avDCC som koplingsreagens i nærvær av HOBT. Det ønskede beskyttede heksapeptidhydrasid Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser (Bzl)-Ser(Bzl)- Glu(OBzl)-HNNH2ble isolert som rent krystalinsk faststoff i et tilfredstillende utbytte. The α-carboxyl group on the terminal glutamic acid residue of the aforementioned hexapeptide was then specifically transferred to the hydrazide function by reaction with an equivalent amount of hydrazine using DCC as the coupling reagent in the presence of HOBT. The desired protected hexapeptide hydrazide Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser (Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH2 was isolated as pure crystalline solid in satisfactory yield.
For syntesen av det beskyttede tetrapeptid Boc-Ile-Thr(Bzl)-Thr '(Bzl)-Lys (Z)-OH ble en lignende trinnvis fremgangsmåte anvendt i For the synthesis of the protected tetrapeptide Boc-Ile-Thr(Bzl)-Thr '(Bzl)-Lys (Z)-OH, a similar stepwise procedure was used in
ved bruk av den N-hydroksysuccinimid aktive ester fremgangsmåten til Anderson et al.,J.Amer. Chem. Soc. 8j5 , 1839 (1964 ). Reaksjonen mellom Boc-Thr(Bzl)-OSu og H-Lys(Z)-OH gav dipeptidet Boc-Thr(Bzl)-Lys(Z)-OH som en olje som ble deprotektertpå a-amino enden og fikk reagere med Boc-Thr(Bzl)-OSu hvilket1 gav tripeptidet Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH som et krystalinsk faststoff. Fjerning av Boc-gruppen og omsetning av det resulterende materiale HC1 • H-Thr(Bzl)-Thr(Bzl)-Lys(Z).OH medBoc-Ile-OSu gav det ønskede beskyttelde tetrapeptid Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH using the N-hydroxysuccinimide active ester method of Anderson et al., J. Amer. Chem. Soc. 8j5 , 1839 (1964 ). The reaction between Boc-Thr(Bzl)-OSu and H-Lys(Z)-OH gave the dipeptide Boc-Thr(Bzl)-Lys(Z)-OH as an oil which was deprotected on the α-amino end and allowed to react with Boc- Thr(Bzl)-OSu which1 gave the tripeptide Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH as a crystalline solid. Removal of the Boc group and reaction of the resulting material HC1 • H-Thr(Bzl)-Thr(Bzl)-Lys(Z).OH with Boc-Ile-OSu gave the desired protected tetrapeptide Boc-Ile-Thr(Bzl)- Thr(Bzl)-Lys(Z)-OH
som en krystalinsk ren forbindelse etter kromatografi på en kisel-gelkolonne. Dette tetrapeptid-fragmentet ble så deprotektert på aminoenden og kondensert med det beskyttede heksapeptidet Boc-Val-Asp (OBzl) -Thr (Bzl) -Ser (Bzl) -Ser (Bzl) -Glu (OBzl) -HNNH2 ved azid-metoden. hvilket gav det beskyttede decapeptidetBoc-Val-Asp(Bzl) hr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)_ as a crystalline pure compound after chromatography on a silica gel column. This tetrapeptide fragment was then deprotected at the amino terminus and condensed with the protected hexapeptide Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH2 by the azide method. which gave the protected decapeptide Boc-Val-Asp(Bzl) hr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)_
OH i godt utbytte.. OH in good profit..
Fjerning av Boc-gruppen fra decapeptid forbindelsen med TFA og etterfølgende kobling med N-terminale tetrapeptid Ac-Ser(Bzl)-Asp Obzl)-Ala-Ala-HNNH2ved acid fremgangsmåten resulterte i dannelsen av det ønskede beskyttede tetradecapeptidét H^C-CO-Ser(Bzl)-Asp (OBzl)-Ala-Ala-Val-Asp(/Bzl)-Thr(Bzl)-SEr(Bzl)-Ser(Bzl).Glu(OBzl)-Ile-Thr(Bzl)-Thr)Bzl)-Lys(Z)-OH. For den endelige kopling ble dette acetyl tetradecapeptid aktivert med DCC og HOBT og den dannede aktive ester fikk så reagere med TFA H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-LYs(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(Obzl)-Ala-Glu(OBzl)-Asn-Obzl som var fremstilt fra den tilsvarende blokerte forbindelsen ved behandling med TFA hvilket gav det beskyttede acetyl-oktakospeptide H3C-CO-Ser(Bzl)-Asp(QBzl)-Ala-Ala-Val-Asp(OBzl)-Thr- (Bzl) -Ser (Bzl) -Ser (Bzl) -Glu (OBzl) -Ile-Thr (Bzl) -Thr (Bzl) -'•Lys (Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-GluOBzl)-Asn-OBzl. Behandling med vannfritt HF fjernet alle beskyttelsesgruppene og rensing og ione-byttekronomatografi gav thymosin a^. Removal of the Boc group from the decapeptide compound with TFA and subsequent coupling with the N-terminal tetrapeptide Ac-Ser(Bzl)-Asp Obzl)-Ala-Ala-HNNH2 by the acid method resulted in the formation of the desired protected tetradecapeptide H^C-CO- Ser(Bzl)-Asp (OBzl)-Ala-Ala-Val-Asp(/Bzl)-Thr(Bzl)-SEr(Bzl)-Ser(Bzl).Glu(OBzl)-Ile-Thr(Bzl)-Thr )Bzl)-Lys(Z)-OH. For the final coupling, this acetyl tetradecapeptide was activated with DCC and HOBT and the active ester formed was then allowed to react with TFA H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-LYs(Z)-Glu(OBzl )-Val-Val-Glu(OBzl)-Glu(Obzl)-Ala-Glu(OBzl)-Asn-Obzl which was prepared from the corresponding blocked compound by treatment with TFA giving the protected acetyl-octacos peptide H3C-CO-Ser (Bzl)-Asp(QBzl)-Ala-Ala-Val-Asp(OBzl)-Thr- (Bzl) -Ser (Bzl) -Ser (Bzl) -Glu (OBzl) -Ile-Thr (Bzl) -Thr ( Bzl) -'•Lys (Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-GluOBzl)-Asn -Obzl. Treatment with anhydrous HF removed all the protecting groups, and purification and ion-exchange chromatography yielded thymosin a^.
På lignende måte fikk man fra det beskyttede tetradecapeptide H3C-CO-Ser(Bzl)-AspOBzlj-Ala-Ala-Val-Asp(OBzl)Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH det fri tetra- In a similar manner, from the protected tetradecapeptide H3C-CO-Ser(Bzl)-AspOBzlj-Ala-Ala-Val-Asp(OBzl)Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)- Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH the free tetra-
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decapeptidet H3C-CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OI-I. the decapeptide H3C-CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OI-I.
Syntesen av [Asn 2]-thymosin a, fulgte det samme skjema som syntesen avtymosin a^. Alle mellom produktene som ble brukt var de samme bortsett fra det N-terminale acetyl tetrapeptidhydrasidet H3C-CO-Ser(Bzl)-Asn-Ala-Ala-HNNH2. For syntesen av denne forbindelsen ble Boc-Ala-Ala-OH først overført til Boc-Ala-Ala-OBzl som ved selektiv fjerning av Boc-gruppen med HC1 gav dipeptidseter-saltet HC1"H-Ala-Ala- OBzl. Dipeptidet ble så koblet med Boc-Asn-OH ved bruk av DCC/HOBT - metoden til Konig and Geiger, Chem. Ber. 103, 788 (1970), hvilket gav det beskyttede dipeptidesteren Boc-Asn-Ala-Ala-OBzl som ble behandlet med HCL i THF for å fjerne Boc-gruppen. Det reulterende produktet HC1■Asri-Ala-Ala-OBzl ble så kondensert (DCC/HOBT-metoden) med H3C-CO-Ser(Bzl)-OH •DCHA hvilket gav det ønskede tetrapeptidet H3C-CO-Ser(Bzl)-Asn-Ala-Ala-OBzl. The synthesis of [Asn 2 ]-thymosin a followed the same scheme as the synthesis of thymosin a^. All intermediate products used were the same except for the N-terminal acetyl tetrapeptide hydrazide H3C-CO-Ser(Bzl)-Asn-Ala-Ala-HNNH2. For the synthesis of this compound, Boc-Ala-Ala-OH was first transferred to Boc-Ala-Ala-OBzl which, by selective removal of the Boc group with HC1, gave the dipeptide ester salt HC1"H-Ala-Ala-OBzl. The dipeptide was then coupled with Boc-Asn-OH using the DCC/HOBT method of Konig and Geiger, Chem. Ber. 103, 788 (1970), affording the protected dipeptide ester Boc-Asn-Ala-Ala-OBzl which was treated with HCL in THF to remove the Boc group.The resulting product HC1■Asri-Ala-Ala-OBzl was then condensed (DCC/HOBT method) with H3C-CO-Ser(Bzl)-OH •DCHA to give the desired tetrapeptide H3C -CO-Ser(Bzl)-Asn-Ala-Ala-OBzl.
Det tilsvarende hydrasinet H3C-CO-Ser(Bzl)-Asn-Ala-Ala-HNNH^erholdtes i godt utbytte ved hydrasinolyse av denne forbindelsen. Fragment-kondensasjonen mellom hydrasidet og decapeptidet TFA H-Val-Asp(OBzl)-thr(Bzl)-Ser(Bzl)-SEr(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH fra det beskyttede tetradecapeptidet H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Glu(/Bzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH. Denne forbindelsen ble så koblet til det deblokerte C--terminalet tetradekapeptide TFA-sal tet som er ovenfornevnt og gav det tilsvarende beskyttede [Asn 2]-thymosin ci£ Fjérning av alle beksyttelses-gruppene ved behandling med vannfri HF fulgt av kolonneione ione-bytte kronomatografi-rensing gav det The corresponding hydrazine H3C-CO-Ser(Bzl)-Asn-Ala-Ala-HNNH^ was kept in good yield by hydrazinolysis of this compound. The fragment condensation between the hydrazide and the decapeptide TFA H-Val-Asp(OBzl)-thr(Bzl)-Ser(Bzl)-SEr(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys (Z)-OH from the protected tetradecapeptide H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Glu(/Bzl)-Ile-Thr (Bzl)-Thr(Bzl)-Lys(Z)-OH. This compound was then coupled to the deblocked C-terminal tetradecapeptide TFA salt mentioned above to give the corresponding protected [Asn 2 ]-thymosin ci£ Removal of all protecting groups by treatment with anhydrous HF followed by column ion exchange chronomatography purification gave it
2 2
ønskede [Asn ]-thymosin mens den samme metoden med det beskyttede tetradecapeptidet H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH gav det acetylerte tetradecapeptidet H3C-CO-Ser-Asn-Ala-Val-Asp-Thr-Ser-Glu-Ile-Thr-Thr-Lys-OH. desired [Asn ]-thymosin while the same method with the protected tetradecapeptide H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Glu(OBzl) -Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH gave the acetylated tetradecapeptide H3C-CO-Ser-Asn-Ala-Val-Asp-Thr-Ser-Glu-Ile-Thr-Thr-Lys -OH.
I et vidre aspekt av foreliggende oppfinnelse er det funnet at flere av mellomprodukt peptidene som brukes i ovenfornevnte syntese av thymosin og [Asn 2]-thmosin a, har aktivitet ved regul-ering og diffrensiering og funksjon av T-celler, nemlig In a further aspect of the present invention, it has been found that several of the intermediate peptides used in the above-mentioned synthesis of thymosin and [Asn 2]-thmosin a have activity in the regulation and differentiation and function of T cells, namely
Thymosin a.^ [Asn 2]-thymosin eller de foran-nevnte nye okta-, undeca- eller tetradecapeptider som danner en del av foreliggende oppfinnelse og deres farmaseutisk fordraglige salter kan gis til varmblodige pattedyr ved partenteral applikasjon enten intraven-øst ,subkutant eller intramuskulært. Disse forbindelsene er sterke immunopotensierende middler ved en daglig dosering i om-rådet fra ca 1-100 mg/kg.kroppsvekt pr dag ved intravinøs administrering. Åpenbart vil den nødvendige dose variere med den spesielle tilstanden som behandles, tilstandens grad og behandling-ens varighet. En egnet doseringsform for farmaseutisk anvendelse er et 1 mgl lyofilisert thymosin a^, [Asn 2]-thymosin a, eller et av de forannevnte peptidfragmenter derav' som skal rekonstitueres før bruk ved tilsetning av sterilt vann eller saltvann. Thymosin a.^ [Asn 2]-thymosin or the above-mentioned new octa-, undeca- or tetradecapeptides which form part of the present invention and their pharmaceutically acceptable salts can be given to warm-blooded mammals by parenteral application either intravenously, subcutaneously or intramuscularly. These compounds are strong immunopotentiating agents at a daily dosage in the range of approx. 1-100 mg/kg body weight per day by intravenous administration. Obviously, the required dose will vary with the particular condition being treated, the degree of the condition and the duration of the treatment. A suitable dosage form for pharmaceutical use is a 1 mgl lyophilized thymosin a^, [Asn 2]-thymosin a, or one of the aforementioned peptide fragments thereof' which must be reconstituted before use by adding sterile water or saline.
Også omfattet av rammen for foreliggende oppfinnelse ligger de farmaseutisk fordraglige salter avthymosin..a^ , [Asn 2]-thymosin og de forannevnte pe<p>tidfragmenter derav. Egnede salter omfatter natrium og kaliumsaltene eller saltene med sterk organisk base som guandin. Dertil kan parrionene til disse kationene slik som klor, bromid, sulfat, fosfat, meleat, acetat, citrat, benzoat, succinat, nalat, ascorbat o.l., innbefattes i fremstillingen.. Also covered by the scope of the present invention are the pharmaceutically acceptable salts of thymosin, [Asn 2 ]-thymosin and the aforementioned peptide fragments thereof. Suitable salts include the sodium and potassium salts or the salts with a strong organic base such as guandine. In addition, the parrions of these cations such as chlorine, bromide, sulphate, phosphate, meleate, acetate, citrate, benzoate, succinate, nalate, ascorbate etc., can be included in the preparation.
Forkortelsene som brukes heri har følgende betydning: Boc=t-butyloksykarbonyl; Bzl=benzyl;DCC=dicycloheksylkarbodimid; DMF-dimetylformamide; THF=tetrahydrofuran; HOSu= N-hydroksysuccin-imide; Triton B=40% metanolløsning av trimetylbenzylammonium hyd-roksyd; NMM=N-metylmorfolin; CHA=cycloheksylamin; DCHA=dicyclohe-ksylamin; Z=benzyloksykarbony1; DMSO=dimetylsulfoksid; TFA=tri-fluoracetik syre; TLC= tynt lag kromatografi; Et3N=trietylamin; HOBT=l-hydroksybenzotriazole. The abbreviations used herein have the following meanings: Boc=t-butyloxycarbonyl; Bzl=benzyl; DCC=dicyclohexylcarbodiimide; DMF-dimethylformamide; THF=tetrahydrofuran; HOSu= N-hydroxysuccinimide; Triton B=40% methanol solution of trimethylbenzylammonium hydroxide; NMM=N-methylmorpholine; CHA=cyclohexylamine; DCHA=dicyclohexylamine; Z=benzyloxycarbonyl; DMSO=dimethylsulfoxide; TFA=tri-fluoroacetic acid; TLC= thin layer chromatography; Et3N=triethylamine; HOBT=1-hydroxybenzotriazole.
be følgende eksempler beskriver i detalj syntesen av thymosin i og [Asn 2]-thymosin a]_- be following examples describe in detail the synthesis of thymosin i and [Asn 2]-thymosin a]_-
Selvom spesifikke beskyttelsés grupper har blitt anvendt i be-skrivelsen av syntesen av thymosin og [Asn 2]-thymosin vil det være åpenbart for enhver fagmann å anvende ekvivalente beskyttelsesgrupper i sådanne synteser. Although specific protecting groups have been used in the description of the synthesis of thymosin and [Asn 2 ]-thymosin, it will be obvious to any person skilled in the art to use equivalent protecting groups in such syntheses.
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Eksempel 1Example 1
A.a) Boc-Asn-OH (11.0 g,47.5 mmol) ble oppløst i 200 ml MeOH ogA.a) Boc-Asn-OH (11.0 g, 47.5 mmol) was dissolved in 200 ml MeOH and
20 ml vann ble tilsatt. Løsningen ble titrert til pH 7,0 med en 20 % vanndigløsning av Cs-jCO^(ca. 55 ml). Blandingen ble inndampet til tørnet og resten ble fordampet 2 ganger fra DMF (120 ml hver, 45°C). Det erholdte faste hvite stoff ble så rørt med 8,9 g benzylbromid (52 mmol) i 120 ml DMF i 6 timer. Ved inndampning 20 ml of water was added. The solution was titrated to pH 7.0 with a 20% aqueous solution of Cs-jCO 3 (approx. 55 ml). The mixture was evaporated to dryness and the residue was evaporated twice from DMF (120 mL each, 45°C). The white solid obtained was then stirred with 8.9 g of benzyl bromide (52 mmol) in 120 ml of DMF for 6 hours. By evaporation
til tørhet og behandling med et stort volum vann, ble produktet øyeblikkelig fast. Det ble oppsamlet ved filtrering , oppløst i etylacetat, vasket med vann, tørket over Na^SO^, inndampet til en fast masse og krystalisert fra et etylacetat med petroleter. Utbyttet 13.8 g (90,3 %) av Boc-Asn-OBzl; m.p. 120-122°C; [ct]p<5>= -17,29° (c=l,DMF) . to dryness and treatment with a large volume of water, the product immediately solidified. It was collected by filtration, dissolved in ethyl acetate, washed with water, dried over Na^SO^, evaporated to a solid mass and crystallized from an ethyl acetate with petroleum ether. Yield 13.8 g (90.3%) of Boc-Asn-OBzl; m.p. 120-122°C; [ct]p<5>= -17.29° (c=1, DMF) .
Boc-Asn-OBzl (13.7 g, 42,4 mmol) ble oppløst i 80 ml THF og behandlet med 500 ml 4N HC1 i THF. Blandingen fikk stå 45 min. hvorunder noe produkt begynte å utfelles. Ved behanling med 1000 ml eter ble et hvitt faststoff øyeblikkelig dannet. Produktet ble filtrert,.vasket med eter og tørket over NaOH-pellets i våkum. Utbyttet: 10.3 g (94 %) av HC1'H-Asn-OBzl; m.p. 122-126°C; [a]^<5>= +6.82°. Boc-Asn-OBzl (13.7 g, 42.4 mmol) was dissolved in 80 mL of THF and treated with 500 mL of 4N HCl in THF. The mixture was allowed to stand for 45 min. below which some product began to precipitate. On treatment with 1000 ml of ether, a white solid formed immediately. The product was filtered, washed with ether and dried over NaOH pellets in vacuum. Yield: 10.3 g (94%) of HC1'H-Asn-OBzl; m.p. 122-126°C; [a]^<5>= +6.82°.
b) H-Glu(OBzl)-OH (7.0 g, 29.5 mmol) ble finknust med en morter og så rørt med 8,88 g (32.3 mmol) av Boc-Ala-OSu i 48 timer i b) H-Glu(OBzl)-OH (7.0 g, 29.5 mmol) was finely crushed with a mortar and then stirred with 8.88 g (32.3 mmol) of Boc-Ala-OSu for 48 h in
250 ml D11F i nærvær av 6 mlNMM. Noe mer enn NMM ble tilsatt for å holde reaksjonen svakt basisk under reaksjonen. Løsningsmiddel-et ble avdampet og resten fordelt mellom 300 ml etylacetat og 500 ml H20 som inneholdt 2 ml av 10% H2S04*Det organiske skiktet ble så vasket 3 ganger med vann, tørket over Na2S04og inndampet til'tørhet. Produktet ble tatt opp i et lite volumeter og behandlet med et stort volum Petroleter. Et hvit amorft faststoff ble erholdt som var homogent i TCL. Utbyttet:11,0 g(91.5%) av . Boc-Ala-Glu(OBzl)-OH;m.p. 84-88°C; ta]^<5>= 8,08°(c=l,DMF. 250 ml D11F in the presence of 6 mlNMM. Slightly more than NMM was added to keep the reaction slightly basic during the reaction. The solvent was evaporated and the residue partitioned between 300 ml of ethyl acetate and 500 ml of H 2 O containing 2 ml of 10% H 2 SO 4 *The organic layer was then washed 3 times with water, dried over Na 2 SO 4 and evaporated to dryness. The product was taken up in a small volumetric flask and treated with a large volume of petroleum ether. A white amorphous solid was obtained which was homogeneous in TCL. The yield: 11.0 g (91.5%) of . Boc-Ala-Glu(OBzl)-OH; m.p. 84-88°C; ta]^<5>= 8.08°(c=1, DMF.
Boc-Ala-Glu(OBzl)-OH (10.4 g,25.4 mmol), HC1 • H-Asn-OBzl(6.56 g, 25.4 mmol) og HOSu (5.9 g,50,8 mmol) ble oppløst i DMF (250 ml, 0°C). DCC (5.7 g,27.6 mmol) ble tilsatt umidelbart etterfulgt av Et3N (3.5 ml). Blandingen ble rørt ved 0°C i to. timer og så ved 25°C i 40 timer hvorunder noe mer Et^N. ble tilsatt fra tid til annen for å holde reaksjonen svakt basisk. De uløslig dannede biproduktene ble frafiltrert og filtratet inndampet- til tørhet. Det resterende oljeaktige materiale størknet ved behandling av vann. Rå-produktet ble tatt opp i CHCl^og vasket med vann (3x), tørket over en Na2SO^og inndampet. til et mindre volum. Noé faststoff som.ble dannet på dette trinnet ble frafiltrert (sterkt forurenset med dicycloheksylurea) og filtratet behandlet med petroleter. Et krystalinsk produkt ble erholdt. Utbytte: 8.0 g (51.4 %) avBoc-Ala-Glu(OBzl)-Asn-OBzl); m.p. 102-105°C; [a]^<5>12.5°(c=l,DMF. Boc-Ala-Glu(OBzl)-OH (10.4 g, 25.4 mmol), HCl • H-Asn-OBzl (6.56 g, 25.4 mmol) and HOSu (5.9 g, 50.8 mmol) were dissolved in DMF (250 mL , 0°C). DCC (5.7 g, 27.6 mmol) was added immediately followed by Et 3 N (3.5 mL). The mixture was stirred at 0°C for two. hours and then at 25°C for 40 hours during which some more Et^N. was added from time to time to keep the reaction slightly basic. The insoluble by-products were filtered off and the filtrate evaporated to dryness. The remaining oily material solidified on treatment with water. The crude product was taken up in CHCl 4 and washed with water (3x), dried over Na 2 SO 4 and evaporated. to a smaller volume. Some solids formed in this step were filtered off (heavily contaminated with dicyclohexylurea) and the filtrate treated with petroleum ether. A crystalline product was obtained. Yield: 8.0 g (51.4%) of Boc-Ala-Glu(OBzl)-Asn-OBzl); m.p. 102-105°C; [a]^<5>12.5°(c=1, DMF.
c) H-Glu(OBzl)-OH (4.74 g, 20 mmol) ble knust i en morter og rørt med Boc-Glu(OBzl)-OSu (0.7 g, 20 mmol) i DMF i 36 timer i c) H-Glu(OBzl)-OH (4.74 g, 20 mmol) was crushed in a mortar and stirred with Boc-Glu(OBzl)-OSu (0.7 g, 20 mmol) in DMF for 36 h in
nærvær av 3.6 ml NMM. Den dannede løsningen ble inndampet til en sirup og behandlet med vann. Det oljeaktige pepitatet ble tatt opp i etylacetatet, vasket suksesivt med 5% HOAc og vann (3x) , tørket over Na2SO^og inndampet til tørhet hvilket gav 14.03 g av en klar olje. Den fikk stå under petroleter. Den resterende oljeaktige Boc-Glu(OBzl)-Glu(OBzl)-OH veide lo.2 g (90%). TLC presence of 3.6 ml NMM. The resulting solution was evaporated to a syrup and treated with water. The oily peptate was taken up in ethyl acetate, washed successively with 5% HOAc and water (3x), dried over Na 2 SO 4 and evaporated to dryness giving 14.03 g of a clear oil. It was allowed to stand under petrol. The remaining oily Boc-Glu(OBzl)-Glu(OBzl)-OH weighed lo.2 g (90%). TLC
25 o 25 o
indikerte at produktet var homogent. q = -7-59 (c=l,DMF).indicated that the product was homogeneous. q = -7-59 (c=1, DMF).
d) Boc-Ala-Glu(OBzl)-Asn-OBzl) (28.2 g; 46 mmol ble behandlet med 1.1 liter 4N HCl i THF i 1 time. Avdampning av løsningsmidd-el og overskuddsyre etterlot en olje som ble inndampet 2 ganger til med friskt THF. Restoljen gikk over til et faststoff ved behandling av et stort volumeter. Det faste HCl • H-Ala-Glu (OBzl)-Asn-OBzl ble rørt med Boc-Glu(OBzl)-Glu(OBzl)-OH 825,6 g, 4 6 mmol) ,HOSu (10.6 g, 92 mmol) og DCC 810,9 g 5 3mmol). i DMF (54 0 ml) ved 0° i en time og så ved 25° i 48 timer. Et^N ble tilsatt for å holde reaksjonen svakt basisk over hele tidsrommet (ca. 16 ml Et^N totalt). De uløslige biproduktent som ble dannet ble frafiltrert og filtratet inndampet til tørhet. Rå-produktet ble oppløst i CHCl^, vasket med vann (3x), tørket over en Na2SO^d) Boc-Ala-Glu(OBzl)-Asn-OBzl) (28.2 g; 46 mmol was treated with 1.1 L of 4N HCl in THF for 1 h. Evaporation of solvent and excess acid left an oil which was evaporated 2 more times with fresh THF. The residual oil turned to a solid on treatment with a large volumet. The solid HCl • H-Ala-Glu (OBzl)-Asn-OBzl was stirred with Boc-Glu(OBzl)-Glu(OBzl)-OH 825 .6 g, 4 6 mmol), HOSu (10.6 g, 92 mmol) and DCC 810.9 g 5 3 mmol). in DMF (540 ml) at 0° for one hour and then at 25° for 48 hours. Et^N was added to keep the reaction slightly basic over the entire period (approx. 16 ml of Et^N in total). The insoluble by-products that formed were filtered off and the filtrate evaporated to dryness. The crude product was dissolved in CHCl^, washed with water (3x), dried over a Na2SO^
og inndampet itl tørhet. Produktet ble fast ved behandling ved petroleter. Omkrystalisasjonen fra isopropanol gav 28.9 g(59.8 %) av Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(obzl)-Asn-OBzl;m.p. 169-175°C; [a]^<5>=-11.78° (c=l,DMF). and evaporated itl dryness. The product solidified on treatment with petroleum ether. The recrystallization from isopropanol gave 28.9 g (59.8 %) of Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(obzl)-Asn-OBzl; m.p. 169-175°C; [α]^<5>=-11.78° (c=1, DMF).
■Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (3.9 g,3.48 mmol) ■Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (3.9 g, 3.48 mmol)
ble behandlet med 15 ml 4N HCl i THF i 30 min. Noe krystalinsk produkt startet og dannes. Eter (210 ml) ble tilsatt.og det felte fastestoff oppsamlet og vasket med eter. Rå-materialet ble krystalisert fra MeOH og eter. Utbyttet: 2.53 g(75.1 %) av HCl-H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl: m.p. 14 8-151°C: was treated with 15 ml of 4N HCl in THF for 30 min. Some crystalline product started and forms. Ether (210 mL) was added and the precipitated solid was collected and washed with ether. The crude material was crystallized from MeOH and ether. Yield: 2.53 g (75.1%) of HCl-H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl: m.p. 14 8-151°C:
[a]p<5>=-3.65° (c = 1, DMF).[α]p<5>=-3.65° (c = 1, DMF).
e) Boc-Val-OSu (12.6 g,40 mmol) og H-Val-OH 84.68 g,40 mmol) ble kondensert i DMF 8250 ml) i 96 timer i nærvær av 2 ml Et^N. e) Boc-Val-OSu (12.6 g, 40 mmol) and H-Val-OH 84.68 g, 40 mmol) were condensed in DMF 8250 ml) for 96 h in the presence of 2 ml Et^N.
Mer Et^N ble tilsatt ved behov for å holde reaksjonen svakt basisk Det resterende uløslige materiale ble fra filtrert og filtratet inndampet til tørhet (45°C). Resten ble fordelt mellom eteren og fortynnet H2S04(ca. 1%) og det organiske skiktet vasket med vann (3x), tørket over en Na2S04og inndampet til en.skummet glatt masse. Produktet ble krystalisert fra eter dg petroliumeter. Utbyttet: 12.2 g(96.4%) av Boc-Val-Val-OH; m.p. 155-158°C; [ct]^<5>= +1.lo° (c = 1, DMF). More Et^N was added as needed to keep the reaction slightly basic. The remaining insoluble material was filtered off and the filtrate was evaporated to dryness (45°C). The residue was partitioned between ether and dilute H2SO4 (approx. 1%) and the organic layer washed with water (3x), dried over Na2SO4 and evaporated to a frothy smooth mass. The product was crystallized from ether and petroleum ether. Yield: 12.2 g (96.4%) of Boc-Val-Val-OH; m.p. 155-158°C; [ct]^<5>= +1.lo° (c = 1, DMF).
Boc-V<p>J.-Val-OH (40,5 g, 128 mmol) ble behandlet me'-"' 1,8 1 av.Boc-V<p>J.-Val-OH (40.5 g, 128 mmol) was treated with'-"' 1.8 1 av.
[N HCl i THF i 60 min. Inndampning for å fjerne overskuddet av syre og løsningsmiddel etterfulgt av behandling med eter gav 34,5 g HCl •H-Val-Val-OH som et hvitt amorft pulver. Det ble behandlet med Boc-Glu(OBzl)-OSu (55,6 g, 128 mmol) i en liter DMF i 24 timer i nærvær av 54 ml Et^N. Reaksjonsblandingen.ble filtrert for å fjerne noe uløslig materiale og filtratet inndampet til tørrhet. Den gjenværende oljeaktive rest ble opptatt i EtOAc (1.5 1) og vasket med 5 % HOAc (2x) etterfulgt av vann (3x) .. [N HCl in THF for 60 min. Evaporation to remove excess acid and solvent followed by treatment with ether gave 34.5 g of HCl •H-Val-Val-OH as a white amorphous powder. It was treated with Boc-Glu(OBzl)-OSu (55.6 g, 128 mmol) in one liter of DMF for 24 hours in the presence of 54 ml of Et₂N. The reaction mixture was filtered to remove any insoluble material and the filtrate evaporated to dryness. The remaining oily residue was taken up in EtOAc (1.5 L) and washed with 5% HOAc (2x) followed by water (3x)..
Det organiske skiktet ble tøket (Na2SO^) og inndampet til tørr-het og gav en fargeløs klar olje som ikke krystaliserte. Den ble så oppløst i 3,2 1 eter og behandlet med CHA (17 ml) inntil blandingens pH var 7.5. De.t faste erholdte salt ble oppsamlet og omkrystalisert fra MeOH og eter. Utbyttet: 58.9 g (72.7%) The organic layer was dried (Na 2 SO 4 ) and evaporated to dryness to give a colorless clear oil which did not crystallize. It was then dissolved in 3.2 L of ether and treated with CHA (17 mL) until the pH of the mixture was 7.5. The solid salt obtained was collected and recrystallized from MeOH and ether. Yield: 58.9 g (72.7%)
av Boc-Glu (OBzl)-Val-Val-OH CHA; m.p. 158 - 160°C; [a]^<5><=>33,4.1° of Boc-Glu (OBzl)-Val-Val-OH CHA; m.p. 158 - 160°C; [a]^<5><=>33.4.1°
.(c = 1, MeOH) ..(c = 1, MeOH) .
Boc-Glu(Obzl)-Val-Val-OH \ CHA (1.69 g, 2 66 mmol) ble opp-, slemmet i vann (40 ml) og etylaminacetat (40 ml), i en skille trakt under tilsetning av 4 ml 1 M H^SO^. Etter kraftig røring bppiøste det faste stoff og det organiske skiktet ble vasket flere ganger med vann, tørket over Na^GO^og inndampet til en ' olje 81,45 g). Det således erholdte fri tripeptridet ble så kondensert med 2.58 g HCl • H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (2.61 mmol) i 15 ml DMF i nærvær av HOSu (0.612 g.5.32 mmol), NMM (0,3 ml,2.66 mmol) og DCC (o,63 g, 3,06 mmol) i løpet av en time ved 0°C og 60-timer ved 25°C. Mer enn NUM ble tilsatt ved behov for å holde reaksjonen svakt basisk. Et uløslélig dannet bi-produkt ble frafiltrert og filtratet inndampet til tørrhet (45°C). Den gjenblivende oljeaktivresten størknet ved behandling med vann. Det faste rå-produktet ble oppløst i DMF (50 ml) og felt med MeOH (300 ml) Utbyttet : 2.25 g 358.7%) Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl; m.p. 277-280°C [a]^5 =-12.43° (c = 1, DMF). Boc-Glu(Obzl)-Val-Val-OH \CHA (1.69 g, 2 66 mmol) was dissolved in water (40 mL) and ethylamine acetate (40 mL), in a separatory funnel, while adding 4 mL of 1 M H^SO^. After vigorous stirring, the solid dissolved and the organic layer was washed several times with water, dried over Na 2 GO 2 and evaporated to an oil (81.45 g). The free tripeptide thus obtained was then condensed with 2.58 g of HCl • H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (2.61 mmol) in 15 ml of DMF in the presence of HOSu (0.612 g .5.32 mmol), NMM (0.3 ml, 2.66 mmol) and DCC (0.63 g, 3.06 mmol) during one hour at 0°C and 60 hours at 25°C. More than NUM was added as needed to keep the reaction slightly basic. An insoluble by-product formed was filtered off and the filtrate evaporated to dryness (45°C). The remaining active oil residue solidified when treated with water. The solid crude product was dissolved in DMF (50 ml) and precipitated with MeOH (300 ml) Yield: 2.25 g 358.7%) Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala -Glu(OBzl)-Asn-OBzl; m.p. 277-280°C [α]^5 =-12.43° (c = 1, DMF).
Dette produktet (0,72 g, 0 49 mmol) ble hydrogenert over 15% Pd/ BaSO^(0,5 g) i 3 timer, ved 3.4 atm i en blandina av 4 0 ml DMF/30 ml MeOH/ 2 ml H^O. Blandingen ble så filtrert og filtratet inndampet til tørrhet. Det ble deretter behandlet med 5 ml' TFA i 3 0 min. og den erholdte rest etter avdampning av syren . ble revet flere ganger med eter. Det dannede faste stoffet ble tatt opp i vann (20 ml) og lyofilisert og gav 0,47 g rå-produkt. Forbindelsen ble satt på en 3 x 32 cm kolonne av et sterkt basisk polysterenharpiks (Bio-Rad AG1-X 2) som var ekvilibrert med pH 8.1 ammoniumacetat buffer (2% HOAc innstilt på pH 3.1 med NH^) .. Kolonnen ble eluert suksesivt med 200 ml av o,o25 M pH 5.5 NH4OAc,0.025 m HOAc, 0,05 M HOAc, 0,25 M HOAcm 0,5 M HOAc, 0,75 M HOAc, IM HOAc. Fraksjoner nå 12 ml ble oppsamlet og eluatet fra hvert rør undersøkt ved TLC. Fraksjonene som inneholdt det ønskede materialet (rørne 225-229) ble slått sammen og lyofilisert 2 ganger og gav 0,223 g (48.1%) av ren Glu-Val-Val--Glu-Glu-Ala^-Glu--Åsn som var homogent i TLC og papir elektroforese. This product (0.72 g, 0.49 mmol) was hydrogenated over 15% Pd/BaSO^(0.5 g) for 3 h, at 3.4 atm in a mixture of 40 mL DMF/30 mL MeOH/2 mL H ^O. The mixture was then filtered and the filtrate evaporated to dryness. It was then treated with 5 ml of TFA for 30 min. and the residue obtained after evaporation of the acid. was torn several times with ether. The solid formed was taken up in water (20 ml) and lyophilized to give 0.47 g of crude product. The compound was applied to a 3 x 32 cm column of a strongly basic polystyrene resin (Bio-Rad AG1-X 2) equilibrated with pH 8.1 ammonium acetate buffer (2% HOAc adjusted to pH 3.1 with NH^). The column was eluted successively with 200 ml of o,o25 M pH 5.5 NH4OAc,0.025 m HOAc, 0.05 M HOAc, 0.25 M HOAcm 0.5 M HOAc, 0.75 M HOAc, IM HOAc. Fractions reaching 12 ml were collected and the eluate from each tube examined by TLC. The fractions containing the desired material (tubes 225-229) were pooled and lyophilized 2 times yielding 0.223 g (48.1%) of pure Glu-Val-Val--Glu-Glu-Ala^-Glu--Åsn which was homogeneous in TLC and paper electrophoresis.
f) Boc-Lys(Z)-OH (15 g, 39.5 mmol) ble rørt med HOSu (5.3 g, 50.5 mmol) og 8.66 g, 42 mmol) i THF (250 ml) i tre f) Boc-Lys(Z)-OH (15 g, 39.5 mmol) was stirred with HOSu (5.3 g, 50.5 mmol) and 8.66 g, 42 mmol) in THF (250 mL) in three
timer. Et uløselig biprodukt ble frafiltrert og filtratet inndampet til tørrhet,. Den gjenværende sirup (24,2 g) ble behandlet med iso-propanol (150 ml), og petroleter (150 ml)' aav et olje aktivt produkt (21.g ) som ikke krystaliserte. Den 1! hours. An insoluble by-product was filtered off and the filtrate evaporated to dryness. The remaining syrup (24.2 g) was treated with iso-propanol (150 ml) and petroleum ether (150 ml) to yield an oil-active product (21 g) which did not crystallize. The 1!
'{rå aktive esteren Boc-Lys (Z)-OSu ble således brukt for kondensasjon med K-Lys(Z)-OH (10,6 g, 33 mmol) i DMF (250 ml) i 72 timer i nærvær av 5.5 ml Et. Mer Et^N ble tilsatt ved leilighetsvis for å holde den røret reaksjonsblandingen svakt basisk. The crude active ester Boc-Lys(Z)-OSu was thus used for condensation with K-Lys(Z)-OH (10.6 g, 33 mmol) in DMF (250 mL) for 72 h in the presence of 5.5 mL One. More Et^N was added occasionally to keep the reaction mixture slightly basic.
En liten mengde uoppløst materiale ble så frafiltrert og filtratet inndampet til tørrhet (45°C). Den tilbakeblivende olje-resten ble behandlet med 1,5 ml 5 % HOAc. Det utfelte produktet ble ekstrahert over i et etylacetat og den organiske fasen vasket "med vann, tørket over en Na^SO^og inndampet til en olje. Den ble krystalisert fra etylacetat (300 ml) som inneholdt DCHA A small amount of undissolved material was then filtered off and the filtrate evaporated to dryness (45°C). The remaining oil residue was treated with 1.5 ml of 5% HOAc. The precipitated product was extracted into ethyl acetate and the organic phase washed with water, dried over Na 2 SO 4 and evaporated to an oil. It was crystallized from ethyl acetate (300 mL) containing DCHA
(10 ml) som et salt. Omkrystalisasjonen fra MeOH og eter gav 22.7 g (72.5 %) av Boc-Lys (Z ) -Lys (Z) -OH * DCHA; m.p.' 160 - 162°C; (10 ml) as a salt. The recrystallization from MeOH and ether gave 22.7 g (72.5%) of Boc-Lys(Z )-Lys(Z)-OH * DCHA; m.p.' 160 - 162°C;
[a]^<5>= - 2.21° (c = l,MeOH).[α]^<5>= - 2.21° (c = 1,MeOH).
Boc-Lys (7.) -OH DCHA (10. g, 12.14 mmol) ble fordelt mellom EtOAc•(1 liter) og'0,1 N H?S04(1 liter). Det or<q>ansiske skiktet ble så vasket.med vann (3x), tørket over et Na2SO^og inndampet til tørrhet (7.9 g). Den frisyren, Boc-Lys(Z)-Lys(Z)-OH som således erholdtes ble behandlet med friskt fremstilt Boc-Lys (7.) -OH DCHA (10.g, 12.14 mmol) was partitioned between EtOAc• (1 L) and 0.1 N H₂SO 4 (1 L). The organic layer was then washed with water (3x), dried over Na 2 SO 4 and evaporated to dryness (7.9 g). The formula, Boc-Lys(Z)-Lys(Z)-OH thus obtained was treated with freshly prepared
4N HCl i THF i 3 0 minutter. Løsningsmiddelet oa syre-overskudd. ble avdampet (30°C) og resten refordampet 2 ganger med THF. 4N HCl in THF for 30 min. The solvent, including excess acid. was evaporated (30°C) and the residue reevaporated 2 times with THF.
Den tilbakeblivende rest størknet ved behandling med eter. Salt et HCl•H.Lys(Z)-Lys(Z)-OH ble oppsamlet ved filtrering og vasket flere ganger med eter og gav 6.7 g hvitt pulver. Det ble opp-løst i DMF (70 ml), kjølet i is-bad og behandlet med Et^N (1.63 ml) fulgt av Boe- Glu(OBzl)-OSu (5.54 g, 12.76 mmol). Blandingen ble rørt. i 0°C i en time og så ved 2 5°C i 2 4 timer. Mer Et-jN ble tilsatt i løpet av denne tiden for å holde reaksjonen nær pH 7.5. Noen ml eddiksyre ble tilsatt for å surgjøre reaksjonen (pH 3.,5) og løsningsmiddelet fjernet ved inndampning. Den dannede resten ble tatt opp i EtOAc, vasket med. vann (3x), tørket over Na2SO^ og inndampet til tørrhet hvorved produktet begynte og bli fast. Det ble revet i eter og rekrystalisert fra etylacetat. Utbyttet: 7.26 g (69.5 %)• av Boc-Glu(OBzl)-Lys (Z)-Lys (Z)-OH; m.p. 153 - 155°C; [a]p<5>= -2.71° (c = 1, THF.. The remaining residue solidified by treatment with ether. Salt et HCl•H.Lys(Z)-Lys(Z)-OH was collected by filtration and washed several times with ether to give 6.7 g of white powder. It was dissolved in DMF (70 ml), cooled in an ice bath and treated with Et^N (1.63 ml) followed by Boe-Glu(OBzl)-OSu (5.54 g, 12.76 mmol). The mixture was stirred. at 0°C for one hour and then at 25°C for 24 hours. More Et-jN was added during this time to keep the reaction close to pH 7.5. A few ml of acetic acid were added to acidify the reaction (pH 3.5) and the solvent removed by evaporation. The residue formed was taken up in EtOAc, washed with water (3x), dried over Na 2 SO 4 , and evaporated to dryness whereupon the product began to solidify. It was triturated in ether and recrystallized from ethyl acetate. The yield: 7.26 g (69.5 %)• of Boc-Glu(OBzl)-Lys (Z)-Lys (Z)-OH; m.p. 153 - 155°C; [a]p<5>= -2.71° (c = 1, THF..
g) Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu (OBzl)-Asn-OBzl (1.7 g, 1.16 mmol ble behandlet med TFA (24 ml) g) Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu (OBzl)-Asn-OBzl (1.7 g, 1.16 mmol) was treated with TFA (24 ml)
i 3 0 minutter. Etter fordampning av syre-overskudd (3 0°c) ble j for 30 minutes. After evaporation of excess acid (30°c), j
ires t-en revet med eter. Det erholdte pulveret ble vasket grundig, med eter og petrol-eter og tørket overNaOH i våkum og gav tri-fluouracetatsaltet av oktapeptidet (1.71 q). Den aktive ester Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSu ble så fremstilt in situ ved ires t-en torn with ether. The powder obtained was washed thoroughly with ether and petroleum ether and dried over NaOH in vacuo to give the trifluoroacetate salt of the octapeptide (1.71 q). The active ester Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSu was then prepared in situ by
å røre Boe—Glu(OBzl)-Lys(Z)-Lys(Z)-OH (0.993 g. 1.16 mmol), HOSu (0.16 g, 1.4 mmol) og DCC (0,274 g,1.33 mmol) i 15 ml DMF to stirring Boe—Glu(OBzl)-Lys(Z)-Lys(Z)-OH (0.993 g, 1.16 mmol), HOSu (0.16 g, 1.4 mmol) and DCC (0.274 g, 1.33 mmol) in 15 mL of DMF
i 0°C i 3 timer. Denne løsningen som inneholdt den aktive tri'at 0°C for 3 hours. This solution which contained the active tri'
-peptid-ester ble tilsatt octapeptid-saltet CF3COOH•H-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)- Ala-Glu(OBzl)-Asn-OBzl (1.71 g) sammen med 0,2 ml Et. Noen flere dråper F,t3N og DMF (15 ml.) ble tilsatt og blandingen rørt 3 dager ved 25°C. En gelaktig halvfast-masse oppstod. Den ble surgjort ved eddiksyre og behandlet med vann. Det hvite faste.presipitat ble oppsamlet og vasket. (H?0,MeOH, eter) og gav 2,2 5 g råprodukt som ble smeltet ved 310-313°C. Det ble op<p>løst i DMF og felt med MeOH. Utbyttet: 1.75 g (63.3 %) av Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) --Asn-OBzl; m.<p>.314-316°C; 2 5 o [a]D = 13.63 (c =1, DMSO); homogeneous i TLC. Boc-Glu (OBzl) -Lys (7.) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl)-Glu(OBzl)-Ala-Glu(Obzl)-Asn-OBzl (0,5 g, 0,226 mmol), ble o<p>pløst i 2 ml TFA og rørt med 15 ml HF ved 0°C i 15 min. Etter avdampning av syre-overskudd (0°C), ble resten oppløst i 5 % vandig HOAc, vasket med eter (3x), inndampet til et lite volum og leofylisert og gav o,34 g rått produkt. Det ble kromato-grafert på ionebytte-kolonnen som er beskrevet ovenfor for oktapeptid og gav 0,13 g (42.1%) av ren Glu-Lys-Lys-Clu-Val-Val-Glu-Glu-Ala-Glu-Asn; [a]^<5>-85.65° (C = 1, H?0). -peptide ester was added to the octapeptide salt CF3COOH•H-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)- Ala-Glu(OBzl)-Asn-OBzl (1.71 g) together with 0, 2 ml Et. A few more drops of F,t3N and DMF (15 ml.) was added and the mixture stirred for 3 days at 25°C. A gel-like semi-solid mass was formed. It was acidified with acetic acid and treated with water. The white solid precipitate was collected and washed. (H?0,MeOH, ether) and gave 2.25 g of crude product which was melted at 310-313°C. It was dissolved in DMF and precipitated with MeOH. Yield: 1.75 g (63.3%) of Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl ) --Asn-OBzl; m.<p>.314-316°C; 2 5 o [α]D = 13.63 (c = 1, DMSO); homogeneous in TLC. Boc-Glu (OBzl) -Lys (7.) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl)-Glu(OBzl)-Ala-Glu(Obzl)-Asn-OBzl (0, 5 g, 0.226 mmol), was dissolved in 2 ml TFA and stirred with 15 ml HF at 0°C for 15 min. After evaporation of excess acid (0°C), the residue was dissolved in 5% aqueous HOAc, washed with ether (3x), evaporated to a small volume and lyophilized to give 0.34 g of crude product. It was chromatographed on the ion exchange column described above for octapeptide and gave 0.13 g (42.1%) of pure Glu-Lys-Lys-Clu-Val-Val-Glu-Glu-Ala-Glu-Asn; [α]^<5>-85.65° (C = 1, H?0).
h) Boc-Leu-OSu (4.0 g, 12 .. 2 mmol) og H-Lys (Z)-OH (3.42 g, 12.2 mmol) ble kondensert i DMF (75 ml) i løpet av 48 timer i h) Boc-Leu-OSu (4.0 g, 12 .. 2 mmol) and H-Lys (Z)-OH (3.42 g, 12.2 mmol) were condensed in DMF (75 mL) over 48 h in
nærvær av Et3N (1,7 ml). Reaksjonsblandingens pH ble holdt ved 7.5 ved tilsetning av Et3N periodisk som vanlig. Det gjenværende uløslige materiale ble frafelt og filtrert og filtratet inndampet til tørrhet. Den dannede skumaktige -glassmassen ble oppløst, i eter (2 00 ml) og blandingen behandlet med 3 ml DCHA for å gi presence of Et 3 N (1.7 mL). The pH of the reaction mixture was maintained at 7.5 by adding Et 3 N periodically as usual. The remaining insoluble material was precipitated and filtered and the filtrate evaporated to dryness. The foamy glass mass formed was dissolved in ether (200 mL) and the mixture treated with 3 mL of DCHA to give
krystalinsk materiale som ble oppsamlet vasket med eter og omkrystalisert fra MeOH og eter. Utbyttet: 5.7.g (69.5%) av Boc-Leu-Lys(Z)-OH-DCHA; m.p. 140-142°C; [a]^<5>= -7.20° (c = l.MeOH). i i' crystalline material which was collected, washed with ether and recrystallized from MeOH and ether. Yield: 5.7 g (69.5%) of Boc-Leu-Lys(Z)-OH-DCHA; m.p. 140-142°C; [α]^<5>= -7.20° (c = 1.MeOH). i i'
3oc-Leu-Lys(Z)-OH-DCHA (2,97 g, 4.4 mmol) ble overført i den fri-syren (fordelt mellom EtOAc og 0,1 N H7S0^) og den erholdte fargeløse olje (2,2 g) ble behandlet med 4N HCl i THF (40 ml) 3oc-Leu-Lys(Z)-OH-DCHA (2.97 g, 4.4 mmol) was taken up in the free acid (partitioned between EtOAc and 0.1 N H 7 SO 4 ) and the obtained colorless oil (2.2 g ) was treated with 4N HCl in THF (40 mL)
i 30 min. Syre overskuddet og løsningsmiddelet ble fordampet (30°C) og resten behandlet med eter. Den gjenblivende oljen for 30 min. The excess acid and solvent were evaporated (30°C) and the residue treated with ether. The remaining oil
ble oppløst i eteret og inndampet 2 ganger mer med friske eter. Resten ble så rørt med Boc-Asp(OBzl)-OSu (1.85 gr 4.4 mmol) i nærvær av Et3N (1.85 ml) natten over. Reaksjonsblandingen ble så inndampet til tørrhet og gav en oljerest som ble tatt opp i etylacetat, vasket med vann (3x), tørket over en Na2S04 og inndampet til tørrhet igjen. Det således erholdte rå-produkt ble krystalisert fra etylacetat og petroleter og gav 1.52 g (49.6%) av ren Boc--Asp (OBzl)-Leu-Lys (Z ) -OH; m.n. 109-111°C; t"^5 -16.14° was dissolved in the ether and evaporated 2 times more with fresh ether. The residue was then stirred with Boc-Asp(OBzl)-OSu (1.85 g 4.4 mmol) in the presence of Et 3 N (1.85 ml) overnight. The reaction mixture was then evaporated to dryness to give an oil residue which was taken up in ethyl acetate, washed with water (3x), dried over Na 2 SO 4 and evaporated to dryness again. The crude product thus obtained was crystallized from ethyl acetate and petroleum ether and gave 1.52 g (49.6%) of pure Boc--Asp (OBzl)-Leu-Lys (Z )-OH; m.n. 109-111°C; t"^5 -16.14°
(c = 1, DMF). (c = 1, DMF).
Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(Obzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (1.2 g,0,545 mmol) ble behandlet med 35 ml TFA i 30 min. Syreoverskuddet ble raskt fordam<p>et og resten revet med eter flere ganger og gav 1.2 g av undecapeptid TFA-saltet som et hvitt pulver. Det ble o<p>nl^st i en blanding av DMF (5 ml) og DMSO (2 ml) og behandlet med Boc-Asp(OBzl)-Leu-Lys (Z) -OSu fremstilt in situ ved av røre Boc-Asp(OBzl)-Leu-Lys (Z)-OH (0,381 g. 0,545 mmol) med HOSu (0,126 g, 1.1 mmol) og DCC (0,124 g, 0,599 mmol) i 3 ml DMF ved 0°C i tre timer. Blandingen som inneholdt den aktive tripeptid esteren og undecapeptid ble rørt ved 0°C i 2 timer og så ved 2 5°C i tre dager hvorunder'Et^N ble tilsatt fra tid til annen for å holde pH svakt basisk. En gelatinaktig substans ble dannet. Den ble revet med 5 % HOAc og det resulterende hvite faste stoff ble filtrert og vasket med vann, MeOH og eter og gav 1,28 g rått-materiale som smeltet ved 325-326°C. Represipitering av'DMF/DMSO (10 ml/5 ml) ogMeOH (230 ml) gav 1.22 g (80.2%) av ren Boc-Asp Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(Obzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (1.2 g, 0.545 mmol) was treated with 35 ml of TFA for 30 min. The excess acid was quickly evaporated and the residue triturated with ether several times to give 1.2 g of the undecapeptide TFA salt as a white powder. It was dissolved in a mixture of DMF (5 ml) and DMSO (2 ml) and treated with Boc-Asp(OBzl)-Leu-Lys (Z)-OSu prepared in situ by stirring Boc- Asp(OBzl)-Leu-Lys (Z)-OH (0.381 g, 0.545 mmol) with HOSu (0.126 g, 1.1 mmol) and DCC (0.124 g, 0.599 mmol) in 3 mL DMF at 0 °C for three hours. The mixture containing the active tripeptide ester and undecapeptide was stirred at 0°C for 2 hours and then at 25°C for three days during which Et 2 N was added from time to time to keep the pH slightly basic. A gelatinous substance was formed. It was triturated with 5% HOAc and the resulting white solid was filtered and washed with water, MeOH and ether to give 1.28 g of crude material melting at 325-326°C. Reprecipitation of DMF/DMSO (10 ml/5 ml) and MeOH (230 ml) gave 1.22 g (80.2%) of pure Boc-Asp
(OBzl) -Leu:-Lys (Z) -Glu (OBzl) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) - Glu (OBzl) -Glu (OBzl) -Ala--Glu (OBzl)-Asn-OBzl) ; m.<p>. 326-327°C; (OBzl) -Leu:-Lys (Z) -Glu (OBzl) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) - Glu (OBzl) -Glu (OBzl) -Ala--Glu (OBzl)-Asn-OBzl); m.<p>. 326-327°C;
[oUq5 ■= -15.71° (c = 1, DMF/DMSO).[oUq5 ■= -15.71° (c = 1, DMF/DMSO).
Dette produktet (1,128 g. 0,404 mmol) ble blandet med 7 ml anisol og behandlet med 25 ml vannfritt HF ved 0°C i 15 min.. Syre- This product (1.128 g, 0.404 mmol) was mixed with 7 ml of anisole and treated with 25 ml of anhydrous HF at 0°C for 15 min.. Acid-
{overskuddet ble fordampet (0°C) og den qjenblivende rest fordelt mellom eter og vann. Vann-skiktet ble op<p>vasket 2 ganger med eter inndampet til halvparten av det opprinnelige volumet og lyofilisert hvilket gav 0,69 g rått materiale. Det ble kronomatografert på den ovenfor beskrevne måte for oktapeptid. Materiale som ble eluert i rørene 101-120 ble oppsamlet og lyofilisert hvilket gav 0,25 g produkt som ble vist å være litt forurenset med mindre forurensninger. Det ble derfor rekomato-grafert på samme kolonne hvilket gav 0,155 g (22%) av ren Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-- Glu-Glu- Ala-Glu Asn. Papirelektroforese indikert-at det var homogent. [a]^<5>= -86,27° The excess was evaporated (0°C) and the remaining residue distributed between ether and water. The water layer was washed 2 times with ether evaporated to half the original volume and lyophilized which gave 0.69 g of crude material. It was chronomatographed in the manner described above for octapeptide. Material eluted in tubes 101-120 was collected and lyophilized yielding 0.25 g of product which was shown to be slightly contaminated with minor impurities. It was therefore rechromatographed on the same column, which gave 0.155 g (22%) of pure Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val--Glu-Glu-Ala-Glu Asn. Paper electrophoresis indicated that it was homogeneous. [a]^<5>= -86.27°
(c = 1, 0,1 N HCl).(c = 1, 0.1N HCl).
B.a) .H-Ser(Bzl)-OH (18.6 g, 95.4 mmol) ble o<pp>last i 45 ml triton B, inndampet til tørrhet og resten re--f ordampet to ganger med DMF (10 0 ml hver). Resten ble så rørt med AcOSu (16.9 g, 95.4 mmol) i 150'ml DMF i 20 timer. N-metylmorfulin ble tilsatt fra tid til annen for å. holdt reaksjonsblaningen svakt basisk. Løsningsmiddelet ble fjernet og ekstraksjon av produktet med EtOAc ble etterfulgt av vask med små mengder 10% HOAc, og H20 (produktet er vannløslig, bruk et lite volum H20). Tørkning over Na-^SO^ og inndamping til tørrhet igjen' gav en kalr olje 14.5 g, Ac-Ser.(Bzl) -OIJ. Forbindelsen krystaliserte ikke. Den ble derfor oppløst i en blanding av 300 ml' MeOH B.a).H-Ser(Bzl)-OH (18.6 g, 95.4 mmol) was dissolved in 45 ml triton B, evaporated to dryness and the residue re-evaporated twice with DMF (100 ml each) . The residue was then stirred with AcOSu (16.9 g, 95.4 mmol) in 150 mL DMF for 20 h. N-methylmorpholine was added from time to time to keep the reaction mixture slightly basic. The solvent was removed and extraction of the product with EtOAc was followed by washing with small amounts of 10% HOAc, and H 2 O (the product is water soluble, use a small volume of H 2 O). Drying over Na-^SO^ and evaporation to dryness again gave a caloric oil 14.5 g, Ac-Ser.(Bzl)-OIJ. The compound did not crystallize. It was therefore dissolved in a mixture of 300 ml of MeOH
og 30 ml H20, titrert til. pH 7.0 med. 20 % Cs„C03og inndampet til en fast masse. Saltet ble refordampet 2 ganger mer med DMF og rørt med benzyl - bromid (15.4 g, 91 mmol) i 250 ml and 30 mL H2O, titrated to pH 7.0 with 20% Cs„C03 and evaporated to a solid mass. The salt was re-evaporated twice more with DMF and stirred with benzyl bromide (15.4 g, 91 mmol) in 250 ml
DMF i 13 timer. Etter fordampningen av løsningsmiddelet ble resten tatt opp i H20 (600 ml) og det dannede oljeaktige produkt ble ekstrahert over EtOAc. Det ble vasket medK20. tørket over Na-jSO^ og fordampet til. en sirup som ved podinge øyeblikkelig krystaliserte. Den ble krystalisert fra EtOAc og petroleum eter hvilket gav 10,42 g (32.2 % totalt) av H3C-CO-Ser(Bzl)-OBzl; m.p. 39-91°C. DMF for 13 hours. After evaporation of the solvent, the residue was taken up in H 2 O (600 mL) and the resulting oily product was extracted over EtOAc. It was washed with K20. dried over Na-jSO^ and evaporated to. a syrup which on inoculation instantly crystallized. It was crystallized from EtOAc and petroleum ether to give 10.42 g (32.2% overall) of H3C-CO-Ser(Bzl)-OBzl; m.p. 39-91°C.
H3C-CO-Ser(Bzl)-OBzl (2.2 g, 6.73 mmol) ble oppløst i 75 mlH3C-CO-Ser(Bzl)-OBzl (2.2 g, 6.73 mmol) was dissolved in 75 mL
EtOH og rørt forsiktig med 5 ml.H2NNHp natten over. Noe uløslig-stoff som presipiterte ble frafiltrert og filtratet ble inndampet til en olje som størknet ved behandling med eter. Produktet ble omkryst.alisert fra et lite volum EtOH og eter hvilket gav j i "i 1,40 g (32,3%) av H^C-CO-Ser(Bzl)-HNNH^; m.p. 128-130 C; +5.30° (c MeOH). D EtOH and stirred gently with 5 ml.H2NNHp overnight. Some insoluble matter which precipitated was filtered off and the filtrate was evaporated to an oil which solidified on treatment with ether. The product was recrystallized from a small volume of EtOH and ether to give 1.40 g (32.3%) of H^C-CO-Ser(Bzl)-HNNH^; m.p. 128-130 C; +5.30 ° (c MeOH).D
b) L-alanin (3.57 g, 40 mmol) ble oppløst i 18,3 ml triton B (40 ml), inndampet til tørrhet og den oljeaktiverest b) L-alanine (3.57 g, 40 mmol) was dissolved in 18.3 ml triton B (40 ml), evaporated to dryness and the oil activated
reinndampet 2 ganger ved -DMF (30 ml hver). Det erholdte salt ble rørt med 11,45 g Boe- Ala- OSu (40 ml) i 40 ml DMF, med 4 ml NMM tilsatt i 20 timer. Løsningsmiddelet ble fjernet og evaporated 2 times with -DMF (30 ml each). The salt obtained was stirred with 11.45 g Boe-Ala-OSu (40 ml) in 40 ml DMF, with 4 ml NMM added for 20 hours. The solvent was removed and
resten tatt opp i 10 % HOAc (100 ml ). Produktet ble ekstrahert i EtOAc (4x100 ml), vasket to ganger med et lite volum H20, tørk-et over Na2S04, inndampet til et lite. volum og behandlet med petroleter inntil det ble uklart. Et krystalinsk produkt ble dannet ved lagring i kjøleskap natten over. Utbyttet : 3.2 g(76.3%) the residue taken up in 10% HOAc (100 ml). The product was extracted into EtOAc (4x100 mL), washed twice with a small volume of H 2 O, dried over Na 2 SO 4 , evaporated to a small. volume and treated with petroleum ether until it became cloudy. A crystalline product was formed by overnight storage in a refrigerator. The yield : 3.2 g (76.3%)
av Boc-Ala-Ala-OH; m.p. 115-113°C,of Boc-Ala-Ala-OH; m.p. 115-113°C,
Boc-Ala~Ala-OH (36.2 g, 139 mmol) ble behandlet med 3 1 4 N HClBoc-Ala~Ala-OH (36.2 g, 139 mmol) was treated with 3 1 4 N HCl
i THF i 30 min. Inndampning og opparbeiding som normalt gav en - oljeaktiv-masse som størknet ved behandling med eter . Produktet ble omkrystalisert fra etanol med eter hvilket gav in THF for 30 min. Evaporation and work-up normally yielded an active oil mass which solidified when treated with ether. The product was recrystallized from ethanol with ether to give
9.1 g (33,3%) av HCl • H-Ala-Ala-OH; m.p. 209-211°C-.HCl•H-Ala-Ala-OH (2,36 g, 12 mmol) ble oppløst i 20 ml DMF,kjølt i et isbad og behandlet med 1,63 ml av Et^N(12 mmol) etterfulgt av Boc-Asp(Bzl)-OSu (12 mmol). Blandingen ble rørt forsiktig ved 0°C i 2 timer også 25°C natten over, hvorunder en ekvivalent mengde av Et3N(12 mmol) ble tilsatt i små porsjoner hvilket holdt reaksjonens pH nær 3.0. Noen ml HOAc ble tilsatt og den surgjorte blanding inndampet til tørrhet.Det dannede produktet ble ekstrahert over i EtOAc, vasket med H^O (3 ganger), tørket over Na^So^, og inndampet til en oljeaktivrest (6 g). Den ble oppløst i EtOAc og titrert til pH 3,0 ved DCHA. Det krystalinske saltet feltes ut- og ble omkrystalisert fra iso-propanol og petroleter hvilket gav 5.1 g (65.7%) av Boc-Asp(OBzl)-Ala-Ala-OH• DCHA;m,p. 138-141°; [ct]^<5>= -13.33° (c = 1, MeOH). 9.1 g (33.3%) of HCl • H-Ala-Ala-OH; m.p. 209-211°C-.HCl•H-Ala-Ala-OH (2.36 g, 12 mmol) was dissolved in 20 ml of DMF, cooled in an ice bath and treated with 1.63 ml of Et^N (12 mmol ) followed by Boc-Asp(Bzl)-OSu (12 mmol). The mixture was stirred gently at 0°C for 2 h also 25°C overnight, during which an equivalent amount of Et 3 N (12 mmol) was added in small portions keeping the pH of the reaction close to 3.0. A few ml of HOAc were added and the acidified mixture was evaporated to dryness. The product formed was extracted into EtOAc, washed with H 2 O (3 times), dried over Na 2 SO 4 , and evaporated to an oily residue (6 g). It was dissolved in EtOAc and titrated to pH 3.0 with DCHA. The crystalline salt precipitated out and was recrystallized from iso-propanol and petroleum ether which gave 5.1 g (65.7%) of Boc-Asp(OBzl)-Ala-Ala-OH• DCHA;m,p. 138-141°; [ct]^<5>= -13.33° (c = 1, MeOH).
Boc-Asp(OBzl)-Ala-Ala-OH'DCHA (3.5 g, 5.4 mmol) ble fordelt mellom 500 ml EtOAc og 350 ml H?0 som inneholdt lo ml 10 % H2S04. Det vandige skiktet ble ekstrahert en gang til EtOAc (250 ml) og det sammenslåtte EtOAC skiktet vasket to ganger med H20; Boc-Asp(OBzl)-Ala-Ala-OH'DCHA (3.5 g, 5.4 mmol) was partitioned between 500 mL EtOAc and 350 mL H 2 O containing 10 mL 10% H 2 SO 4 . The aqueous layer was extracted once into EtOAc (250 mL) and the combined EtOAC layer was washed twice with H 2 O;
tørket over Na2S04, og inndampet til tørrhet hvilket gav et I dried over Na2S04, and evaporated to dryness giving an I
én glassaktig masse av Boc-Asp(OBzl)-Ala-Ala-OH (2.5 g).one glassy mass of Boc-Asp(OBzl)-Ala-Ala-OH (2.5 g).
Dette materialet ble behandlet med 200 ml friskt fremstilt 4NThis material was treated with 200 ml of freshly prepared 4N
HCl i THF i 30 min., inndampet ved 32°C til en sirup og reinndampet to ganger til med THF. Den oljeaktige resten ble fast ved behandling med eter. Dette HCl•H-Asp(OBzl)-Ala-Ala-OH (1.93 g, 4.83 mmol) ble brukt i den neste reaksjonen som innebefattet azid-kobling med H^C-CO-Ser(Bzl)-N3 som var fremstilt fra' 1.24 g H3C-CO-Ser(Bzl)-HNNH2(4.9 mmol) i 25 ml DMF (-25°C) med 7.42 ml 3.3 N HCl i THF (24.5 mmol) og 0,99 ml i-amylnitrit (7.35 mmol) rørt i.-30°C i 30 min. Azid-løsningen som var fremstilt ble kjølt ned til -35°C, blandet med 4.1 ml Et3N og så behandlet med det hvite pulveret av HCl H-Asp(OBzl)-Ala-Ala-OH (1.93 g) HCl in THF for 30 min., evaporated at 32°C to a syrup and re-evaporated twice more with THF. The oily residue solidified on treatment with ether. This HCl•H-Asp(OBzl)-Ala-Ala-OH (1.93 g, 4.83 mmol) was used in the next reaction involving azide coupling with H^C-CO-Ser(Bzl)-N3 prepared from ' 1.24 g H3C-CO-Ser(Bzl)-HNNH2 (4.9 mmol) in 25 ml DMF (-25°C) with 7.42 ml 3.3 N HCl in THF (24.5 mmol) and 0.99 ml i-amyl nitrite (7.35 mmol ) stirred at -30°C for 30 min. The azide solution which was prepared was cooled down to -35°C, mixed with 4.1 ml of Et3N and then treated with the white powder of HCl H-Asp(OBzl)-Ala-Ala-OH (1.93 g)
som var fremstilt ovenfor. Blandingen ble rørt ved -20°C i 30 min og så ved 4°C i to dager. Noe mer Et3N ble tilsatt for å holde reaksjonen svakt basisk. Normal opparbeiding av en krystalinsk masse som ble krystalisert fra THF og petroleter hvilket gav 1.85 g (65.6 %) av' H3C-CO-Ser (Bzl)-Asp (OBzl)-Ala-Ala-OH m.p. 167-170°C; [a]^<3>= 18.91° (c = 1, DMSO). which was presented above. The mixture was stirred at -20°C for 30 min and then at 4°C for two days. Some more Et3N was added to keep the reaction slightly basic. Normal work-up of a crystalline mass which was crystallized from THF and petroleum ether gave 1.85 g (65.6%) of H3C-CO-Ser (Bzl)-Asp (OBzl)-Ala-Ala-OH m.p. 167-170°C; [α]^<3>= 18.91° (c = 1, DMSO).
'H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH (0,825 g. 1,41 mmol)'ble oppløst i 4 ml DMF og avkjølt til 0°C i et isbad. Løsningen ble tilsatt H2NNH2(54.3 mg ; 1.69 mmol) etterfulg av HOBT- 'H 3 C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH (0.825 g, 1.41 mmol)' was dissolved in 4 ml of DMF and cooled to 0°C in an ice bath. The solution was added H2NNH2 (54.3 mg ; 1.69 mmol) followed by HOBT-
H20 (0,475 g,3.10 mmol) og -DCC (0,32 g. 1,55 mmol). Blanding-H 2 O (0.475 g, 3.10 mmol) and -DCC (0.32 g, 1.55 mmol). Mixture-
en ble justert til pH 7.5 med NMM og rørt ved 0°C i'2 timer etterfulgt av 17 timer ved 25°C, Reakajorismassen ble en gel i løpet av denne tiden.. Den ble fortynnet med MeOH og det faste materialet som ble tilbake ble oppsamlet på et sugefilter og vasket grundig MeOH, eter og petroleter hvilket gav et materiale som smeltet ved 2 29-2 32°C. Produktet ble så felt fra DMF og MeOH hvilket gav 0,51 g (61.0%) av H3C-CO~Ser(Bzl)-Asp(OBzl)-. Ala-Ala-HNNH2; m.p. 230-232°C; [ a]* 5 = -17.94° (c = 1. DMSO). one was adjusted to pH 7.5 with NMM and stirred at 0°C for 2 hours followed by 17 hours at 25°C, the reaction mass became a gel during this time. It was diluted with MeOH and the solid material remaining was collected on a suction filter and washed thoroughly with MeOH, ether and petroleum ether which gave a material melting at 2 29-2 32°C. The product was then precipitated from DMF and MeOH giving 0.51 g (61.0%) of H3C-CO~Ser(Bzl)-Asp(OBzl)-. Ala-Ala-HNNH2; m.p. 230-232°C; [α]* δ = -17.94° (c = 1. DMSO).
c) H-Glu(OBzl)-OH (39.4 g, 166 mmol) ble rørt med Boc-Ser(Bzl)-OSu (65.0 g, 166 mmol) i 900 ml DMF natten over i nær-var av Et3N(2.3 ml, 165 mmol). Mer Et3N ble tilsatt i løpet av denne tiden for å holde reaksjonsblandingen svakt basisk. Den klare løsningen ble inndampet til tørrhet og den oljeaktige resten fordelt mellom EtOAc (1,5 1) og 5% HOAc (2 1). Det organiske skiktet ble vasket med H20 (2 ganger), tørket over Na2S04 og konsentrert til en klar olje (90,0 g) som ble tatt opp i 3 liteif eter og behandlet med 25 ml cycloheksylamin. Det faste stoffet som ble danned ble rekrystalisert fra MeOH og eter. Utbyttet; 76.2 g (74.8%) Boc-Ser (Bzl) --Glu (OBzl) -OH-CHA; m.p. 154-156°C; [a]^5 =■+ 6.32° (c = 1, MeOH). c) H-Glu(OBzl)-OH (39.4 g, 166 mmol) was stirred with Boc-Ser(Bzl)-OSu (65.0 g, 166 mmol) in 900 mL DMF overnight in the presence of Et3N (2.3 mL , 165 mmol). More Et 3 N was added during this time to keep the reaction mixture slightly basic. The clear solution was evaporated to dryness and the oily residue partitioned between EtOAc (1.5 L) and 5% HOAc (2 L). The organic layer was washed with H 2 O (2x), dried over Na 2 SO 4 and concentrated to a clear oil (90.0 g) which was taken up in 3 liters of ether and treated with 25 mL of cyclohexylamine. The solid that formed was recrystallized from MeOH and ether. The dividend; 76.2 g (74.8%) Boc-Ser(Bzl)--Glu(OBzl)-OH-CHA; m.p. 154-156°C; [α]^5 =■+ 6.32° (c = 1, MeOH).
Boc-Ser(Bzl)-Glu(OBzl)-OH-CHA (76.2 g, 124 mmol) ble oppslemmet i en blanding av.1.5 liter av H20-og EtOAc hver. Denne blandingen ble tilsatt 10 % ^SO^ inntil den ble sur (pH ca. 2,5) og det faste'stoffet gikk i oppløsning. Det organiske skiktet som inneholdt den fri dipaptid-syren ble vasket med K90 ( 2 ganger), tørket og inndampet til tørrhet hvilket gav en klar olje (63,5 g)-. Den ble behandlet med 3 liter friskt fremstilt 4.1 N HCl i THF Boc-Ser(Bzl)-Glu(OBzl)-OH-CHA (76.2 g, 124 mmol) was slurried in a mixture of 1.5 L each of H 2 O and EtOAc. To this mixture was added 10% ^SO^ until it became acidic (pH about 2.5) and the solid dissolved. The organic layer containing the free dipeptide acid was washed with K90 (2 times), dried and evaporated to dryness to give a clear oil (63.5 g). It was treated with 3 liters of freshly prepared 4.1 N HCl in THF
i 45 minutter og inndampet til en oljeaktivrest som ble reinndampet to ganger til med THF. Resten HCl • II-Ser (Bzl)-Glu (OBzl)-OH ble oppløst i DMF (500 ml),kjølt til 0°C, og behandlet med Boc-Ser(Bzl)-OSu (43.66 g, 124 mmol) øyeblikkelig etterfulgt for 45 minutes and evaporated to an oily residue which was re-evaporated twice more with THF. The residue HCl • II-Ser(Bzl)-Glu(OBzl)-OH was dissolved in DMF (500 mL), cooled to 0°C, and treated with Boc-Ser(Bzl)-OSu (43.66 g, 124 mmol) immediately followed
av 27 ml Et-^N. Blandingen ble rørt natten over ved 25°C hvor under mer Et^N ble tilsatt leilighetsvis for å holde reaskjons-blandingen svakt basisk. Noen små mengder av uorganisk stoff ;: ble fjernet ved filtrering og filtratet ble inndampet til en '•. olje som ble tatt opp i EtOAc, vasket med 5 % HOAc og H,,0, tørk-et over Na2S04og igjen fordampet til tørrhet. Produktet ble krystalisert fra EtOAc med petroleter. Utbyttet: 71.8 g(33.7%) av Boc-Ser (Bzl)-Ser (Bzl)- Glu (OBzl)-OH ; m.p. 112-113°C; [a<]>^<5>=. +17,91° (c =1, THF). of 27 ml Et-^N. The mixture was stirred overnight at 25°C during which time more Et₂N was added occasionally to keep the reaction mixture slightly basic. Some small amounts of inorganic matter ;: were removed by filtration and the filtrate was evaporated to a '•. oil which was taken up in EtOAc, washed with 5% HOAc and H 2 O, dried over Na 2 SO 4 and again evaporated to dryness. The product was crystallized from EtOAc with petroleum ether. The yield: 71.8 g (33.7%) of Boc-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH; m.p. 112-113°C; [a<]>^<5>=. +17.91° (c = 1, THF).
Boc-Ser (Bzl)-Ser (Bzl)-Glu (OBzl)-OH .(71.6 g,104 mmol) ble behandlet med 2. 7 1 friskt fremstilt 3.9 N HCl i THF i 45 min.. Blandingen ble inndampet til tørrhet og resten inndampet 2 ganger til med THF hvilket gav en fast masse (59,3 g, m.p. 161-165°C). Den ble oppsamlet og vasket med eter og rørt i 500 ml DMF med Boc-Thr(Bzl)-OSu (38.2 g, 94 ,mmolj i nærvær av Et3N Boc-Ser (Bzl)-Ser (Bzl)-Glu (OBzl)-OH .(71.6 g, 104 mmol) was treated with 2.7 L of freshly prepared 3.9 N HCl in THF for 45 min. The mixture was evaporated to dryness and the residue evaporated 2 more times with THF which gave a solid mass (59.3 g, m.p. 161-165°C). It was collected and washed with ether and stirred in 500 mL DMF with Boc-Thr(Bzl)-OSu (38.2 g, 94 mmol) in the presence of Et3N
(25 il) i 0 C i 1 time også ved 25 C i 15 timer. Mer Et^N(25 µl) at 0 C for 1 hour also at 25 C for 15 hours. More Et^N
(14.5 ml) ble tilsatt i flere porsjoner i løpet av denne tiden for å holde reaksjonen svakt basisk. Noe dannet uløslige materiale frafiltrert og filtratet inndampet til en olje som ble oppløst i EtOAc (1,5 1), vasket med 5% H20 (2x), tørket over Na2SO^og inndampet til én fast masse. Produktet ble omkrystalisert fra EtOAc og petroleter hvilket gav 64.8 g (73.1%) av Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH:m.p. 115-113°C; (14.5 mL) was added in several portions during this time to keep the reaction slightly basic. Some insoluble material formed was filtered off and the filtrate evaporated to an oil which was dissolved in EtOAc (1.5 L), washed with 5% H 2 O (2x), dried over Na 2 SO 4 and evaporated to a solid. The product was recrystallized from EtOAc and petroleum ether to give 64.8 g (73.1%) of Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH: m.p. 115-113°C;
.Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (54.5 g, 61,7 mmol) ble behandlet med HCl i THF (1,5 liter; 4.1 N) og opparbeidet . som vanlig hvilket gav HCl H-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)- Glu( .Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (54.5 g, 61.7 mmol) was treated with HCl in THF (1.5 L; 4.1 N) and worked up . as usual which gave HCl H-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)- Glu(
(OBzl)-OH (46.3 g, 56.6 mmol) som et hvitt pulver. Det ble så rørt i DMF (500 ml) med Boc-Asp(OBzl)-OSu (23.7 g, 56 mmol) i 0°C i 2 timer i nærvær av Et-^N (16 ml). Balndingen blé vidre rørt i 25°C i 15 timer hvorunder ytteligere 7.4 liter Et^N (OBzl)-OH (46.3 g, 56.6 mmol) as a white powder. It was then stirred in DMF (500 mL) with Boc-Asp(OBzl)-OSu (23.7 g, 56 mmol) at 0°C for 2 h in the presence of Et-N (16 mL). The mixture was further stirred at 25°C for 15 hours during which an additional 7.4 liters of Et^N
ble tilsatt. Produktet ble opparbeidet som vanlig krystalisert fra CH2C12og petroleter. Utbyttet: 50.35 g (82.7%) av Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)Ser(Bzl)-Glu(OBzl)-OH; m.p. 111-113°C; [alp<5>= +7,21° ( c = 1,DMSO). was added. The product was worked up as usual, crystallized from CH2C12 and petroleum ether. Yield: 50.35 g (82.7%) of Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)Ser(Bzl)-Glu(OBzl)-OH; m.p. 111-113°C; [αlp<5>= +7.21° ( c = 1.DMSO).
Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (50.0 g,Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (50.0 g,
46 mmol) ble avmasket med HCl (4.15 N) i THF og opparbeidet som vanlig hvilket gav 45,'4 g hvitt fast stoff. Den ble oppløst i THF (1,5 liter) og behandlet med eter (7 liter). Ved én stand på 0°C natten over erholdtes et hvitt fast nulver (44,0 g, m.p- 179-184°C) . Deler av dette materialet HCl • H-Asp (OBzl).-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (43.7 g, 42,7 mmol) 46 mmol) was demasked with HCl (4.15 N) in THF and worked up as usual which gave 45.4 g of white solid. It was dissolved in THF (1.5 L) and treated with ether (7 L). At 0°C overnight, a white solid was obtained (44.0 g, m.p. 179-184°C). Parts of this material HCl • H-Asp (OBzl).-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (43.7 g, 42.7 mmol)
ble så oppløst i 500 ml DMF, avkjølt til 0°C og behandlet med Et^NdO ml). Blandingen ble rørt i 15 timer hvorunder mer Et^N (7,9 ml) ble tilsatt i flere porsjoner for å holde reaksjonen svakt basisk. Det uløslige stoffet ble fjernet ved filtrering og filtratet inndampet til tørhet. Den oljeaktive resten ble, oppløst i CH2C12, vasket med 5 % KOAc, H-jO, tørket over Na2S04, og inndampet til et mindre volum (0,5 liter) under tilsetning av petroleter. Produktet krystaliserte langsomt ved henstand natten over. Det ble omkrystalisert fra THF og isopropanol hvilket gav 26.3' g(51.4%) av Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(Obzl)-OH; m,p. 174-177°C: [a]^<5>= +0,34° (c 1, THF). was then dissolved in 500 ml DMF, cooled to 0°C and treated with Et^NdO ml). The mixture was stirred for 15 hours during which more Et₂N (7.9 mL) was added in several portions to keep the reaction slightly basic. The insoluble material was removed by filtration and the filtrate evaporated to dryness. The oil-active residue was dissolved in CH 2 Cl 2 , washed with 5% KOAc, H 2 O, dried over Na 2 SO 4 , and evaporated to a smaller volume (0.5 liter) while adding petroleum ether. The product crystallized slowly on standing overnight. It was recrystallized from THF and isopropanol which gave 26.3 g (51.4%) of Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(Obzl)-OH; m.p. 174-177°C: [α]^<5>= +0.34° (c 1, THF).
Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)- OH (13.0 g; 10.94 mmol) ble oppløst i DMF (50 ml) avkjølt til 0°C og så behandlet med H,NNH2(0,421 g; 13,. 14 mmol) HOBT (3.683 g; 24.1.mmol) og DCC (2,48 g; 12,04 mmol), NMM ble så tilsatt inntil reaksjonen viste pH 7.5. Blandingen ble rørt 13 timer og-fUt-rert for å fjerne uløslige biprodukter. Filtratet ble inndampet til tørhet og resten behandlet med H20. Det dannede faste stoff ble oppsamlet og krystalisert fra DMF og isonronanol hvilket gav 8.7 g (66.4%) av Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu (OBzl)-HNNH2; m.p. 215-213°C; [ a]* 5 = [37.62° (c = 1 DMSO). Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (13.0 g; 10.94 mmol) was dissolved in DMF (50 mL) cooled to 0 °C and then treated with H,NNH 2 (0.421 g; 13.14 mmol) HOBT (3.683 g; 24.1 mmol) and DCC (2.48 g; 12.04 mmol), NMM was then added until the reaction showed pH 7.5. The mixture was stirred for 13 hours and filtered to remove insoluble byproducts. The filtrate was evaporated to dryness and the residue treated with H 2 O. The solid formed was collected and crystallized from DMF and isonronanol yielding 8.7 g (66.4%) of Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu (OBzl)- HNNH2; m.p. 215-213°C; [α]*δ = [37.62° (c = 1 DMSO).
d) Boc-Thr(Bzl)-Lys(Z) OH (14.0 g, 24.5 mmol) ble behandlet med 500 ml 4,0 N HCl i THF i 30 min,, inndam<p>et til tør-het og g jeninndampet 2 ganger med friskt TIIF. Denne oljeaktivresten størknet ved behandling med eter. Det størkede pulvere (11,4 g ,21.6 mmol) av dipeptid-hydrokl.oridsaltet ble så opp-løst i 140 ml DMF,' avkjølet til 0°C og behandlet med Boc-Thr(Bzl)-OSu (8,8 g, 21.6 mmol) etterfulgt av 3.5 ml Et^N. Noen dråper Et^N ble tilsatt å holde reaksjonsbetingelsene svakt basiske mens blandingen ble rørt i ytteligere 24 timer ved 25°C. Den ble surgjort med 5 ml HOAc og så fortynnet med et stort volum vann. Det faste rå-produktet som utfeltes ble oppsamlet, oppløst i EtOAc, vasket méd n^CK tørket over Na^SO^ , og inndampet til tørhet hvilket gav en glassaktig fast masse. Krystal-isasjonen fra EtOAc og petroleter gav 13.8 g (83.7%) av Boc-Thr (Bzl ) -Thr (Bzl ) -Lys ( Z ) -OH ; m.D. 110-112°C; [a]25 = +19,45° d) Boc-Thr(Bzl)-Lys(Z)OH (14.0 g, 24.5 mmol) was treated with 500 ml of 4.0 N HCl in THF for 30 min, evaporated to dryness and re-evaporated 2 times with fresh TIIF. This oil active residue solidified on treatment with ether. The solidified powder (11.4 g, 21.6 mmol) of the dipeptide hydrochloride salt was then dissolved in 140 ml of DMF, cooled to 0°C and treated with Boc-Thr(Bzl)-OSu (8.8 g , 21.6 mmol) followed by 3.5 ml Et^N. A few drops of Et^N were added to keep the reaction conditions slightly basic while the mixture was stirred for an additional 24 hours at 25°C. It was acidified with 5 mL of HOAc and then diluted with a large volume of water. The solid crude product that precipitated was collected, dissolved in EtOAc, washed with n^CK dried over Na^SO^ , and evaporated to dryness which gave a glassy solid mass. The crystallization from EtOAc and petroleum ether gave 13.8 g (83.7%) of Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH; m.d. 110-112°C; [a]25 = +19.45°
D D
(c = 1, EtOAc).(c = 1, EtOAc).
Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (41.5 g, 54 mmol) ble behandlet med 500 ml friskt fremstilt 3.55 N HCl i THF i 25 min..ved inndampning til. en sirup som ble gjeninndampet 2 ganger med friskt THF. Den oljeaktige resten størknet ved behandling med eter. Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (41.5 g, 54 mmol) was treated with 500 ml of freshly prepared 3.55 N HCl in THF for 25 min..by evaporation to. a syrup which was re-evaporated twice with fresh THF. The oily residue solidified on treatment with ether.
Den ble oppsamlet og vasket med eter hvilket gav 37.4 g rått-hydrokloridsalt av tripeptidet oppløst i 500 ml DMF avkjølt til 0°C og behandlet med Boc-Ile-OSu 817.4 g, 53 mmol) etterfulgt av 16 ml Et^N. Blandingen ble rørt ved 25°C natten over hvorunder mer Et^N ble tilsatt i små porsjoner ( 6.2 ml totalt ) for å holde svakt basiske betingelser. Den resulterende blandingen ble filtrert og filtratet inndampet til en olje som ble ekstrahert over i EtOAc, vasket med 5% HOAc, H?0 , tørket over Na-jSO^, og inndampet hvilket gav en gulaktig olje. Den ble krystalisert. fra EtOAc og petroleter. Det således erholdte rå faste stoffet (39,6 , m.p. 140 - 142°C) ble funnet å være forurenset med flere små forurensninger. Materialet ble så kromat-ografert på kisel gel kolonne (70-230 mesh, 4.7 x 67 cm) ved bruk av CHCl^/MeOH (95:5. v/v) som eluent. Fraksjonene som innef holdt det ønskede produkt ( Kontrolert ved TC1 ) ble slått sammen og inndampet hvilket gav et oljeaktivt produkt som ble krystalisert fra CHC13 og petroleter. Utbytte: 19.1 g(41,2%) It was collected and washed with ether to give 37.4 g crude hydrochloride salt of the tripeptide dissolved in 500 ml DMF cooled to 0°C and treated with Boc-Ile-OSu 817.4 g, 53 mmol) followed by 16 ml Et^N. The mixture was stirred at 25°C overnight during which time more Et^N was added in small portions (6.2 ml total) to maintain slightly basic conditions. The resulting mixture was filtered and the filtrate evaporated to an oil which was extracted into EtOAc, washed with 5% HOAc, H 2 O , dried over Na 2 SO 4 , and evaporated to give a yellowish oil. It was crystallized. from EtOAc and petroleum ether. The crude solid thus obtained (39.6 , m.p. 140 - 142°C) was found to be contaminated with several small impurities. The material was then chromatographed on a silica gel column (70-230 mesh, 4.7 x 67 cm) using CHCl 2 /MeOH (95:5. v/v) as eluent. The fractions containing the desired product (Controlled by TC1) were pooled and evaporated which gave an oil-active product which was crystallized from CHCl 3 and petroleum ether. Yield: 19.1 g (41.2%)
av Boe-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH: m.p. 144-146°C: [a]2<5>= +2.40° (c = 1, CHC13). of Boe-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH: m.p. 144-146°C: [α]2<5>= +2.40° (c = 1, CHCl 3 ).
Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (0,439 g 0,496 mmol) ble behandlet med 4 N HCl i THF i 3o min. og opparbeidet som vanlig hvilket gav 0,39 g av HCl • H-Ile-Thr (Bzl) -Thr (Bzl.) -Lys (Z) -OH. Boc-Val-Asp (OBzl) -Thr (Bzl).-Ser (Bzl) -Ser (Bzl) -Glu (OBzl) -HNNH^ Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (0.439 g 0.496 mmol) was treated with 4 N HCl in THF for 30 min. and worked up as usual which gave 0.39 g of HCl • H-Ile-Thr (Bzl)-Thr (Bzl.)-Lys (Z)-OH. Boc-Val-Asp (OBzl) -Thr (Bzl).-Ser (Bzl) -Ser (Bzl) -Glu (OBzl) -HNNH^
(o,59 g. 0,492 mmol) ble så oppløst i 6 ml av DMF, avkjølt til - 2<5>°C.og behandlet med 0,5 7 ml 4,3 N HCl i THF (2.46 mmol) umiddelbart fulgt av 0,1 ml i-amylnitrit (0,74 mmol). Etter røring ved -20° - -25°C i 30 min ble temperaturen senket til -35°C hvoretter 0,42 ml Et3N og HCl•H-ILe-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (0,39 g, fremstilt som ovenfor) bie tilsatt. Blandingen ble . rørt til -20°C i 30 min og så ved 4°C i 48 timer hvorunder Et3N ble tilsatt fra tid.til annen for å holdt pH på ca 7.5. Blandingen ble så fortynnet med 250 ml 5% HOAc og det dannede faste produktet ble oppsamlet og vasket med H20, MeOH, eter og tørket over NaOH-pellets i våkum hvilket gav 0,32 g rått materiale (m.p. 244-254°C). (0.59 g. 0.492 mmol) was then dissolved in 6 mL of DMF, cooled to -2<5>°C. and treated with 0.57 mL of 4.3 N HCl in THF (2.46 mmol) immediately followed by 0.1 ml of i-amyl nitrite (0.74 mmol). After stirring at -20° - -25°C for 30 min, the temperature was lowered to -35°C, after which 0.42 ml of Et3N and HCl•H-ILe-Thr(Bzl)-Thr(Bzl)-Lys(Z)- OH (0.39 g, prepared as above) bee added. The mixture became . stirred at -20°C for 30 min and then at 4°C for 48 hours during which Et3N was added from time to time to keep the pH at about 7.5. The mixture was then diluted with 250 ml of 5% HOAc and the solid product formed was collected and washed with H 2 O, MeOH, ether and dried over NaOH pellets in vacuo to give 0.32 g of crude material (m.p. 244-254°C).
Det ble.oppløst DMSO og presipitert ved tilsetning av MeOH.. Utbyttet:0,698 g (81,7%) av Boc-Val-Asp(Bzl)-Thr(Bzl)-Ser(Bzl)-Ser (Bzl)-Glu(OBzl)-ILe-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH; m.p. 263-271°C. DMSO was dissolved and precipitated by addition of MeOH. Yield: 0.698 g (81.7%) of Boc-Val-Asp(Bzl)-Thr(Bzl)-Ser(Bzl)-Ser (Bzl)-Glu( OBzl)-ILe-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH; m.p. 263-271°C.
H3C-CO-Ser (Bzl)-Asp (OBzl)-Ala-Ala-HNNH2 (0,4.08.g, 0,63.mmol) oppslemmet i 10 ml DMF ble avkjølet til -20°C og behandlet med frisk fremstilt 5.43 N, KCl i THF (0,627 ml, 3.4 mmol) etter-, fulgt av 10% i-amylnitrit i DMF (1,39 ml, 1,03 mmol).Etter 30 min. røring ble den avkjølt ned til -30°C hvoretter Et-^N (0,476 ml, 3.4 mmol) ble tilsatt etterfulgt av TFA-saltet av decapeptidet H-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr (Bzl ) -Ly s ( Z ) -OH ( 1, 334 g, 0,63 mmol). Blandingen ble rørt H3C-CO-Ser (Bzl)-Asp (OBzl)-Ala-Ala-HNNH2 (0.4.08.g, 0.63.mmol) slurried in 10 ml DMF was cooled to -20°C and treated with freshly prepared 5.43 N, KCl in THF (0.627 mL, 3.4 mmol) after, followed by 10% i-amyl nitrite in DMF (1.39 mL, 1.03 mmol). After 30 min. stirring, it was cooled down to -30°C after which Et-^N (0.476 mL, 3.4 mmol) was added followed by the TFA salt of the decapeptide H-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)- Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Lys(Z)-OH (1.334 g, 0.63 mmol). The mixture was stirred
i -20°C i 30 min og så ved 4°C i 5 dager hvorunder mer Et^N®9DMSO ble tilsatt for å holde reaksjonen under svakt basiske betingelser og forhindre at gelen ble dannet. Reaksjonsblandingen ble helt i 5% HOAc (300 ml) og det faste dannede<p>resipitatet ble oppsamlet vasket med H20,MeOH, eter, og tørket hvilket gav i at -20°C for 30 min and then at 4°C for 5 days during which more Et^N®9DMSO was added to keep the reaction under slightly basic conditions and prevent the gel from forming. The reaction mixture was poured into 5% HOAc (300 mL) and the solid formed<p>precipitate was collected, washed with H 2 O, MeOH, ether, and dried to give
1,49 g materiale som smeltet ved 296-299°C. Produktet ble re-presipitert fra DMSO med MeOH. Utbyttet: 1,4 0 g (35.37%) av H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-LYs(Z)-OM; [a]2<5>= +6.37° 1.49 g of material which melted at 296-299°C. The product was re-precipitated from DMSO with MeOH. Yield: 1.40 g (35.37%) of H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl )-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-LYs(Z)-OM; [a]2<5>= +6.37°
(c = l,DMSO).(c = 1,DMSO).
Det erholdte tetradecapeptidet (1,35 g.0,558 mmol) ble rørt med HOBT-H20 (o,18S g,l,23 mmol) noen få minutter i en blanding av The resulting tetradecapeptide (1.35 g.0.558 mmol) was stirred with HOBT-H 2 O (0.18S g.1.23 mmol) for a few minutes in a mixture of
15 ml av hver av DMF og DMSO. Blandingen ble.så kjølet i et is-bad hvoretter DCC (0,126 g,0,614 mmol) ble tilsatt og røring fortsatte i 2 4 timer i samme temperatur. I en adskilt kolbe ble det. Boc--Asn (OBzl) -Leu-Lys (Z) -Glu (OBzl) -Lys (Z ) --Lys.(Z) -Glu (OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (4,0 g, 1,43 mmol) behandlet med 40 ml TFA, i 25 min. og TFA-saltet av det dannede tetradecapeptidet raskt felt under tilsetning av volum-eter. Det dannede faste stoff onpsamlet og vasket grundig med eter hvilket gav 3.74' g. hvitt pulver (TFA-saltet av C-terminalt tetradecapeptid). Deler av dette materialet (1,567 g, 0,5533 mmol) ble satt til den aktive esteren som stammet fra amino terminalt tetradecapeptid som fremstilt ovenfor' i en DMFDMSO blanding. Noen få dråper N.MM ble tilsatt for å bringe reaksjonens pH til 7.5-8,0 og røring ble fortsatt en time ved 0°C 15 ml each of DMF and DMSO. The mixture was then cooled in an ice bath after which DCC (0.126 g, 0.614 mmol) was added and stirring continued for 24 hours at the same temperature. In a separate flask it was. Boc--Asn (OBzl) -Leu-Lys (Z) -Glu (OBzl) -Lys (Z ) --Lys.(Z) -Glu (OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl) -Ala-Glu(OBzl)-Asn-OBzl (4.0 g, 1.43 mmol) treated with 40 mL TFA, for 25 min. and the TFA salt of the tetradecapeptide formed quickly precipitated with the addition of bulk ether. The solid formed was collected and washed thoroughly with ether to give 3.74 g of white powder (the TFA salt of C-terminal tetradecapeptide). Portions of this material (1.567 g, 0.5533 mmol) were added to the active ester derived from amino terminal tetradecapeptide as prepared above in a DMFDMSO mixture. A few drops of N.MM were added to bring the pH of the reaction to 7.5-8.0 and stirring was continued for one hour at 0°C
og så fem dager ved 25°C.Reaksjonsblandingen ble så helt i 1 5 1 5% eddiksyre. Det presioiterte produkt ble vasket grundig med H20, MeOH, DMF, MeOH og eter hvilket gav 2.2.1 g av det ønskede produkt, H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(BZL)-Ser (Bzl),-Ser (Bzl) -Thr (Bzl) - Ile-Thr (Ezl) -Thr (Bzl) -Lys (Z) -Asn (OBzl) - Leu-LYs(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl som smeltet over 300°C. and then five days at 25°C. The reaction mixture was then poured into 1 5 1 5% acetic acid. The precipitated product was washed thoroughly with H 2 O, MeOH, DMF, MeOH and ether to give 2.2.1 g of the desired product, H 3 C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl )-Thr(BZL)-Ser (Bzl),-Ser (Bzl) -Thr (Bzl) - Ile-Thr (Ezl) -Thr (Bzl) -Lys (Z) -Asn (OBzl) - Leu-LYs(Z )-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl which melted above 300 °C.
Det beskyttede oktacosapentidet (2.21 g,0,435 mmol) ble oppløstThe protected octacosapentide (2.21 g, 0.435 mmol) was dissolved
i 8 ml TFA, blandet med 4 ml anisol og rørt med vannfritt. iHFin 8 ml TFA, mixed with 4 ml anisole and stirred with anhydrous. iHF
ved 0°C ved 30 min. Syrene ble fjernet ved 0°C (vakum-destila-sjonen) og den faste resten som ble tilbake ble onnløst i 200 at 0°C at 30 min. The acids were removed at 0°C (vacuum distillation) and the solid residue that remained was dissolved in 200
ml H90, vasket 2 ganaer med eter, inndamnet til halvparten av volumet og leofylisert ' hvilket gav 1.1 g rå-produkt. Det ble renset ved en gang gjennom en Sefakex<R>'G-1<0>kolonne ( 3 x 30 cm; 0,2M HOAc) og så en DEAE-Sefadex^kolonne (3 x 75 cm) under elu-ering med økende konsentrasjon av ammonium acetat (pH 7.0 ,0,025.M, 0,25M) etterfulgt av fortynnet eddiksyre. Fraksjonene som inne-! ml H90, washed 2 g with ether, reduced to half the volume and lyophilized to give 1.1 g of crude product. It was purified once through a Sephakex<R>'G-1<0> column (3 x 30 cm; 0.2M HOAc) and then a DEAE-Sefadex^ column (3 x 75 cm) eluting with increasing concentration of ammonium acetate (pH 7.0 ,0.025.M, 0.25M) followed by dilute acetic acid. The factions that in-!
holdt det ønskede materialet ble slått sammen og leofylisert hvilket gav 0,231 g H3C"CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn [tymosin a^] som et amort hvitt pulver. kept the desired material was pooled and lyophilized yielding 0.231 g of H3C"CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys -Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn [thymosin a^] as an off-white powder.
På akrylamid gel isoelektrisk fokus beveger det syntetiske materialet seg identisk med det naturlige tymosin a, isolert On acrylamide gel isoelectric focus, the synthetic material moves identically to the natural thymosin a, in isolation
fra bovintymus-kjertel (Proe.Nati. Acad. Sei. (USA). 7 4, 725 (1977)). from bovine thymus gland (Proe.Nati. Acad. Sei. (USA). 7 4, 725 (1977)).
Eksempel 2Example 2
Boc-Ala-Ala-OH (7,23 g, 27,3 mmol) ble opnløst i en blandingBoc-Ala-Ala-OH (7.23 g, 27.3 mmol) was dissolved in a mixture
av 200 ml MeOH'og 20 ml H20. Cs2C03løsning (20% vanndig) ble tilsatt -inntil pH nådde 7.0' ( ca 30 ml) og den resulterende nøytrale løsningen ble inndampet til tørhet. Resten ble reinndampet 2 ganger med DMF (15 0 ml) og det gelantiløse faste'stoffet som ble tilbake ble rørt i 120 ml DMF med benzylbromid of 200 ml MeOH' and 20 ml H 2 O. Cs 2 CO 3 solution (20% aqueous) was added -until the pH reached 7.0' (about 30 ml) and the resulting neutral solution was evaporated to dryness. The residue was evaporated twice with DMF (150 ml) and the gel-antifree solid that remained was stirred in 120 ml of DMF with benzyl bromide
(7,2 g, 4 2 mmol) i 15 timer. Løsningsmiddelet ble så fjernet ved inndampning og resten behandlet med 500 ml vann. Produkt- (7.2 g, 42 mmol) for 15 h. The solvent was then removed by evaporation and the residue treated with 500 ml of water. Product-
et presipiterte som en vandig olje som gradvis størknet ved ' henstand. Den ble tatt opp i EtOAc (400 ral) vasket med H2'0 a precipitated as a watery oil which gradually solidified on ' standing. It was taken up in EtOAc (400 ral) washed with H 2 O
(3x), tørket over Na^So^q^<i>nndam<p>et til en sirup som ved behandling med petroleter begynte og krystalisere. Produktet ble omkrystalisert fra EtOAc og petroleter hvilket gav 7.2 g. (3x), dried over Na^So^q^<i>nndam<p>et to a syrup which on treatment with petroleum ether began to crystallize. The product was recrystallized from EtOAc and petroleum ether giving 7.2 g.
(73,8%) Boc-Ala-Ala-OBzl; .m.p. 71-73°C.(73.8%) Boc-Ala-Ala-OBzl; .m.p. 71-73°C.
Boc-Ala-Ala-OBzl (6.0 g, 17,15 mmol) ble -behandlet med 380 ml frisk fremstilt 4 N HCl i THF i 30 min, Syre-overskudd og løsn-ingsmiddelet ble avdampet og den tilbakeblivende sirup gjennin-dampet 2 ganger med friskt THF. Resten størknet øyeblikkelig ved behandling med eter. Den ble omkrystalisert fra MeOH og eter hvilket gav 4.30 g (76,4%) HCl•H-Ala-Ala-OBz1; [a]^<5>=-38,86° Boc-Ala-Ala-OBzl (6.0 g, 17.15 mmol) was -treated with 380 ml of freshly prepared 4 N HCl in THF for 30 min, Excess acid and the solvent were evaporated and the remaining syrup re-evaporated 2 times with fresh THF. The residue solidified immediately on treatment with ether. It was recrystallized from MeOH and ether to give 4.30 g (76.4%) HCl•H-Ala-Ala-OBz1; [a]^<5>=-38.86°
(c = 1 , MeOH).(c = 1, MeOH).
HCl'H-Ala-Ala-OBzl (4.25 g 14,83 mmol) ble oppløst i DMF (60 ml) avkjølet til 0°C og rørt med Boc-Asn-OII (3,45 g, 14.33 mmol) HOBT (4,02 g, 28,6 mmol), NMM (2ml) og DCC (3.37 g, 16.35 mmol) ved 0°C i to timer og så ved 25°C i 20 timer. Mer NMM ble tisatt fra tid til annen for å holde reaksjonen svakt basisk. De u— j HCl'H-Ala-Ala-OBzl (4.25 g 14.83 mmol) was dissolved in DMF (60 mL) cooled to 0°C and stirred with Boc-Asn-OII (3.45 g, 14.33 mmol) HOBT (4 .02 g, 28.6 mmol), NMM (2 ml) and DCC (3.37 g, 16.35 mmol) at 0°C for two hours and then at 25°C for 20 hours. More NMM was added from time to time to keep the reaction slightly basic. They u— j
. løslig dannede biproduktene ble frafiltrert og filtratet inndampet til tørrhet. Den oljeaktive resten størknet umiddelbart etter behandling med vann. Den ble tatt onn i EtOAc, vasket med H"20 (3x) , tørket over Na^SO^og inndampet til et mindre volum hvorved produktet begynte å krystalisere. Det samme volum petroleter ble tilsatt og blandingen fikk stå natten over. Rå-produktet ble omkrystalisert fra THF og petroleter hvilket gav 5.2 g(75.6%) Boc-Asn-Ala-Ala-OBzl; m.p. 153-155.°C; [«lp<5>=-55.61° . the soluble by-products were filtered off and the filtrate evaporated to dryness. The oil-active residue solidified immediately after treatment with water. It was taken up in EtOAc, washed with H"2O (3x), dried over Na^SO^ and evaporated to a small volume whereupon the product began to crystallize. The same volume of petroleum ether was added and the mixture was allowed to stand overnight. The crude product was recrystallized from THF and petroleum ether which gave 5.2 g (75.6%) Boc-Asn-Ala-Ala-OBzl; m.p. 153-155.°C; [«lp<5>=-55.61°
(c = 1, MeOH).(c = 1, MeOH).
Boc-Asn-Ala-Ala-OBzl (2.2 g, 4.75 mmol) ble behandlet med 220Boc-Asn-Ala-Ala-OBzl (2.2 g, 4.75 mmol) was treated with 220
ml 4.0 n HCl i THF i 30 min. Syreoverskudd og løsningsmiddelet ble avdampet og resten igjeninndampet to ganger med friskt THF. Det oljeaktive produktet størknet ved behandling av eter. Det ble revet med mere frisk eter og oppsamlet hvilket gav et hvitt pulver (1,85 g). Det således erholdte hydrokloridsaltet ble oppløst i 4 0 ml DMf og avkjølt til 0°C ved tilsetning av H^C-Co-SER-Ser(Bz1)-OH•DCHA (1.97 g, 4.75 mmol). Etter røring ml 4.0 n HCl in THF for 30 min. Excess acid and the solvent were evaporated and the residue reevaporated twice with fresh THF. The oily active product solidified on treatment with ether. It was triturated with more fresh ether and collected to give a white powder (1.85 g). The hydrochloride salt thus obtained was dissolved in 40 ml of DMf and cooled to 0°C by addition of H^C-Co-SER-Ser(Bz1)-OH•DCHA (1.97 g, 4.75 mmol). After stirring
ved 0°C i 30 min. kunne utfelling■av DCHA-HCl konstateres og HOBT (1,19 g) lbe tilsatt etterfulgt av DCC(1,03 g, 5.23 mmol). Reaksjonen ble så justert til pH 7.5' med noen få dråper NMM og rørt ved 0°C i 2 timer og så ved 2 5°C natten over. Det uløs-lige biproduktet ble fjernet ved filtrering og filtratet inndampet til en svakt farget fast masse. Den ble grundig vasket med H-jO og EtOAc hvilket gav et brungult farget pulver som ble krystalisert fra DMF (40 ml) og isopropanol (500ml). Utbyttet: 1,57 g(56,9%) H3C-CO-Ser (Bzl) --Asn-Ala-Ala-OBz 1 ;m p. 213-215°C; at 0°C for 30 min. precipitation of DCHA-HCl could be observed and HOBT (1.19 g) was added followed by DCC (1.03 g, 5.23 mmol). The reaction was then adjusted to pH 7.5' with a few drops of NMM and stirred at 0°C for 2 hours and then at 25°C overnight. The insoluble by-product was removed by filtration and the filtrate evaporated to a faintly colored solid mass. It was thoroughly washed with H 2 O and EtOAc to give a tan colored powder which was crystallized from DMF (40 mL) and isopropanol (500 mL). Yield: 1.57 g (56.9%) H3C-CO-Ser (Bzl) --Asn-Ala-Ala-OBz 1 ; m p. 213-215°C;
[a]25 =-23.11° (c = 1, DMSO). [α]25 = -23.11° (c = 1, DMSO).
D D
H3C--CO-Ser (Bzl)--Asn-Ala-Ala-OBzl (1,57 g,2.69 mmol). ble oppløstH3C--CO-Ser(Bzl)--Asn-Ala-Ala-OBzl (1.57 g, 2.69 mmol). was dissolved
i 20 ml DMF og rørt med 2 ml H2NNH2i 13 timer. Det dannede faste produktet ble oppsamlet og vasket grundig med.DMF,EtOH, in 20 ml DMF and stirred with 2 ml H2NNH2i 13 hours. The solid product formed was collected and washed thoroughly with DMF, EtOH,
og eter hvilket gav 1 22g(89,6%) H3C-CO-Ser(Bzl)-Asn-Ala-Ala-HNNH2, m.p. 262-264°C; [a]^5 =-26,7 (c = 1, DMSO). and ether which gave 1 22g(89.6%) H3C-CO-Ser(Bzl)-Asn-Ala-Ala-HNNH2, m.p. 262-264°C; [α]^5 = -26.7 (c = 1, DMSO).
Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-ILe-Thr (Bzl) -Thr (Bzl) -Lys ( Z ) -OH (0,698 g, 0,353 mmol). ble behandlet med 10 ml TFA i 30 min. og peptidsaltet ble felt med eter. Det . ble samlet på et sugefilter, vasket med eter og tørket hvilket gav 0,652 g materiale .(0,333 mmol som TFA-salt),I en adskilt > flaske ble , H^C-CO-Ser(Bzl)-Asn-Ala-Ala-HNNH,,(0,17 g, 0,335 mmol) oppslemmet i 7 ml DMF og behandlet med 0,27 ml 6.13 N HCl i THF ved -20°C. Blandingen ble tilsatt o,63 ml 10%i-amylnitrit i DMF og løsningen rørt med samme temperatur i 3 0- min. Temperaturen ble senket til -30°C hvoretter 0,234 ml Et3N (1,67 mmol) ble tilsatt etterfulgt av TFA-saltet av decapeptidet (o,652 g) som ovenfor fremstilt. Blandingen ble fortynnet med 3 ml DMSO ved -20°C og justert svakt basisk (pH 7.5) med noen får dråper Et^N. Den ble rørt ved -2 0°C i 3 0 min og så ved 4°C i 5 dager. Mer DMSO (5 ml) og Et^N ble tilsatt i løpet av denne perioden for å opprettholde det svakt basiske betingelsene og hindre reaksjonsblandingen i en gel. Hele løsningen ble så helt i 5% HOAc (250 ml) hvilket gav en hvitfelning som ble oonsamlet, vasket med H20, MeOH, eter og tørket. Rå-produktet (0,702 g, m.p. 290-291°C ble gjenfelt fra DMSO med MeOH. Utbyttet: 0,348 g (42.0%) H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser (Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH; m.p. 296-293°C (dec.);[a]2<5>= +3.77° (c = 1, DMSO). Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-ILe-Thr (Bzl) -Thr (Bzl) -Lys ( Z ) -OH (0.698 g , 0.353 mmol). was treated with 10 ml of TFA for 30 min. and the peptide salt was precipitated with ether. That. was collected on a suction filter, washed with ether and dried to give 0.652 g of material (0.333 mmol as TFA salt), In a separate bottle, HNNH,, (0.17 g, 0.335 mmol) slurried in 7 mL DMF and treated with 0.27 mL 6.13 N HCl in THF at -20°C. To the mixture was added 0.63 ml of 10% i-amyl nitrite in DMF and the solution stirred at the same temperature for 30 minutes. The temperature was lowered to -30°C after which 0.234 ml Et 3 N (1.67 mmol) was added followed by the TFA salt of the decapeptide (0.652 g) as prepared above. The mixture was diluted with 3 ml of DMSO at -20°C and adjusted slightly basic (pH 7.5) with a few drops of Et^N. It was stirred at -20°C for 30 min and then at 4°C for 5 days. More DMSO (5 mL) and Et₂N were added during this period to maintain slightly basic conditions and prevent the reaction mixture from gelling. The entire solution was then poured into 5% HOAc (250 mL) which gave a white precipitate which was collected, washed with H 2 O, MeOH, ether and dried. The crude product (0.702 g, m.p. 290-291°C) was reprecipitated from DMSO with MeOH. Yield: 0.348 g (42.0%) H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)- Thr(Bzl)-Ser (Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH; mp 296-293°C (dec.);[a]2< δ>= +3.77° (c = 1, DMSO).
Det erholdte produkt (0,8 66 g, 0,37 3 mmol) ble rørt med HOBT. H20 (0,126 g, 0,32 mmol) i en blanding av DMSO ( 8 ml) og The product obtained (0.866 g, 0.373 mmol) was stirred with HOBT. H 2 O (0.126 g, 0.32 mmol) in a mixture of DMSO (8 mL) and
DMF (6 ml) og avkjølt i et isbad. Blandingen ble behandlet med DCC (0,035 g, 0,411 mmol) og så rørt ved 0° i 24 timer . Den ble så blandet med TFA-saltet av tetradecapeptidet som var fremstilt som.i eks. IB.d( 1,05 g, 0, 373 mmol) og ytteligere 2 ml DMSO. Noen.dråper NMM ble tilsatt for å bringe pH til 7,5-3,0 og røringen ble fortsatt ved 0°C i 1 time og så ved 25°C i 5 dager. Opparbeiding som beskrevet i eks. IB.d, gav 1 ,5775 g av det fullstendig beskyttede octacosapeptidet. H3C-CO-Ser(Bzlj-Asn-Ala--Ala-Val-Asp (OBzl) -Thr (Bzl) -Ser (Bzl) -Ser (Bzl) -Glu (OBzl) - Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)Lys (Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl. DMF (6 mL) and cooled in an ice bath. The mixture was treated with DCC (0.035 g, 0.411 mmol) and then stirred at 0° for 24 h. It was then mixed with the TFA salt of the tetradecapeptide which had been prepared as in ex. IB.d (1.05 g, 0.373 mmol) and an additional 2 ml of DMSO. A few drops of NMM were added to bring the pH to 7.5-3.0 and stirring was continued at 0°C for 1 hour and then at 25°C for 5 days. Processing as described in ex. IB.d, yielded 1.5775 g of the fully protected octacosapeptide. H3C-CO-Ser(Bzlj-Asn-Ala--Ala-Val-Asp (OBzl) -Thr (Bzl) -Ser (Bzl) -Ser (Bzl) -Glu (OBzl) - Ile-Thr(Bzl)-Thr (Bzl)-Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)Lys (Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Ala -Glu(OBzl)-Asn-OBzl.
Det fullsteridig beskyttede octacosapeptidet (1,57 g, 0,32 mmol) ble oppløst i lo ml TFA som inneholdt 3 ml anisol.Blandingen ble rørt med 45 ml HF ved 0°C i 30.min og så opparbeidet som beskrevet i eks. IB.d. Rensning og Sefadex<R>G-10 og DEAE-sefadex<R>kolonnen som ovenfor beskrevet gav 0,238 g H3C-CO-SEr-Asn--Ala-Ala-Val-Asp-Thr-SEr-Ser-Glu-Ile-THr.Thr-Lys-Asp<->Seu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn [(Asn 2)-tymosin ct^]som i hvitt amorft produkt. Forbindelsen beveget seg i stillingen litt mindre sur enn naturlig tymosin på akrylamid gelen med isoelektrisk fokus i overenstemmelse med forskjellen i struk-tur. The fully sterically protected octacosapeptide (1.57 g, 0.32 mmol) was dissolved in 10 ml of TFA containing 3 ml of anisole. The mixture was stirred with 45 ml of HF at 0°C for 30 min and then worked up as described in ex. IB.d. Purification and the Sefadex<R>G-10 and DEAE-sefadex<R>column as described above yielded 0.238 g of H3C-CO-SEr-Asn--Ala-Ala-Val-Asp-Thr-SEr-Ser-Glu-Ile- THr.Thr-Lys-Asp<->Seu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn [(Asn 2 )-thymosin ct^]as in white amorphous product . The compound moved to the position slightly less acidic than native thymosin on the acrylamide gel with isoelectric focus consistent with the difference in structure.
Claims (17)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US78989877A | 1977-04-22 | 1977-04-22 | |
| US05/871,563 US4116951A (en) | 1977-04-22 | 1978-01-23 | [Asn2 ]-thymosin α1 and analogs thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| NO781404L true NO781404L (en) | 1978-10-24 |
Family
ID=27120967
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO781404A NO781404L (en) | 1977-04-22 | 1978-04-21 | PROCEDURE FOR THE PREPARATION OF THYMOSIN ALFA-1 AND AN ANALOG COMPOSITION |
| NO821608A NO148924C (en) | 1977-04-22 | 1982-05-13 | ANALOGY PROCEDURE FOR THE PREPARATION OF IMMUNPOTENCING Peptides. |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO821608A NO148924C (en) | 1977-04-22 | 1982-05-13 | ANALOGY PROCEDURE FOR THE PREPARATION OF IMMUNPOTENCING Peptides. |
Country Status (18)
| Country | Link |
|---|---|
| JP (1) | JPS53137914A (en) |
| AT (1) | AT364470B (en) |
| CA (1) | CA1113088A (en) |
| CH (3) | CH641152A5 (en) |
| DE (1) | DE2817082A1 (en) |
| DK (1) | DK147918C (en) |
| ES (1) | ES469016A1 (en) |
| FI (1) | FI781241A7 (en) |
| FR (2) | FR2401134A1 (en) |
| GB (1) | GB1590668A (en) |
| GR (1) | GR71886B (en) |
| HU (1) | HU180783B (en) |
| IT (1) | IT1113134B (en) |
| LU (1) | LU79488A1 (en) |
| NL (1) | NL7804364A (en) |
| NO (2) | NO781404L (en) |
| PT (1) | PT67937B (en) |
| SE (1) | SE447262B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4116951A (en) * | 1977-04-22 | 1978-09-26 | Hoffmann-La Roche Inc. | [Asn2 ]-thymosin α1 and analogs thereof |
| DE2919592A1 (en) * | 1979-05-15 | 1981-01-15 | Max Planck Gesellschaft | METHOD FOR PRODUCING THYMOSINE ALPHA 1 AND DERIVATIVES THEREOF |
| EP0033384B1 (en) * | 1980-01-18 | 1984-02-15 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Medicaments containing fragments of thymosin-alpha-1 with immunostimulating activity, and thymosin-alpha-1 fragments |
| US4339427A (en) * | 1980-04-14 | 1982-07-13 | Hoffmann-La Roche Inc. | Radioimmunoassay of thymosinα |
| EP0056594B1 (en) * | 1981-01-14 | 1984-09-12 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Thymosin-alpha-1 fragments and pharmaceutical compositions with immunoregulating action containing them |
| CN1058500C (en) * | 1993-02-03 | 2000-11-15 | 施塞克龙药品公司 | Thymosin alpha-1 derivatives |
| US6262230B1 (en) * | 1994-01-28 | 2001-07-17 | Sciclone Pharmaceuticals Inc. | Analogs of thymosin α1 |
-
1978
- 1978-04-17 CH CH408878A patent/CH641152A5/en not_active IP Right Cessation
- 1978-04-18 DK DK169478A patent/DK147918C/en not_active IP Right Cessation
- 1978-04-19 DE DE19782817082 patent/DE2817082A1/en not_active Ceased
- 1978-04-20 GR GR56036A patent/GR71886B/el unknown
- 1978-04-20 FR FR7811686A patent/FR2401134A1/en active Granted
- 1978-04-21 ES ES469016A patent/ES469016A1/en not_active Expired
- 1978-04-21 GB GB1574/78A patent/GB1590668A/en not_active Expired
- 1978-04-21 CA CA301,643A patent/CA1113088A/en not_active Expired
- 1978-04-21 NO NO781404A patent/NO781404L/en unknown
- 1978-04-21 IT IT22629/78A patent/IT1113134B/en active
- 1978-04-21 PT PT67937A patent/PT67937B/en unknown
- 1978-04-21 JP JP4673978A patent/JPS53137914A/en active Pending
- 1978-04-21 LU LU79488A patent/LU79488A1/en unknown
- 1978-04-21 SE SE7804612A patent/SE447262B/en not_active IP Right Cessation
- 1978-04-21 AT AT0286178A patent/AT364470B/en not_active IP Right Cessation
- 1978-04-21 FI FI781241A patent/FI781241A7/en not_active Application Discontinuation
- 1978-04-21 HU HU78HO2067A patent/HU180783B/en unknown
- 1978-04-24 NL NL7804364A patent/NL7804364A/en not_active Application Discontinuation
- 1978-12-15 FR FR7835404A patent/FR2405926A1/en active Granted
-
1982
- 1982-05-13 NO NO821608A patent/NO148924C/en unknown
- 1982-11-05 CH CH644982A patent/CH640218A5/en not_active IP Right Cessation
- 1982-11-05 CH CH645082A patent/CH641153A5/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| ATA286178A (en) | 1981-03-15 |
| FR2401134B1 (en) | 1983-09-30 |
| HU180783B (en) | 1983-04-29 |
| FR2405926A1 (en) | 1979-05-11 |
| NL7804364A (en) | 1978-10-24 |
| CH641153A5 (en) | 1984-02-15 |
| NO148924C (en) | 1984-01-11 |
| NO821608L (en) | 1978-10-24 |
| FI781241A7 (en) | 1978-10-23 |
| JPS53137914A (en) | 1978-12-01 |
| FR2405926B1 (en) | 1983-09-09 |
| PT67937B (en) | 1980-04-07 |
| DK147918C (en) | 1985-08-19 |
| ES469016A1 (en) | 1980-01-01 |
| CA1113088A (en) | 1981-11-24 |
| CH640218A5 (en) | 1983-12-30 |
| PT67937A (en) | 1978-05-01 |
| LU79488A1 (en) | 1979-05-25 |
| GR71886B (en) | 1983-08-04 |
| IT7822629A0 (en) | 1978-04-21 |
| SE447262B (en) | 1986-11-03 |
| AT364470B (en) | 1981-10-27 |
| DK147918B (en) | 1985-01-07 |
| FR2401134A1 (en) | 1979-03-23 |
| DK169478A (en) | 1978-10-23 |
| NO148924B (en) | 1983-10-03 |
| GB1590668A (en) | 1981-06-03 |
| CH641152A5 (en) | 1984-02-15 |
| SE7804612L (en) | 1978-12-20 |
| DE2817082A1 (en) | 1978-11-02 |
| IT1113134B (en) | 1986-01-20 |
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