CA3186364A1 - Solid phase peptide synthesis via side chain attachment - Google Patents
Solid phase peptide synthesis via side chain attachmentInfo
- Publication number
- CA3186364A1 CA3186364A1 CA3186364A CA3186364A CA3186364A1 CA 3186364 A1 CA3186364 A1 CA 3186364A1 CA 3186364 A CA3186364 A CA 3186364A CA 3186364 A CA3186364 A CA 3186364A CA 3186364 A1 CA3186364 A1 CA 3186364A1
- Authority
- CA
- Canada
- Prior art keywords
- resin
- trt
- protecting group
- group selected
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000007790 solid phase Substances 0.000 title claims abstract description 35
- 238000010647 peptide synthesis reaction Methods 0.000 title claims abstract description 29
- 229920005989 resin Polymers 0.000 claims abstract description 245
- 239000011347 resin Substances 0.000 claims abstract description 245
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 137
- -1 hydroxy amino acid amides Chemical class 0.000 claims abstract description 31
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 31
- 125000002349 hydroxyamino group Chemical group [H]ON([H])[*] 0.000 claims abstract description 12
- 108010061784 Peptaibols Proteins 0.000 claims abstract description 8
- ULYMTCOVLNBXIE-HEXJONDASA-N (2s)-2-[[(2s,4r)-1-[2-[[(2s)-2-[[2-[[(2s,3r)-2-[[(2s,3s)-2-[[2-[[(2s)-2-[[(2s,3s)-2-[[(2s)-2-acetamido-3-(1h-indol-3-yl)propanoyl]amino]-3-methylpentanoyl]amino]-5-amino-5-oxopentanoyl]amino]acetyl]amino]-3-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]- Chemical class C([C@@H](C(=O)NC(C)(C)C(=O)NC(C)(C)C(=O)NC(C)(C)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NC(C)(C)C(=O)NC(C)(C)C(=O)N1C[C@H](O)C[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@](C)(CC)C(=O)N1[C@@H](C[C@@H](O)C1)C(=O)NCC(=O)NC(CO)CC=1C=CC=CC=1)C(C)C)NC(C)=O)C1=CC=CC=C1.O=C([C@@H]1CCCN1C(=O)C(C)(C)NC(=O)[C@@H]1C[C@@H](O)CN1C(=O)C(C)(C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1C[C@@H](O)CN1C(=O)C(C)(C)NC(=O)[C@H](CC(C)C)NC(=O)C(C)(C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(C)=O)[C@@H](C)CC)[C@@H](C)CC)NC(CO)CC1=CC=CC=C1 ULYMTCOVLNBXIE-HEXJONDASA-N 0.000 claims abstract description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 96
- 239000012634 fragment Substances 0.000 claims description 39
- 125000006239 protecting group Chemical group 0.000 claims description 38
- 239000002253 acid Substances 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 37
- 150000001413 amino acids Chemical class 0.000 claims description 31
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 25
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 19
- 238000009833 condensation Methods 0.000 claims description 19
- 230000005494 condensation Effects 0.000 claims description 19
- 238000010511 deprotection reaction Methods 0.000 claims description 19
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 19
- 238000003776 cleavage reaction Methods 0.000 claims description 18
- 230000007017 scission Effects 0.000 claims description 18
- OIRCOABEOLEUMC-GEJPAHFPSA-N bivalirudin Chemical group C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 OIRCOABEOLEUMC-GEJPAHFPSA-N 0.000 claims description 16
- 108010011459 Exenatide Proteins 0.000 claims description 14
- HTQBXNHDCUEHJF-XWLPCZSASA-N Exenatide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 HTQBXNHDCUEHJF-XWLPCZSASA-N 0.000 claims description 14
- 229960001519 exenatide Drugs 0.000 claims description 14
- 108010055460 bivalirudin Proteins 0.000 claims description 13
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical group N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 claims description 13
- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 claims description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 12
- NRKVKVQDUCJPIZ-MKAGXXMWSA-N pramlintide acetate Chemical compound C([C@@H](C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCCCN)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 NRKVKVQDUCJPIZ-MKAGXXMWSA-N 0.000 claims description 12
- 108010068072 salmon calcitonin Proteins 0.000 claims description 12
- 108010091893 Cosyntropin Proteins 0.000 claims description 11
- 108010016076 Octreotide Proteins 0.000 claims description 11
- 229960001500 bivalirudin Drugs 0.000 claims description 11
- 229960003773 calcitonin (salmon synthetic) Drugs 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 229960002700 octreotide Drugs 0.000 claims description 11
- 229960003611 pramlintide Drugs 0.000 claims description 10
- 108010029667 pramlintide Proteins 0.000 claims description 10
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 10
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- 229910052740 iodine Inorganic materials 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 8
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 8
- 239000011630 iodine Substances 0.000 claims description 8
- 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 claims description 8
- PUDHBTGHUJUUFI-SCTWWAJVSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-n-[(2s,3r)-1-amino-3-hydroxy-1-oxobutan-2-yl]-19-[[(2r)-2-amino-3-naphthalen-2-ylpropanoyl]amino]-16-[(4-hydroxyphenyl)methyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-7-propan-2-yl-1,2-dithia-5,8,11,14,17-p Chemical compound C([C@H]1C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](N)CC=1C=C2C=CC=CC2=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(N)=O)=O)C(C)C)C1=CC=C(O)C=C1 PUDHBTGHUJUUFI-SCTWWAJVSA-N 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 108010021336 lanreotide Proteins 0.000 claims description 7
- 229960002437 lanreotide Drugs 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 6
- 101500025354 Homo sapiens Insulin B chain Proteins 0.000 claims description 5
- 150000001414 amino alcohols Chemical class 0.000 claims description 5
- BUQICHWNXBIBOG-LMVFSUKVSA-N Ala-Thr Chemical group C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)N BUQICHWNXBIBOG-LMVFSUKVSA-N 0.000 claims description 4
- 108010069514 Cyclic Peptides Proteins 0.000 claims description 4
- 102000001189 Cyclic Peptides Human genes 0.000 claims description 4
- 102000004877 Insulin Human genes 0.000 claims description 4
- 108090001061 Insulin Proteins 0.000 claims description 4
- XGDCYUQSFDQISZ-BQBZGAKWSA-N Leu-Ser Chemical group CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(O)=O XGDCYUQSFDQISZ-BQBZGAKWSA-N 0.000 claims description 4
- 150000001408 amides Chemical class 0.000 claims description 4
- 125000003545 alkoxy group Chemical group 0.000 claims description 3
- ZZVUWRFHKOJYTH-UHFFFAOYSA-N diphenhydramine Chemical group C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1 ZZVUWRFHKOJYTH-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 150000007513 acids Chemical class 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 125000005518 carboxamido group Chemical group 0.000 claims 13
- RAXXELZNTBOGNW-UHFFFAOYSA-N 1H-imidazole Chemical group C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims 12
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine group Chemical group NC(=N)N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 claims 11
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 claims 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims 6
- 230000001590 oxidative effect Effects 0.000 claims 5
- 238000004587 chromatography analysis Methods 0.000 claims 4
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical group C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 claims 4
- 150000008574 D-amino acids Chemical class 0.000 claims 3
- 239000003570 air Substances 0.000 claims 3
- 239000007800 oxidant agent Substances 0.000 claims 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 2
- 150000004820 halides Chemical class 0.000 claims 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical group N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims 2
- 239000003960 organic solvent Substances 0.000 claims 2
- 150000003573 thiols Chemical group 0.000 claims 2
- 125000000008 (C1-C10) alkyl group Chemical group 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 150000001350 alkyl halides Chemical class 0.000 claims 1
- 238000011097 chromatography purification Methods 0.000 claims 1
- HRKQOINLCJTGBK-UHFFFAOYSA-N dihydroxidosulfur Chemical compound OSO HRKQOINLCJTGBK-UHFFFAOYSA-N 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 claims 1
- 229940125396 insulin Drugs 0.000 claims 1
- 125000001424 substituent group Chemical group 0.000 claims 1
- 150000003512 tertiary amines Chemical class 0.000 claims 1
- 125000005039 triarylmethyl group Chemical group 0.000 claims 1
- 229920000642 polymer Polymers 0.000 abstract description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 122
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 84
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 51
- 230000015572 biosynthetic process Effects 0.000 description 22
- 239000000203 mixture Substances 0.000 description 22
- 238000003786 synthesis reaction Methods 0.000 description 22
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 18
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 13
- SWZCTMTWRHEBIN-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(O)C=C1 SWZCTMTWRHEBIN-QFIPXVFZSA-N 0.000 description 11
- 238000011068 loading method Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 230000002378 acidificating effect Effects 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- UKAUYVFTDYCKQA-UHFFFAOYSA-N homoserine Chemical compound OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 7
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- 238000005859 coupling reaction Methods 0.000 description 6
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- 238000010561 standard procedure Methods 0.000 description 4
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- AYMLQYFMYHISQO-QMMMGPOBSA-N (2s)-3-(1h-imidazol-3-ium-5-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CN=CN1 AYMLQYFMYHISQO-QMMMGPOBSA-N 0.000 description 3
- 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 3
- YOKDHMTZJSRRIQ-KZULUSFZSA-N 9h-fluoren-9-ylmethyl n-[(2r,3r)-1,3-dihydroxybutan-2-yl]carbamate Chemical compound C1=CC=C2C(COC(=O)N[C@H](CO)[C@H](O)C)C3=CC=CC=C3C2=C1 YOKDHMTZJSRRIQ-KZULUSFZSA-N 0.000 description 3
- OQROAIRCEOBYJA-UHFFFAOYSA-N bromodiphenylmethane Chemical compound C=1C=CC=CC=1C(Br)C1=CC=CC=C1 OQROAIRCEOBYJA-UHFFFAOYSA-N 0.000 description 3
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- ZYJPUMXJBDHSIF-LLVKDONJSA-N (2r)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](C(O)=O)CC1=CC=CC=C1 ZYJPUMXJBDHSIF-LLVKDONJSA-N 0.000 description 1
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 1
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- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 description 1
- IZKGGDFLLNVXNZ-KRWDZBQOSA-N (2s)-5-amino-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)N)C(O)=O)C3=CC=CC=C3C2=C1 IZKGGDFLLNVXNZ-KRWDZBQOSA-N 0.000 description 1
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 description 1
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- IVBOWQJZGWLABY-UHFFFAOYSA-N 1-[bromo(phenyl)methyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Br)C1=CC=CC=C1 IVBOWQJZGWLABY-UHFFFAOYSA-N 0.000 description 1
- RUXWULLFBASCJB-UHFFFAOYSA-N 1-[bromo(phenyl)methyl]-4-methylbenzene Chemical compound C1=CC(C)=CC=C1C(Br)C1=CC=CC=C1 RUXWULLFBASCJB-UHFFFAOYSA-N 0.000 description 1
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- XZFYRXDAULDNFX-UWVGGRQHSA-N Cys-Phe Chemical compound SC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UWVGGRQHSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- 239000012448 Lithium borohydride Substances 0.000 description 1
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- KNPVDQMEHSCAGX-UWVGGRQHSA-N Phe-Cys Chemical compound SC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KNPVDQMEHSCAGX-UWVGGRQHSA-N 0.000 description 1
- NYQBYASWHVRESG-MIMYLULJSA-N Phe-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 NYQBYASWHVRESG-MIMYLULJSA-N 0.000 description 1
- DZHDVYLBNKMLMB-ZFWWWQNUSA-N Trp-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 DZHDVYLBNKMLMB-ZFWWWQNUSA-N 0.000 description 1
- GRSCONMARGNYHA-PMVMPFDFSA-N Trp-Lys-Phe Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O GRSCONMARGNYHA-PMVMPFDFSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 125000003710 aryl alkyl group Chemical group 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- VBEGHXKAFSLLGE-UHFFFAOYSA-N n-phenylnitramide Chemical compound [O-][N+](=O)NC1=CC=CC=C1 VBEGHXKAFSLLGE-UHFFFAOYSA-N 0.000 description 1
- 101150009274 nhr-1 gene Proteins 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- HBEJJYHFTZDAHZ-QMMMGPOBSA-N tert-butyl (2s)-2-amino-4-methylpentanoate Chemical compound CC(C)C[C@H](N)C(=O)OC(C)(C)C HBEJJYHFTZDAHZ-QMMMGPOBSA-N 0.000 description 1
- XJJBXZIKXFOMLP-ZETCQYMHSA-N tert-butyl (2s)-pyrrolidine-2-carboxylate Chemical compound CC(C)(C)OC(=O)[C@@H]1CCCN1 XJJBXZIKXFOMLP-ZETCQYMHSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
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- 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
- C07K14/585—Calcitonins
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- 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
- C07K14/605—Glucagons
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- 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
- C07K14/61—Growth hormones [GH] (Somatotropin)
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- 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
- C07K14/62—Insulins
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- 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
- C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- C07K14/695—Corticotropin [ACTH]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
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- 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
Abstract
The present application discloses peptides and peptaibols of high purity may be obtained by solid phase peptide synthesis using as the starting resin hydroxy amino acids, hydroxy amino acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino acids attached to polymers through their side chain.
Description
SOLID PHASE PEPTIDE SYNTHESIS
VIA SIDE CHAIN ATTACHMENT
SUMMARY:
[0001] Peptides and peptaibols of high purity were obtained by solid phase peptide synthesis using as the starting resin hydroxy amino acids, hydroxy amino acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino acids attached to polymers through their side chain.
Definitions and Abbreviations:
VIA SIDE CHAIN ATTACHMENT
SUMMARY:
[0001] Peptides and peptaibols of high purity were obtained by solid phase peptide synthesis using as the starting resin hydroxy amino acids, hydroxy amino acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino acids attached to polymers through their side chain.
Definitions and Abbreviations:
[0002] "Hya" or "hydroxyl amino acid(s)" means amino acids that contain a hydroxyl (-OH) group.
[0003] N-terminus or amino terminus is the first amino acid in a peptide chain.
[0004] C-terminus or carboxy terminus is the last amino acid in the peptide chain as shown below.
amino terminal carboxy terminal or N-terminal or C-terminal amino acid amino acid A
r A __ , r , NH2-CHR-CO-Aai-AA2 ................................ AA-NH-CHR-CO2H
peptide
amino terminal carboxy terminal or N-terminal or C-terminal amino acid amino acid A
r A __ , r , NH2-CHR-CO-Aai-AA2 ................................ AA-NH-CHR-CO2H
peptide
[0005] "P" or "solid support" or "resin" means an insoluble material containing a functional group(s) suitable to react and link with an amino acid or peptide. The solid support or resins are well known in the art.
[0006] "Alkyl" such as Ci_malkyl or C1_6alkyl, means a branched or unbranched fully saturated acyclic aliphatic hydrocarbon group (i.e. composed of carbon and hydrogen containing no double or triple bonds). In some embodiments, alkyls may be substituted or unsubstituted.
Alkyls may include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like, and in some embodiment, each of which may be optionally substituted. Non-exclusive alkyl substituents may include C1_3alkoxy, halo (F, Cl, Br or I), nitro, amino, -SH and -OH.
Alkyls may include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like, and in some embodiment, each of which may be optionally substituted. Non-exclusive alkyl substituents may include C1_3alkoxy, halo (F, Cl, Br or I), nitro, amino, -SH and -OH.
[0007] "Attachment" means the linking of an amino acid or a peptide or peptide derivative to an insoluble support.
[0008] "Hse" means homoserine; "Hnv" means hydroxylnorvaline.
[0009] "SPPS" or "solid phase peptide synthesis" means the synthesis of a peptide with the use of a resin as described herein.
Date Recue/Date Received 2023-01-12
Date Recue/Date Received 2023-01-12
[0010] "pNA" means 4-nitro anilide.
[0011] "DME" means dimethoxy ethane.
[0012] "Acid sensitive resin" means an insoluble material or resin containing a functional group(s) suitable to react and link with an amino acid or peptide, which may be cleaved from the peptide by acidic treatment.
[0013] "Acid sensitive protecting group" means a protecting group which may be cleaved from the amino acid or peptide or peptide derivative by acidic treatment or under acidic condition.
[0014] "Peptaibol" means a peptide which contain at its C-terminal position an amino alcohol instead of an amino acid or an amino acid amide.
[0015] "Step-by-step" means the method of peptide synthesis where any of the amino acids contained in the peptide chain is introduced individually and sequentially.
The method may or may not involve an intermediate purification step.
The method may or may not involve an intermediate purification step.
[0016] "Protected peptide" means the peptide with all functional groups blocked or protected by protecting groups.
[0017] "Partially protected peptide" means the peptide which contains at least one functional group blocked or protected by a protecting group.
[0018] Solid phase peptide synthesis is traditionally performed by the attachment of the C-terminal amino acid through its a-carboxyl function on a suitable solid support and elongating the peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. Several hundred thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals.
[0019] In contrary to the attachment of the C-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side chain on suitable resins and their application in SPPS is described very briefly, in particular in less than 30 publication and patents. Most of these publications describe the attachment of the amino acids through a side chain carboxyl function of Asp and Glu. To our knowledge, the reports of the side chain attachment of amino acids through a side chain hydroxyl function and application in peptide synthesis are limited:
The side chain attachment of Fmoc-Hya-pNA (Formula Al) [A. Bernhardt, M.
Drewello and M. Schutkowski, The solid-phase synthesis of side-chain-phosphorylated peptide-nitroanilides J. Peptide Res. 50, 1997. 143-1521 and their use for the synthesis of short nitroanilide substrates, the synthesis of Fmoc-Hya-Oallyl esters (Formula A2 [L. Rizzi, K.
Cendic, N. Vaiana, S. Romeo, Alcohols immobilization onto 2-chlorotritylchloride resin under microwave irradiation, Tetrahedron Letters 52 (2011) 2808-28111) on 2-chlorotrityl resin Date Recue/Date Received 2023-01-12 with the aid of microwaves for application in the preparation of cyclic peptides and the synthesis of Fmoc-Tyr-O-methyl ester (Formula A3 [C. Cabrele, M. Langer and A.
G. Beck-Sickinger, Amino Acid Side Chain Attachment Approach and Its Application to the Synthesis of Tyrosine-Containing Cyclic Peptides, .1 Org. Chem. 1999, 64, 4353-43611), attached on resins of the benzyl-type by the Mitsunobu redox-alkylation of the Tyr-phenoxy function and their application for the synthesis of short cyclic peptides. To our knowledge, the side chain attachment of Hse and Hyp have never been disclosed. In addition, the application of side chain attached Hya on acid sensitive resins for the solid phase synthesis of protected peptides, protected peptide fragments and of protected peptide amides and peptaibols have not been reported.
'P
Al A2 A3 CI CI *
ill X
0 Fmoc-HN Fmoc-HN 0 Fmoc-HN CO2CH3 HN . NO2 0¨-Solid Phase Peptide Synthesis
The side chain attachment of Fmoc-Hya-pNA (Formula Al) [A. Bernhardt, M.
Drewello and M. Schutkowski, The solid-phase synthesis of side-chain-phosphorylated peptide-nitroanilides J. Peptide Res. 50, 1997. 143-1521 and their use for the synthesis of short nitroanilide substrates, the synthesis of Fmoc-Hya-Oallyl esters (Formula A2 [L. Rizzi, K.
Cendic, N. Vaiana, S. Romeo, Alcohols immobilization onto 2-chlorotritylchloride resin under microwave irradiation, Tetrahedron Letters 52 (2011) 2808-28111) on 2-chlorotrityl resin Date Recue/Date Received 2023-01-12 with the aid of microwaves for application in the preparation of cyclic peptides and the synthesis of Fmoc-Tyr-O-methyl ester (Formula A3 [C. Cabrele, M. Langer and A.
G. Beck-Sickinger, Amino Acid Side Chain Attachment Approach and Its Application to the Synthesis of Tyrosine-Containing Cyclic Peptides, .1 Org. Chem. 1999, 64, 4353-43611), attached on resins of the benzyl-type by the Mitsunobu redox-alkylation of the Tyr-phenoxy function and their application for the synthesis of short cyclic peptides. To our knowledge, the side chain attachment of Hse and Hyp have never been disclosed. In addition, the application of side chain attached Hya on acid sensitive resins for the solid phase synthesis of protected peptides, protected peptide fragments and of protected peptide amides and peptaibols have not been reported.
'P
Al A2 A3 CI CI *
ill X
0 Fmoc-HN Fmoc-HN 0 Fmoc-HN CO2CH3 HN . NO2 0¨-Solid Phase Peptide Synthesis
[0020] In one embodiment, there is provided an improved synthesis of peptide acids, peptide amides, and peptaibols of pharmaceutical interest.
P P
( X \
/ %z X
\ H
\d- 0 -1) Y I V Y I
Pr'-Hya-A Pr'-Hya- A
P P
( X \
/ %z X
\ H
\d- 0 -1) Y I V Y I
Pr'-Hya-A Pr'-Hya- A
[0021] Formula I Formula ll
[0022] In one aspect of the present application, the peptides were produced very efficiently in high yield and purity by attaching a hydroxy amino acid through its amino acid side chain, or a small peptide which contain in its sequence a hydroxy amino acid on a resin of the trityl or benzhydryl-type, resulting in amino acid-resin conjugates or peptide resin conjugates of Formula I-TV, wherein P is a solid-phase support selected from the supports used in solid phase peptide synthesis, Pr' is H or an amino protecting group selected from Fmoc, Boc, Trt, Dde and Alloc, wherein pe is an acid sensitive hydroxyl protecting group selected from Trt, Clt, Mmt, Mtt, Dpm and tBu, wherein Hya is a hydroxy amino acid selected from D- or L-Ser, Thr, Tyr, Hse, Hyp, Hnv etc ., and A is OH, an acid sensitive alkoxy group selected from OTrt, 0C1t, OMmt, OW ODpm and OtBu, NH2, NHR1, NR1Ic-r.2 wherein RI- and R2 are Date Recue/Date Received 2023-01-12 independently an alkyl group a protected or semi protected peptide containing 1-10 amino acids in its sequence.
[0023] In another embodiment, we disclose that peptaibols, such as octreotide, were obtained by solid phase synthesis using the resin-bound amino alcohols of the Formula III-VI selected from amino alcohols which are derived from the naturally occurring hydroxy amino acids, wherein P. X, V. Z and Pr' are as defined above, wherein R3, le are alkyl, aryl or aralkyl groups, and Pr' is an acid sensitive protecting group of the trityl, benzhydryl or benzyl type.
X
/ %Z
-1=1 Prl¨N Prl¨N
Formula III Formula IV
X
I--1=7 H
V V
Pr' Prr Formula V Formula VI
X
/ %Z
-1=1 Prl¨N Prl¨N
Formula III Formula IV
X
I--1=7 H
V V
Pr' Prr Formula V Formula VI
[0024] In addition we disclose for the first time that peptides prepared with the application of resins of the Formula I-TV, where the peptides are attached through the side chain hydroxyl function of a hydroxyamino acid on resins of the trityl type, may be cleaved from the resin by mild acidic treatment and wherein the side chain protecting groups of the tBu and benzyl-type remain intact. In one aspect, the cleavage from the resin occurs by the treatment with 1-3 %
acid solutions, such as TFA, diluted HC1 solutions, optionally adding scavengers, in a solvent.
In another aspect, the cleavage may be performed in a solvent such as DCM or acetone. Such partially protected peptides have been found to be useful in the synthesis of longer peptides by fragment condensation in solution or on solid phase. The present method expands the versatility of the application of the resins described herein, and also results in significantly improving the purity of the resulting pharmaceutical peptides, and at the same time, substantially reducing the cost of their synthesis.
Date Recue/Date Received 2023-01-12
acid solutions, such as TFA, diluted HC1 solutions, optionally adding scavengers, in a solvent.
In another aspect, the cleavage may be performed in a solvent such as DCM or acetone. Such partially protected peptides have been found to be useful in the synthesis of longer peptides by fragment condensation in solution or on solid phase. The present method expands the versatility of the application of the resins described herein, and also results in significantly improving the purity of the resulting pharmaceutical peptides, and at the same time, substantially reducing the cost of their synthesis.
Date Recue/Date Received 2023-01-12
[0025] Several peptides of pharmaceutical interest were produced as representative of the new process described herein, either by the step-by-step procedure or by fragment condensation in solution and on solid phase; or a combination thereof. The examples below are representative and do not limit their application in any way to other peptides.
Lanreotide:
Lanreotide:
[0026] In one embodiment, Lanreotide was produced by solid phase synthesis using resin-bound Thr-amide as shown below:
P
= Br-Resin Frnoc-Thr(Resin)-NH2 -4 DIPEA Fmoc-Thr-NH2 + H OCH3 Br ISequential deprotection and coupliing steps, 7 cycles Boc-D-2-Nal-Cys(Trt)-Tyr(C1t)-D-Trp-Lys(Mtt)-Val-Cys(Trt)-Thr(Resin)-N H2 deprotection, cleavage from the resin and simultaneous iodine oxidation I I
H-D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-N H2 Lanreotide Human insulin B-chain:
P
= Br-Resin Frnoc-Thr(Resin)-NH2 -4 DIPEA Fmoc-Thr-NH2 + H OCH3 Br ISequential deprotection and coupliing steps, 7 cycles Boc-D-2-Nal-Cys(Trt)-Tyr(C1t)-D-Trp-Lys(Mtt)-Val-Cys(Trt)-Thr(Resin)-N H2 deprotection, cleavage from the resin and simultaneous iodine oxidation I I
H-D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-N H2 Lanreotide Human insulin B-chain:
[0027] Optionally the human insulin B chain was synthesized by SPPS. In one aspect, the synthesis begins from the resin-bound Thr-t-butyl ester as described in the example, using the 4-methoxy benzhydryl resin. Optionally the synthesis may also be performed on solid phase by condensing the 1-8 partially protected Boc-Phe-Val-Asn(TrO-Gln(TrO-His(Trt)-Leu-Cys(Trt)-Gly-OH fragment with the resin-bound 9-30 fragment; or after the selective cleavage of the partially protected 9-30 fragment from the resin with condensation in solution of the 1-8 and 9-30 fragments.
P
= Br-Resin Fmoc-Thr(Resin)-0tBu . DIPEA Fmoc-Thr-OtBu + H OCH3 Br 1 Sequential deprotection and coupliing steps, 22 cycles H-Phel-Val-Asn(Trt)-Gln(Trt)-His(Trt)-Leu-Cys(Trt)-Gly-Ser(tBu)-His(Trt)-Leu-Val-Glu(tBu)-Ala-Leu-Tyr(tBu)-Leu-Val-Cys(Trt)-Gly-Glu(tBu)-Arg(Pbf)-Gly-Phe-Phe-Tyr(tBu)-Thr(tBu)-Pro-Lys(Boc)-Thr(Resin)3 -0tBu 1 deprotection and cleavage from the resin H-Phel-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr3 -OH human Insulin B-Chain Date Recue/Date Received 2023-01-12 Salmon Calcitonin:
P
= Br-Resin Fmoc-Thr(Resin)-0tBu . DIPEA Fmoc-Thr-OtBu + H OCH3 Br 1 Sequential deprotection and coupliing steps, 22 cycles H-Phel-Val-Asn(Trt)-Gln(Trt)-His(Trt)-Leu-Cys(Trt)-Gly-Ser(tBu)-His(Trt)-Leu-Val-Glu(tBu)-Ala-Leu-Tyr(tBu)-Leu-Val-Cys(Trt)-Gly-Glu(tBu)-Arg(Pbf)-Gly-Phe-Phe-Tyr(tBu)-Thr(tBu)-Pro-Lys(Boc)-Thr(Resin)3 -0tBu 1 deprotection and cleavage from the resin H-Phel-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr3 -OH human Insulin B-Chain Date Recue/Date Received 2023-01-12 Salmon Calcitonin:
[0028] Optionally salmon calcitonin may be produced starting the synthesis from resin bound Fmoc-Thr-Pro-NH2. The peptide chain is then elongated using Fmoc-amino acids.
P
= Br-Resin Fmoc-Thr(Resin DIPEA)-Pro-NH, _.., Fmoc-Thr-Pro-NH2 + H
OCH, Sequential deprotection and coupliing steps, 22 cycles Br H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr(Resin)-Pro-NH2 32 I I
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-OH
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr(Resin)-Pro-NH2 H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Salmon Calcitonin
P
= Br-Resin Fmoc-Thr(Resin DIPEA)-Pro-NH, _.., Fmoc-Thr-Pro-NH2 + H
OCH, Sequential deprotection and coupliing steps, 22 cycles Br H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr(Resin)-Pro-NH2 32 I I
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-OH
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr(Resin)-Pro-NH2 H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Salmon Calcitonin
[0029] Optionally the resin-bound salmon calcitonin is produced by fragment condensation on the resin as shown above, for example, or in solution as shown below using 2-4 fragments.
P
= Br-Resin ...
Fmoc-Thr(Resin)-Pro-NH2 _DIPEAFmoc-Thr-Pro-NH2 + H OCH3 Sequential deprotection and coupliing steps, 22 cycles Br H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr(Resin)-Pro-NH2 32 2% TFA/TES
H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr-Pro-I I
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-OH
1 In v Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Tr)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr-Pro-NH2 H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Salmon Calcitonin Date Recue/Date Received 2023-01-12 Octreotide:
P
= Br-Resin ...
Fmoc-Thr(Resin)-Pro-NH2 _DIPEAFmoc-Thr-Pro-NH2 + H OCH3 Sequential deprotection and coupliing steps, 22 cycles Br H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr(Resin)-Pro-NH2 32 2% TFA/TES
H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr-Pro-I I
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-OH
1 In v Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Tr)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr-Pro-NH2 H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Salmon Calcitonin Date Recue/Date Received 2023-01-12 Octreotide:
[0030] In another embodiment, octreotide was efficiently synthesized by the attachment of Fmoc-threoninol-OTrt to the 4-methoxybenzhydryl resin through the side chain of threoninol as shown below, followed by the octreotide chain assembly using Fmoc-amino acids and finally cleaving octreotide from the resin with subsequent or simultaneous Cys-oxidation.
Fmoc-threoninol-OTrt is much easier to be produced than the Fmoc-Thr(tBu)-ol which may be attached onto the resin through the hydroxymethyl group of threoninol on a suitable resin.
This is because H-Thr(OtBu)-ol, used as the starting material for the production of Fmoc-Thr(tBu)-ol, is much more difficult to be produced than Fmoc-threoninol-OTrt used in the attachment of threoninol through its side chain onto the resin.
= Resin-Br OH OH Fmoc-HN H P 0-ResIn Br + Trt CI DIPEA Frn. FINCH, CH, _________________________________________________ Fmoc-HN
¨OH O-Trt DIPEA O-Trt 0-Resin H D Phe Cys(Mmt) Phe D Trp Lys(Mtt) Phe Thr(Trt)-Cys(Mmt) ¨HN¨
0-Txt ITFA/DCM/TIPS
OH
_CH3 H D Pile Cys Phe D Trp Lys Phe Thr Cys HN
1 oxidation OH
OH
HCH, H-D-Phe-Cys-Phe-D-Trp-Lys-Phe-Thr-Cys ¨N Octreotide OH
Exenatide:
Fmoc-threoninol-OTrt is much easier to be produced than the Fmoc-Thr(tBu)-ol which may be attached onto the resin through the hydroxymethyl group of threoninol on a suitable resin.
This is because H-Thr(OtBu)-ol, used as the starting material for the production of Fmoc-Thr(tBu)-ol, is much more difficult to be produced than Fmoc-threoninol-OTrt used in the attachment of threoninol through its side chain onto the resin.
= Resin-Br OH OH Fmoc-HN H P 0-ResIn Br + Trt CI DIPEA Frn. FINCH, CH, _________________________________________________ Fmoc-HN
¨OH O-Trt DIPEA O-Trt 0-Resin H D Phe Cys(Mmt) Phe D Trp Lys(Mtt) Phe Thr(Trt)-Cys(Mmt) ¨HN¨
0-Txt ITFA/DCM/TIPS
OH
_CH3 H D Pile Cys Phe D Trp Lys Phe Thr Cys HN
1 oxidation OH
OH
HCH, H-D-Phe-Cys-Phe-D-Trp-Lys-Phe-Thr-Cys ¨N Octreotide OH
Exenatide:
[0031] In another example, Fmoc-Ser-NH2 was attached through its side chain on trityl resin and used for the synthesis of exenatide. The synthesis may be performed by the step-by-step manner or by fragment condensation in solution after cleavage a partially protected exenatide fragment from the resin by mild acidic treatment or on solid phase, as described below.
According to this method, most impurities typically formed during the synthesis of many Pro and Gly residues containing peptides are completely avoided and peptides of high purity are obtained. The method also allows the complete avoidance of impurities originating from the cleavage of peptides from peptide amide linkers using other methods known in the art, which significantly reduce the yields and purity of the peptide.
Date Recue/Date Received 2023-01-12 OH 0-Resin Fmoc-HN +
¨ DIPEA
______________________________________ Fmoc-HN
_ CI P = Fmoc-Ser(Resin)-NH2 " ___ o = Resin-CI 0 step-by-step 38 cycles r H-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-X-Lys(Boc)-Gln(Trt)-Met-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Val-Arg(Pbe-Leu-Phe-Ile-Glu(tBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 X = Leu-Ser(tBu) or Leu-LIJSer 1 Deprotection H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-N H2 Exenatide
According to this method, most impurities typically formed during the synthesis of many Pro and Gly residues containing peptides are completely avoided and peptides of high purity are obtained. The method also allows the complete avoidance of impurities originating from the cleavage of peptides from peptide amide linkers using other methods known in the art, which significantly reduce the yields and purity of the peptide.
Date Recue/Date Received 2023-01-12 OH 0-Resin Fmoc-HN +
¨ DIPEA
______________________________________ Fmoc-HN
_ CI P = Fmoc-Ser(Resin)-NH2 " ___ o = Resin-CI 0 step-by-step 38 cycles r H-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-X-Lys(Boc)-Gln(Trt)-Met-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Val-Arg(Pbe-Leu-Phe-Ile-Glu(tBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 X = Leu-Ser(tBu) or Leu-LIJSer 1 Deprotection H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-N H2 Exenatide
[0032] In one aspect, exenatide may be produced by cleavage of the partially protected peptide 12-39 from the resin and condensing it in solution as shown below with the partially protected 1-11 fragment. Alternatively, the condensation to obtain protected exenatide may be performed with the fragments 1-13 and 14-39.
OH 0-Resin Fmoc-HN + C P DIPEA Fmoc-HN
-.1_ = Fmoc-Ser(Resin)-N H2 I
0 I = Resin-CI 0 1step-by-step H-Gln(Trt)-Met-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(tBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-1 Boc-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-X-Lys(Boc)-OH
condensation X= Leu-Ser(tBu) or Leu-N,Ser Boc-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-X-Lys(Boc)-Gln(Trt)-Met-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(tBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-N H2 1 deprotection, cleavage from the resin H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-N H2 Exenatide Pramlintide:
OH 0-Resin Fmoc-HN + C P DIPEA Fmoc-HN
-.1_ = Fmoc-Ser(Resin)-N H2 I
0 I = Resin-CI 0 1step-by-step H-Gln(Trt)-Met-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(tBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-1 Boc-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-X-Lys(Boc)-OH
condensation X= Leu-Ser(tBu) or Leu-N,Ser Boc-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-X-Lys(Boc)-Gln(Trt)-Met-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(tBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-N H2 1 deprotection, cleavage from the resin H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-N H2 Exenatide Pramlintide:
[0033] The method is also highly effective in the production of amylin peptides. In one aspect, the side chain attachment may be performed using one of the C-terminal Ser, Thr or Tyr residues of amylin or its derivatives such as pramlintide. The synthesis may be performed in the step-by-step manner or by fragment condensation in solution or on solid phase. By Date Recue/Date Received 2023-01-12 incorporating pseudoprolines OP, see Mutter et al, Peptide Res. (1995 8, 145) into the growing peptide chain, the synthesis is accelerated and the purity of the peptide obtained is improved.
Fmoc-Tyr(Resin)-NH2 step-by-step Boc-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Y-Cys(Trt)-Y-Gin(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(Resin)-N H2 Resin = 2-chlorotrityl resin 1%-TFA/DCM/12 Boc-Lys(Boc)-Cys-Asn(Trt)-Thr(tBu)-Y-Cys-Y-Gin(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr-NH2 deprotection I I
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gin-Arg-Leu-Ala-Asn-Phe-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-N
pramlintide X = Ser(tBu)-Ser(tBu) or Ser(tBu)-LIJSer Y = Ala-Thr(tBu) or Ala-q-/Thr = pseudoprolin
Fmoc-Tyr(Resin)-NH2 step-by-step Boc-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Y-Cys(Trt)-Y-Gin(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(Resin)-N H2 Resin = 2-chlorotrityl resin 1%-TFA/DCM/12 Boc-Lys(Boc)-Cys-Asn(Trt)-Thr(tBu)-Y-Cys-Y-Gin(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr-NH2 deprotection I I
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gin-Arg-Leu-Ala-Asn-Phe-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-N
pramlintide X = Ser(tBu)-Ser(tBu) or Ser(tBu)-LIJSer Y = Ala-Thr(tBu) or Ala-q-/Thr = pseudoprolin
[0034] Alternatively, the synthesis of pramlintide may be performed in liquid phase with equal success concerning the purity and the yield of the obtained pramlintide. In one embodiment, the protected peptide which is bound on the resin through the side chain of Fmoc-Tyr-NH2 may be quantitatively cleaved from the resin with the side chain protecting groups of the tBu-type remaining intact, using mild acidic treatment at various positions of the peptide chain. In one example, as shown below the partially protected 1-10 fragment prepared on the 2-chlorotrityl resin in the step by step manner was condensed successfully with the partially protected 11-37 fragment amide.
[0035] Pramlintide:
Fmoc-Tyr(Resin)-NH2 lir step-by-step H-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(Resin)-N H2 Resin = 2-chlorotrityl resin I %-TFA/DCM/TES
H-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tB ll )-Asn(Trt)-Thr(tB1-1)-Tyr-N H2 I I
X = Ser(tBu)-Ser(tBu) or Ser(tBu)--PSer Boc-Lys(Boc)-Cys-Asn(Trt)-Thr(tBu)-Y-Cys-Y-Gln-OH
Y = Ala-Thr(tBu) or Ala-LliThr condensation deprotection pramlintide Date Recue/Date Received 2023-01-12 Tetracosactide (ACTH 1-24):
Fmoc-Tyr(Resin)-NH2 lir step-by-step H-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(Resin)-N H2 Resin = 2-chlorotrityl resin I %-TFA/DCM/TES
H-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tB ll )-Asn(Trt)-Thr(tB1-1)-Tyr-N H2 I I
X = Ser(tBu)-Ser(tBu) or Ser(tBu)--PSer Boc-Lys(Boc)-Cys-Asn(Trt)-Thr(tBu)-Y-Cys-Y-Gln-OH
Y = Ala-Thr(tBu) or Ala-LliThr condensation deprotection pramlintide Date Recue/Date Received 2023-01-12 Tetracosactide (ACTH 1-24):
[0036] In another example, ACTH 1-24 was effectively prepared starting from resin-bound Fmoc-Tyr-Pro-OtBu by the step by step procedure or by condensing the 1-10 partially protected fragment in solution with the 11-24 fragment or with the resin-bound fragment, as shown below.
Fmoc-Tyr-OH + H-Pro-OtBu coupling Fmoc-Tyr-Pro-OtBu =
CI-Resin I Resin-CUDIPEA CI P
CI
Fmoc-Tyr(Resin)-Pro-OtBu 1 step-by-step H-Lys(Boc)-Pro- Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(Resin)-Pro-OtBu Boc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(tBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-OH
condensation step-by-step then deprotection and cleavage from the resin Boc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(tBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-OH-Lys(Boc)-Pro- Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(Resin)-Pro-OtBu deprotection, cleavage from the resin .
H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-OH
Tetracosactide (ACTH 1-24) Bivalirudin:
Fmoc-Tyr-OH + H-Pro-OtBu coupling Fmoc-Tyr-Pro-OtBu =
CI-Resin I Resin-CUDIPEA CI P
CI
Fmoc-Tyr(Resin)-Pro-OtBu 1 step-by-step H-Lys(Boc)-Pro- Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(Resin)-Pro-OtBu Boc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(tBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-OH
condensation step-by-step then deprotection and cleavage from the resin Boc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(tBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-OH-Lys(Boc)-Pro- Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(Resin)-Pro-OtBu deprotection, cleavage from the resin .
H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-OH
Tetracosactide (ACTH 1-24) Bivalirudin:
[0037] In another example, bivalirudin was produced in high yield and high purity starting from resin-bound Fmoc-Tyr-Leu-OtBu, extending the peptide chain in the step-by-step manner with Fmoc-amino acids and finally deprotecting and cleaving the peptide from the resin as shown below.
[0038] Alternatively, bivalirudin was obtained by the condensation of protected fragments on the resin or by cleaving a partially protected peptide which contain 4-15 amino acid residues from the resin and condensing it in solution with a bivalirudin fragment which contain 5-16 amino acids. The bivalirudin synthesis by fragment condensation on the resin of the 1-10 partially protected bivalirudin fragment with the resin-bound 11-20 partially protected bivalirudin fragment is described below.
Date Recue/Date Received 2023-01-12 coupling _,...
Fmoc-Tyr-OH + H-Leu-OtBu Fmoc-Tyr-Leu-OtBu = CI-Resin Resin-Cl/DIPEA I
CI CI P
Fmoc-Tyr(Resin)-Leu-OtBu 1 step-by-step H-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-1Ie-Pro-Glu(tBu)-Glu(tBu)-Tyr(Resin)-Leu-OtBu Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-OH
condensation step-by-step then deprotection and cleavage from the resin Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-1Ie-Pro-Glu(tBu)-Glu(tBu)-Tyr(Resin)-Leu-OtBu deprotection, cleavage from the resin 1' H-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH
Bivalirudin EXAMPLES
Example 1:
Date Recue/Date Received 2023-01-12 coupling _,...
Fmoc-Tyr-OH + H-Leu-OtBu Fmoc-Tyr-Leu-OtBu = CI-Resin Resin-Cl/DIPEA I
CI CI P
Fmoc-Tyr(Resin)-Leu-OtBu 1 step-by-step H-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-1Ie-Pro-Glu(tBu)-Glu(tBu)-Tyr(Resin)-Leu-OtBu Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-OH
condensation step-by-step then deprotection and cleavage from the resin Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-1Ie-Pro-Glu(tBu)-Glu(tBu)-Tyr(Resin)-Leu-OtBu deprotection, cleavage from the resin 1' H-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH
Bivalirudin EXAMPLES
Example 1:
[0039] Preparation of Fmoc-Thr(4-methoxybenzhydryl polystyry1)-0tBu P
= Br-Resin Fmoc-Thr-OtBu + H Br DIPEA OCH3 ,-- Fmoc-Thr(Resin)-0tBu
= Br-Resin Fmoc-Thr-OtBu + H Br DIPEA OCH3 ,-- Fmoc-Thr(Resin)-0tBu
[0040] 30 mmol Fmoc-Thr-OtBu prepared from H-Thr-OtBu by its reaction with Fmoc-OSu following conventional methods were reacted with 20 g (30 mmol) of 4-methoxybenzhydryl polystyrene resin (product of CBL-Patras) and 60 mmol DIPEA in 250 ml THF for 10 h at RT.
To the mixture were then added 60 mmol methanol and the mixture was shaken for additional 4 h. The resin was filtered and washed 3X with THF/Me0H/DIPEA (85:10:5), 6X
DMF, 4X
IPA, 3X DEE and dried in vacuum to constant weight. 29 g of resin-bound Fmoc-Thr-OtBu were obtained with a loading of 0.95 mmol/g resin.
Example 2
To the mixture were then added 60 mmol methanol and the mixture was shaken for additional 4 h. The resin was filtered and washed 3X with THF/Me0H/DIPEA (85:10:5), 6X
DMF, 4X
IPA, 3X DEE and dried in vacuum to constant weight. 29 g of resin-bound Fmoc-Thr-OtBu were obtained with a loading of 0.95 mmol/g resin.
Example 2
[0041] Fmoc-Thr(4-methoxybenzhydryl poly styry1)-0-Clt P
= Br-Resin Trt-Thr-OMe + H Br DIPEA OCH3 ..- Trt-Thr(Resin)-0Me Date Recue/Date Received 2023-01-12 10/ TFA,. 1N-LiOH
______________________ H-Thr(Resin)-0Me __ i.- H-Thr(Resin)-OH
Fmoc-OSu Clt-CI . Fmoc-Thr(Resin)-OH > Fmoc-Thr(Resin)-0-Clt DIPEA
= Br-Resin Trt-Thr-OMe + H Br DIPEA OCH3 ..- Trt-Thr(Resin)-0Me Date Recue/Date Received 2023-01-12 10/ TFA,. 1N-LiOH
______________________ H-Thr(Resin)-0Me __ i.- H-Thr(Resin)-OH
Fmoc-OSu Clt-CI . Fmoc-Thr(Resin)-OH > Fmoc-Thr(Resin)-0-Clt DIPEA
[0042] 30 mmol Trt-Thr-OMe prepared from H-Thr-OMe by its reaction with Trt-C1/Me3SiC1 and DIPEA following conventional methods were reacted with 20 g (30 mmol) of 4-methoxy 4'-polystyryl benzhydryl bromide resin (product of CBL-Patras) and 60 mmol DIPEA in 250 ml THF for 10 h at RT. To the mixture were then added 60 mmol methanol and the mixture was shaken for additional 4 h. The resin was filtered and washed 3X with THF/Me0H/DIPEA
(85:10:5), 3X DCM, 3X 1% TFA in DCM, 4X THF, 3X 1N-LiOH in THF/Water/Methanol (70:15:15), 3X THF/Water (75:25) 4X DMF and then reacted for 2 hat RT with 60 mmol Fmoc-OSu and 30 mmol DIPEA, washed 3X DMF, 3X DCM and then reacted for 3h at RT
with 50 mmol Trt-Cl and 50 mmol DIPEA, washed 4X DMF, 6X DEE and dried in vacuum to constant weight. 32.3 g of resin-bound Fmoc-Thr-OtBu were obtained with a loading of 0.78 mmol/g resin.
Example 3
(85:10:5), 3X DCM, 3X 1% TFA in DCM, 4X THF, 3X 1N-LiOH in THF/Water/Methanol (70:15:15), 3X THF/Water (75:25) 4X DMF and then reacted for 2 hat RT with 60 mmol Fmoc-OSu and 30 mmol DIPEA, washed 3X DMF, 3X DCM and then reacted for 3h at RT
with 50 mmol Trt-Cl and 50 mmol DIPEA, washed 4X DMF, 6X DEE and dried in vacuum to constant weight. 32.3 g of resin-bound Fmoc-Thr-OtBu were obtained with a loading of 0.78 mmol/g resin.
Example 3
[0043] Fmoc-Throl(4-methoxy benzhydryl polystyry1)-0-Clt
[0044] A) Starting from Fmoc-threoninol = Resin-Br OH OH H P 0-Resin _CH3 +
DIPEA )¨CH, Br _CH3 Fmoc-HN at-CI Fmoc-HN_ Fmoc-HN
OH 0-CR DIPEA 0-Clt
DIPEA )¨CH, Br _CH3 Fmoc-HN at-CI Fmoc-HN_ Fmoc-HN
OH 0-CR DIPEA 0-Clt
[0045] 50 mmol commercial Fmoc-threoninol (CBL-Patras) in 350 ml DCM were reacted with 55 mmol monomeric CU-CI and 55 mmol DIPEA for 4 h at RT. The obtained mixture was extracted as usual with water and the DCM phase was dried over anhydrous sodium sulphate and filtered. To the resulting solution 30 g of 4-methoxy, 4-polystyryl benzhydryl bromide (CBL-Patras) were added and 50 mmol DIPEA and the resulting mixture was stirred for 4 h at RT. The resin was filtered and washed 6XDMF, 4XIPA and 4X DEE and dried in vacuum to constant weight. 38.4 g of resin-bound Fmoc-threoninol were obtained with a loading of 0.82 mmol/g.
[0046] B) Starting from Trt-Thr(Resin)-0Me Date Recue/Date Received 2023-01-12 Me 0-resin LiBH4 Me 0-resin 1% TFA Me 0-resin Me 0-resin Fmoc-OSu CR-CI Me 0-resin Fmoc¨N DIPEA
[0047] 30 mmol Trt-Thr-OMe prepared from H-Thr-OMe by its reaction with Trt-C1/Me3SiC1 and DIPEA following conventional methods were reacted with 20 g (30 mmol) of 4-methoxy 4'-polystyryl benzhydryl bromide resin (product of CBL-Patras) and 60 mmol DIPEA in 250 ml THF for 10 h at RT. To the mixture were then added 60 mmol methanol and the mixture was shaken for additional 4 h. The resin was filtered and washed 3X with THF/Me0H/DIPEA
(85:10:5), 5X THF, and then reacted with 30 mmol LiBILI in THF. The resin was then filtered and washed 6X THF, 4X DCM, 6X 1% TFA in DCM, 3X with DMF/DIPEA (97:3) and then reacted for 2 h at RT with 60 mmol Fmoc-OSu and 30 mmol DIPEA, washed 3X DMF, DCM and then reacted for 3h at RT with 50 mmol CU-CI and 50 mmol DIPEA, washed DMF, 6X IPA and 6X DEE and dried in vacuum to constant weight. 34.7 g of resin-bound Fmoc-Throl-O-Clt were obtained with a loading of 0.74 mmol/g resin.
Example 4
(85:10:5), 5X THF, and then reacted with 30 mmol LiBILI in THF. The resin was then filtered and washed 6X THF, 4X DCM, 6X 1% TFA in DCM, 3X with DMF/DIPEA (97:3) and then reacted for 2 h at RT with 60 mmol Fmoc-OSu and 30 mmol DIPEA, washed 3X DMF, DCM and then reacted for 3h at RT with 50 mmol CU-CI and 50 mmol DIPEA, washed DMF, 6X IPA and 6X DEE and dried in vacuum to constant weight. 34.7 g of resin-bound Fmoc-Throl-O-Clt were obtained with a loading of 0.74 mmol/g resin.
Example 4
[0048] Fmoc-Ser(trityl resin)-NH2
[0049] 50 mmols of Fmoc-Ser-NH2, prepared according to standard procedures known in the art, were dissolved in 0.5 liter of DCM. To the suspension 30 g of Trityl chloride resin (36 mmol) were added and 65 mmol DIPEA and the mixture was stirred for 6 h at RT.
Then and then 25 ml methanol and 30 mmol DIPEA were added and the mixture was stirred for additional 2h at RT. The resin was then filtered and washed 3X with DCM/Me0H/DIPEA
(90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 41.1 g of Fmoc-Ser-NH2-containing resin were obtained with a loading of 0.71 mmol/g.
Example 5
Then and then 25 ml methanol and 30 mmol DIPEA were added and the mixture was stirred for additional 2h at RT. The resin was then filtered and washed 3X with DCM/Me0H/DIPEA
(90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 41.1 g of Fmoc-Ser-NH2-containing resin were obtained with a loading of 0.71 mmol/g.
Example 5
[0050] Fmoc-Tyr(2-chlorotrityl resin)-NH2
[0051] Following the above procedure, 50 mmol Fmoc-Tyr-NH2 and 30 g 2-CTC
chloride resin gave 43.7 g resin with a loading of 0.81 g Tyr/g resin.
Example 6
chloride resin gave 43.7 g resin with a loading of 0.81 g Tyr/g resin.
Example 6
[0052] Fmoc-Hyp(4-methyl benzhydryl resin)-NH2
[0053] Following the above procedure 50 mmol Fmoc-Hyp-NH2 and 30 g 4-methyl benzhydryl bromide resin gave 39.8 g resin with a loading of 0.49 g Hyp/g resin.
Date Recue/Date Received 2023-01-12 Example 7
Date Recue/Date Received 2023-01-12 Example 7
[0054] Fmoc-Thr(4-methoxybenzhydryl resin)-Pro-N}12
[0055] 50 mmols of Fmoc-Thr-Pro-NI-12 prepared from coupling of Fmoc-Thr(tBu)-OH with H-Pro-NH2 according to standard procedures known in the art, were dissolved in 0.5 liter of DME. To the resulting solution 30 g of 4-methoxy benzhydryl bromide resin (45 mmol) were added and 65 mmol DIPEA and the mixture was stirred for 6 h at RT. Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred for additional 2h at RT. The resin was then filtered and washed 3X with DME/Me0H/DIPEA (90:5:5), 5X DMF, 4X
IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Thr-Pro-NH2 containing resin with a loading of 0.77 mmol/g was obtained.
Example 8
IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Thr-Pro-NH2 containing resin with a loading of 0.77 mmol/g was obtained.
Example 8
[0056] Fmoc-Tyr(2-chlorotrityl resin)-Pro-OtBu
[0057] 50 mmols of Fmoc-Tyr-Pro-OtBu were prepared according to standard procedures known in the art, were dissolved in 0.5 liter of DCM. To the resulting solution 30 g of 2-chlorotrityl chloride resin (48 mmol) were added and 65 mmol DIPEA and the mixture was stirred for 12 h at RT. Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred for additional 2h at RT. The resin was then filtered and washed 3X
with DCM/Me0H/DIPEA (90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Tyr-Pro-OtBu containing resin with a loading of 0.64 mmol/g was obtained.
Example 9
with DCM/Me0H/DIPEA (90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Tyr-Pro-OtBu containing resin with a loading of 0.64 mmol/g was obtained.
Example 9
[0058] Fmoc-Tyr(2-chlorotrityl resin)-Leu-OtBu
[0059] 50 mmols of Fmoc-Tyr-Leu-OtBu, prepared according to standard procedures known in the art, were dissolved in 0.5 liter of THF. To the resulting solution 30 g of 2-CTC chloride resin (48 mmol) were added and 65 mmol DIPEA and the mixture was stirred for 12 h at 60 C.
Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred for additional 2 h at RT. The resin was then filtered and washed 3X with DCM/Me0H/DIPEA
(90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Tyr-Leu-OtBu-containing resin with a loading of 0.64 mmol/g was obtained.
Example 10
Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred for additional 2 h at RT. The resin was then filtered and washed 3X with DCM/Me0H/DIPEA
(90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Tyr-Leu-OtBu-containing resin with a loading of 0.64 mmol/g was obtained.
Example 10
[0060] Solid-phase synthesis of peptides and protected peptide segments.
General procedure.
General procedure.
[0061] Al. Preparation of loaded 2-chlorotrityl resins, general procedure Date Recue/Date Received 2023-01-12
[0062] 2-Chlorotrityl chloride resin (CTC-C1) (100 g; loading 1.6 mmol/g) of CBL-Patras, is placed in a 2 L peptide synthesis reactor and is swollen with 700 mL
dichloromethane (DCM):dimethylformamide (DMF) 1:1 for 30 min at 25 C. The resin is filtered and a solution of 100 mmol Fmoc-amino acid and 300 mmol diisopropylethylamine (DIEA) in 500 mL DCM
is added. The mixture is stirred under nitrogen for 2 hours at 25 C. Then, the remaining active sites of the 2-CTC resin are neutralised by adding 10 mL of methanol (Me0H) and reacting for 1 hour. The resin is filtered and washed twice with 400 mL DMF.
The resin is filtered and treated twice with 500 mL 25% by volume of piperidine in DMF for 30 min. The resin is then washed four times with 500 mL DMF. The resin is deswelled with 3 washes with 500 mL of isopropanol (IPA). The resin is dried to constant weight. On the resin was bound the 70-95% of the mmol of the used amino acid.
dichloromethane (DCM):dimethylformamide (DMF) 1:1 for 30 min at 25 C. The resin is filtered and a solution of 100 mmol Fmoc-amino acid and 300 mmol diisopropylethylamine (DIEA) in 500 mL DCM
is added. The mixture is stirred under nitrogen for 2 hours at 25 C. Then, the remaining active sites of the 2-CTC resin are neutralised by adding 10 mL of methanol (Me0H) and reacting for 1 hour. The resin is filtered and washed twice with 400 mL DMF.
The resin is filtered and treated twice with 500 mL 25% by volume of piperidine in DMF for 30 min. The resin is then washed four times with 500 mL DMF. The resin is deswelled with 3 washes with 500 mL of isopropanol (IPA). The resin is dried to constant weight. On the resin was bound the 70-95% of the mmol of the used amino acid.
[0063] B. Solid-phase synthesis, a general protocol
[0064] The solid-phase synthesis was performed at 24 C with 1.0 g amino acid or peptide esterified to the resin of the trityl or benzhydryl type or attached through its side chain as described in Part A or in the examples Example 1. The following protocol was used in the synthesis.
[0065] Bl. Swelling of the resin
[0066] The resin was placed in a 15 ml reactor and treated twice with 7 mL
NMP, followed by filtration.
NMP, followed by filtration.
[0067] B2. Activation of the amino acid
[0068] The amino acid (3.0 equiv.) and 1-hydroxybenzotriazole (4.0 equiv.) was weighted and dissolved in a reactor with 2.5 their volume in NMP and cooled to 0 C. DIC
was then added (3.0 equiv.) and the mixture was stirred for 15 min.
was then added (3.0 equiv.) and the mixture was stirred for 15 min.
[0069] B3. Coupling
[0070] The solution which was prepared in B2 was then added to the B1 reactor.
The reactor was washed once with one volume of DCM and was added to the reactor which was stirred for 1-3 h at 25 -30 C. In a sample the Kaiser Test was performed to determine the completion of the reaction. If the coupling reaction was not completed after 3 h (positive Kaiser Test), the reaction mixture was filtered and recoupled with a fresh solution of activated amino acid.
After completion of the coupling the reaction mixture was filtered and washed 4 times with NMP (5 volumes per wash).
The reactor was washed once with one volume of DCM and was added to the reactor which was stirred for 1-3 h at 25 -30 C. In a sample the Kaiser Test was performed to determine the completion of the reaction. If the coupling reaction was not completed after 3 h (positive Kaiser Test), the reaction mixture was filtered and recoupled with a fresh solution of activated amino acid.
After completion of the coupling the reaction mixture was filtered and washed 4 times with NMP (5 volumes per wash).
[0071] B4. Removal of the Fmoc-group Date Recue/Date Received 2023-01-12
[0072] The resulting resin in B3 was filtered and then treated for 30 min with 5 mL of a solution which contained 25% by volume of piperidine. The resin is then washed three times with 5 mL NMP.
[0073] B5. Elongation of the peptide chain
[0074] After the incorporation of each amino acid the steps Bl-B5 were repeated until the completion of the peptide chain.
[0075] For the introduction of each individual amino acid the following Fmoc-amino acids were used: Fmoc-Ala-OH, Fmoc-Arg(Pb0-0H, Fmoc-Asn-OH, Fmoc-Asn(TrO-OH, Fmoc-D-Cys(TrO-OH, Fmoc-Cys(TrO-OH, Fmoc-Gln-OH, Fmoc-Gln(TrO-OH, Fmoc-Glu(tBu)-0H, Fmoc-Gly-OH, Fmoc-His(TrO-OH, Fmoc-Hyp(tBu)-0H, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-D-Phe-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-0H, Fmoc-Ser(TrO-OH, Fmoc-Thr(tBu)-0H, Fmoc-Ser(TrO-OH, Fmoc-D-Trp-OH, Fmoc-Trp-OH, Fmoc-D-Trp(Boc)-0H, Fmoc-Trp(Boc)-0H, Fmoc-Tyr(tBu)-0H, Fmoc-Tyr(C10-0H, Fmoc-Val-OH, Boc-D-Cys(TrO-OH, Boc-His(TrO-OH, Boc-Lys(Boc)-0H, Boc-D-2-Nal-OH, Boc-D-Phe-OH, Boc-Ser(tBu)-0H.
[0076] C. General method for the acidic cleavage from the CTC- resin of peptides and of protected peptide segments, which contain Fmoc- or Boc-groups on their N-terminus.
[0077] The resin-bound peptide or peptide segment which was produced as described above in B1-B5 was washed 4 times with 5 mL NMP, 3 times with 5 ml IPA and finally 5 times with 7 ml DCM to remove completely any residual NMP or other basic components. The resin was then cooled to 0 C, filtered from DCM and was treated twice with a solution of 10 mL 1-2%
TFA/DCM at 5 C. The mixture is then stirred 20 min at 0 C and filtered. The resin is then washed three times with 10 mL DCM. Pyridine is then added to the filtrates (1.3 equiv.
relative to TFA) to neutralize the TFA. The cleavage solution in DCM is then mixed with an equal volume of water. The resulting mixture is distilled at reduced pressure to remove DCM
(350 ton at 28 C). The peptide or peptide segment precipitated after the removal of DCM.
The resulting peptide is washed then with water and dried at 30-35 C under 15 Ton vacuum.
Example 11
TFA/DCM at 5 C. The mixture is then stirred 20 min at 0 C and filtered. The resin is then washed three times with 10 mL DCM. Pyridine is then added to the filtrates (1.3 equiv.
relative to TFA) to neutralize the TFA. The cleavage solution in DCM is then mixed with an equal volume of water. The resulting mixture is distilled at reduced pressure to remove DCM
(350 ton at 28 C). The peptide or peptide segment precipitated after the removal of DCM.
The resulting peptide is washed then with water and dried at 30-35 C under 15 Ton vacuum.
Example 11
[0078] Synthesis of resin-bound protected peptides by the condensation of an N-terminal protected fragment with a resin-bound C-terminal protected fragment.
General procedure
General procedure
[0079] To a solution of 0.15 mmol/ml of an N-terminal protected peptide fragment in DMSO/DCM (95:5) are added 0.2 mmol HOBt and the resulting solution is cooled to 5 C.
Then 0.14 mmol DIC were added and the mixture is stirred for 20 min at 15 C
and added then Date Recue/Date Received 2023-01-12 to 0.1 mmol of a resin-bound C-terminal fragment and stirred for additional 6 h at RT. The completion of the condensation reaction is checked by the Kaiser test. In the cases where the Kaiser test remained blue a second condensation was performed in order to drive the condensation into completion.
Example 12
Then 0.14 mmol DIC were added and the mixture is stirred for 20 min at 15 C
and added then Date Recue/Date Received 2023-01-12 to 0.1 mmol of a resin-bound C-terminal fragment and stirred for additional 6 h at RT. The completion of the condensation reaction is checked by the Kaiser test. In the cases where the Kaiser test remained blue a second condensation was performed in order to drive the condensation into completion.
Example 12
[0080] Synthesis of partially protected peptides by the condensation of an N-terminal protected fragment with a C-terminal protected fragment in solution.
General procedure
General procedure
[0081] To a solution of 0.15 mmol/ml of an N-terminal protected fragment in DCM are added 0.2 mmol HOBt and the resulting solution is cooled to 5 C. Then 0.15 mmol EDAC were added and the mixture is stirred for 20 min at 15 C and added then to 0.15 mmol of a C-terminal protected fragment and stirred for additional 2-5 h at RT. The completion of the condensation reaction is checked by HPLC. In the cases where an incomplete condensation was observed an additional portion of 0.015 mmol EDAC was added and the reaction was left to proceed for an additional hour at RT.
Example 13
Example 13
[0082] Deprotection and simultaneous cleavage from the resin of peptides.
General method
General method
[0083] 1.00 g of the protected resin-bound peptide, produced as described above is treated with 20 mL TFA/DTT/water (90:5:5) for 3 h at 5 C and for 1 h at 15 C. The resin is then washed 3X with the cleavage solution and the combined filtrates are then concentrated in vacuum and crude peptide is precipitated by the addition of ether, washed several times with ether and dried in vacuum until constant weight over KOH.
Example 14
Example 14
[0084] Peptide Deprotection General method
[0085] 1.00 g of the protected peptide, produced as described above was treated with 20 mL
TFA/DTT/water (90:5:5) for 3 h at 5 C and for 1 h at 15 C. The resulting solution is concentrated in vacuum and then the deprotected peptide was precipitated by the addition of diisopropylether and washed three times with 10 mL diisopropylether. The resulting solid was dried in vacuum (25 C, 15 Ton) until constant weight under KOH.
Example 15
TFA/DTT/water (90:5:5) for 3 h at 5 C and for 1 h at 15 C. The resulting solution is concentrated in vacuum and then the deprotected peptide was precipitated by the addition of diisopropylether and washed three times with 10 mL diisopropylether. The resulting solid was dried in vacuum (25 C, 15 Ton) until constant weight under KOH.
Example 15
[0086] Purification of crude peptides. Isolation of peptides.
Date Recue/Date Received 2023-01-12 General procedure
Date Recue/Date Received 2023-01-12 General procedure
[0087] The solution of the peptides obtained as described above was concentrated in vacuum and ice water and ether were added. After separation of the organic layer the remaining water solution of the peptide was extracted for additional two times with ether and the resulting solution was sparged with nitrogen or helium, filtered and directly loaded on a semipreparative column 10x25 cm, LichrospherTM 100, RP-18, 12 micron (Merck); Phase A
= 1%-TFA in acetonitrile, phase B = 1%-TFA in water; or Kromasil. HPLC fractions containing the purified peptide were concentrated in vacuum to remove as much as possible the contained acetonitrile and lyophilized using a standard lyophilisation program. prepare the listed compounds.
= 1%-TFA in acetonitrile, phase B = 1%-TFA in water; or Kromasil. HPLC fractions containing the purified peptide were concentrated in vacuum to remove as much as possible the contained acetonitrile and lyophilized using a standard lyophilisation program. prepare the listed compounds.
[0088] Examples 16 to 23, as noted below, were performed using the above procedures to prepare the listed compounds.
[0089] Example 16: Lanreotide
[0090] Example 17: Insulin B-chain
[0091] Example 18: Salmon calcitonin
[0092] Example 19: Octreotide
[0093] Example 20: Exenatide
[0094] Example 21: Pramlintide
[0095] Example 22: Tetracosactide (ACTH 1-24)
[0096] Example 23: Bivalirudin Date Recue/Date Received 2023-01-12
Claims (42)
1. A resin conjugate of the formula I or II, wherein:
\
H
Y I V Y
Pi '-Hya-A PP-Hya-A
Formula I Formula II
Hya is the residue of a hydroxy amino acid;
Pr' is H, or an amino protecting group which is orthogonal to the resin and to other protecting groups;
A is a hydroxyl group or a acid sensitive hydroxyl protecting group selected from tBu, Trt, Clt, or Nlele wherein le and le are H or C1_10 alkyl, or peptide ester or peptide amide or a peptaibol containing 0-30 amino acids;
X, Y, Z and V are each independently a substituent on the ortho, meta or para positions and is selected from H, CI, F, Ci_io alkyl or Ci_io alkoxy; and P is an insoluble solid support or an insoluble linker-resin conjugate suitable for the solid phase synthesis of peptides.
\
H
Y I V Y
Pi '-Hya-A PP-Hya-A
Formula I Formula II
Hya is the residue of a hydroxy amino acid;
Pr' is H, or an amino protecting group which is orthogonal to the resin and to other protecting groups;
A is a hydroxyl group or a acid sensitive hydroxyl protecting group selected from tBu, Trt, Clt, or Nlele wherein le and le are H or C1_10 alkyl, or peptide ester or peptide amide or a peptaibol containing 0-30 amino acids;
X, Y, Z and V are each independently a substituent on the ortho, meta or para positions and is selected from H, CI, F, Ci_io alkyl or Ci_io alkoxy; and P is an insoluble solid support or an insoluble linker-resin conjugate suitable for the solid phase synthesis of peptides.
2. A resin conjugate of the formula III-VI, wherein:
k,Z
H
-1) Pri¨N7\,-0-Pr2 Pr1¨N
Formula III Formula IV
H
V
O-Pr2 Prr Prr Formula V Formula VI
Date Recue/Date Received 2023-01-12 Pr' is H or an amino protecting group, which is orthogonal to the resin and to other protecting groups;
Pr2 is H or a hydroxyl protecting group which is orthogonal to the resin;
R3 and R4 are each independently H or C1-10 alkyl;
X, Y, Z and V are each independently on the ortho, meta or para positions selected from H, CI, F, Ci_io alkyl or C1_10 alkoxy; and P is an insoluble solid support or an insoluble linker-resin conjugate suitable for the solid phase synthesis of peptides.
k,Z
H
-1) Pri¨N7\,-0-Pr2 Pr1¨N
Formula III Formula IV
H
V
O-Pr2 Prr Prr Formula V Formula VI
Date Recue/Date Received 2023-01-12 Pr' is H or an amino protecting group, which is orthogonal to the resin and to other protecting groups;
Pr2 is H or a hydroxyl protecting group which is orthogonal to the resin;
R3 and R4 are each independently H or C1-10 alkyl;
X, Y, Z and V are each independently on the ortho, meta or para positions selected from H, CI, F, Ci_io alkyl or C1_10 alkoxy; and P is an insoluble solid support or an insoluble linker-resin conjugate suitable for the solid phase synthesis of peptides.
3. A method for the preparation of the resin conjugates of the formulae I-VI comprising the following steps:
preparing a hydroxyl containing amino acid or amino alcohol or peptide derivative that is unprotected on at least one of the side chains of the contained hydroxyl amino acids or of the contained amino alcohol or selectively deprotecting the hydroxyl amino acid or the hydroxyl amino alcohol or a peptide derivative at the side chain of the hydroxyl amino acid or the hydroxyl amino alcohol and then attaching it to a suitable resin by its reaction with a resin halide, wherein the resin is selected from the group of the trityl type resins and linkers or the benzhydryl type resins or the benzyl-type resins; and an alcohol or thioalcohol is added to mask any unreacted resin halide.
preparing a hydroxyl containing amino acid or amino alcohol or peptide derivative that is unprotected on at least one of the side chains of the contained hydroxyl amino acids or of the contained amino alcohol or selectively deprotecting the hydroxyl amino acid or the hydroxyl amino alcohol or a peptide derivative at the side chain of the hydroxyl amino acid or the hydroxyl amino alcohol and then attaching it to a suitable resin by its reaction with a resin halide, wherein the resin is selected from the group of the trityl type resins and linkers or the benzhydryl type resins or the benzyl-type resins; and an alcohol or thioalcohol is added to mask any unreacted resin halide.
4. A process for the preparation of monoalkylated Fmoc-amino di-alcohols, the process comprising the reaction of the Fmoc-di-amino alcohol with alkylhalide in the presence of a base such as a tertiary amine base in an organic solvent, such as dichloromethane; and wherein alkyl is a triarylmethyl selected from trityl, 4-methyltrityl, 4-methoxytrityl and 2-chlorotrityl.
5. A process for the solid phase synthesis of biologically active free or partially protected peptides, cyclic peptides and peptaibols, wherein the process comprises the use of the resin conjugates of Claim 1 or Claim 2 as functionalized resins in the solid phase peptide synthesis.
6. A peptide of the formula E-D-2-Nal-Cys(A)-Tyr(C)-D-Trp(F)-Lys(E)-Val-Cys(A)-Thr(Resin)-NH2 wherein:
D- designates the chirality of the amino acid that follows as a D-amino acid;
Date Recue/Date Received 2023-01-12 each A is independently a thiol protecting group selected from Trt, Mmt, Acm or StBu;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
F is H or Boc;
E is an amino protecting group selected from Mtt, Mmt, Trt or Boc; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
D- designates the chirality of the amino acid that follows as a D-amino acid;
Date Recue/Date Received 2023-01-12 each A is independently a thiol protecting group selected from Trt, Mmt, Acm or StBu;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
F is H or Boc;
E is an amino protecting group selected from Mtt, Mmt, Trt or Boc; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
7. The peptide of Claim 6, wherein the peptide is a resin-bound lanreotide.
8. A process for preparing lanreotide comprising:
treating the peptide resin conjugate of Claim 7 with a mild acid selected from solutions of trifluoracetic acid optionally containing scavengers; and oxidizing the resulting peptide using an oxidizing agent selected from air, hydrogen peroxide, DMSO or iodine.
treating the peptide resin conjugate of Claim 7 with a mild acid selected from solutions of trifluoracetic acid optionally containing scavengers; and oxidizing the resulting peptide using an oxidizing agent selected from air, hydrogen peroxide, DMSO or iodine.
9. A process for preparing lanreotide comprising treating the peptide resin conjugate of Claim 7 with a mild acid such as a solution of trifluoracetic acid containing iodine.
10. A peptide of the formula E-Phe-Val-Asn(A)-Gln(A)-His(A)-Leu-Cys(B)-Gly-Ser(C)-His(A)-Leu-Val-Glu(C)-Ala-Leu-Tyr(C)-Leu-Val-Cys(A)-Gly-Glu(C)-Arg(D)-Gly-Phe-Phe-Tyr(C)-Thr(C)-Pro-Lys(E)-Thr(Resin)-0-C wherein:
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
B is a Cys protecting group selected from Mmt, Trt, Acm or StBu;
C is a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
D is a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
B is a Cys protecting group selected from Mmt, Trt, Acm or StBu;
C is a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
D is a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
11. The peptide of Claim 10, wherein the peptide is human insulin B-chain.
12. A process for preparing an insulin B-chain, wherein the process comprises contacting the resin-bound peptide of Claim 10 with an acid solution, such as trifluoroacetic acid solutions in dichloromethane, optionally containing scavengers.
Date Recue/Date Received 2023-01-12
Date Recue/Date Received 2023-01-12
13. A peptide of the formula E-Ser(C)-His(A)-Leu-Val-Glu(C)-Ala-Leu-Tyr(C)-Leu-Val-Cys(B)-Gly-Glu(C)-Arg(D)-Gly-Phe-Phe-Tyr(C)-Thr(C)-Pro-Lys(E)-Thr(Resin)-0-wherein:
A is a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
B is a Cys protecting group selected from Mmt, Trt, Acm or StBu;
Each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A is a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
B is a Cys protecting group selected from Mmt, Trt, Acm or StBu;
Each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
14. The peptide of Claim 13, wherein the peptide corresponds to the partially protected 9-30 fragment of human insulin B-chain.
15. A process for the preparation of insulin B-chain, the process comprising:
condensation of the peptide of Claim 13 with a 1-8 protected insulin fragment Boc-Phe-Val-Asn(A)-Gln(A)-His(A)-Leu-Cys(B)-Gly-OH, wherein;
each A is independently a carboxamido or imidazol protecting group selected from Trt, Mtt and Mmt; and B is a Cys protecting group selected from Mmt, Trt, Acm or StBu.
condensation of the peptide of Claim 13 with a 1-8 protected insulin fragment Boc-Phe-Val-Asn(A)-Gln(A)-His(A)-Leu-Cys(B)-Gly-OH, wherein;
each A is independently a carboxamido or imidazol protecting group selected from Trt, Mtt and Mmt; and B is a Cys protecting group selected from Mmt, Trt, Acm or StBu.
16. .. A peptide of the formula E-Cys(B)-Ser(C)-Asn(A)-Leu-Ser(C)-Thr(C)-Cys(B)-Val-Leu-Gly-Lys(E)-Leu-Ser(C)-Gln(A)-Glu(C)-Leu-His(A)-Lys(E)-Leu-Gln(A)-Thr(C)-Tyr(C)-Pro-Arg(D)-Thr(C)-Asn(A)-Thr(C)-Gly-Ser(C)-Gly-Thr(Resin)-Pro-NH2, wherein:
each A is independently a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or StBu;
each C is independently a hydroxyl, carboxy or phenoxy protecting group selected from of tBu, Trt or Clt;
D is a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Date Recue/Date Received 2023-01-12
each A is independently a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or StBu;
each C is independently a hydroxyl, carboxy or phenoxy protecting group selected from of tBu, Trt or Clt;
D is a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Date Recue/Date Received 2023-01-12
17. The peptide of Claim 16, wherein the peptide is salmon calcitonin.
18. A process for producing salmon calcitonin, the process comprising:
treating the peptide of Claim 16 with a mild acid such as a solution of trifluoracetic acid optionally containing scavengers;
oxidizing the obtained peptide solution using a suitable oxidizing agent selected from air, hydrogen peroxide, DMSO or iodine;
deprotecting the peptide;
purifying the peptide by chromatography; and lyophilizing the peptide.
treating the peptide of Claim 16 with a mild acid such as a solution of trifluoracetic acid optionally containing scavengers;
oxidizing the obtained peptide solution using a suitable oxidizing agent selected from air, hydrogen peroxide, DMSO or iodine;
deprotecting the peptide;
purifying the peptide by chromatography; and lyophilizing the peptide.
19. A process for producing salmon calcitonin, the process comprising:
treating the peptide resin conjugate of Claim 16 with a mild acid such as a solution of trifluoracetic acid;
oxidizing the peptide with iodine;
deprotecting the peptide by treatment with acids such as a solution of trifluoroacetic or hydrochloric acid optionally containing scavengers; and purifying by chromatography and lyophilizing the salmon calcitonin peptide.
treating the peptide resin conjugate of Claim 16 with a mild acid such as a solution of trifluoracetic acid;
oxidizing the peptide with iodine;
deprotecting the peptide by treatment with acids such as a solution of trifluoroacetic or hydrochloric acid optionally containing scavengers; and purifying by chromatography and lyophilizing the salmon calcitonin peptide.
20. A process for the preparation of the peptide of Claim 17, the process comprises condensing the partially protected 11-32 fragment of salmon calcitonin of the formula H-Lys(E)-Leu-Ser(C)-Gln(A)-Glu(C)-Leu-His(A)-Lys(E)-Leu-Gln(A)-Thr(C)-Tyr(C)-Pro-Arg(D)-Thr(C)-Asn(A)-Thr(C)-Gly-Ser(C)-Gly-Thr(Resin)-Pro-NH2 with a 1-10 partially protected fragment of salmon calcitonin of the formula E-Cys(B)-Ser(C)-Asn(A)-Leu-Ser(C)-Thr(C)-Cys(B)-Val-Leu-Gly-OH; wherein each A is independently a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or StBu;
each C is independently a hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Date Recue/Date Received 2023-01-12 Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or StBu;
each C is independently a hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Date Recue/Date Received 2023-01-12 Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
21. A partially protected peptide of the formula:
LO-Resin A-D-Phe-Cys(B)-Phe-D-Trp(C)-Lys(A)-Phe-Thr(E)-Cys(B)-NH
O-E
wherein:
A is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt or Mmt;
D- designates the chirality of the amino acid that follows as a D-amino acid;
B is a thiol protecting group selected from Trt, Mmt, Acm or StBu;
C is H or Boc;
E is a hydroxy protecting group selected from Clt, Trt or tBu; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
LO-Resin A-D-Phe-Cys(B)-Phe-D-Trp(C)-Lys(A)-Phe-Thr(E)-Cys(B)-NH
O-E
wherein:
A is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt or Mmt;
D- designates the chirality of the amino acid that follows as a D-amino acid;
B is a thiol protecting group selected from Trt, Mmt, Acm or StBu;
C is H or Boc;
E is a hydroxy protecting group selected from Clt, Trt or tBu; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
22. The peptide of Claim 21, wherein the peptide is a resin bound octreotide,
23. A process for preparing octreotide, the process comprising:
treating the peptide resin conjugate of Claim 22 with a mild acid, such as a solution of trifluoracetic acid optionally containing scavengers; and oxidizing the obtained peptide solution using a suitable oxidizing agent selected from air, hydrogen peroxide, DMSO or iodine.
treating the peptide resin conjugate of Claim 22 with a mild acid, such as a solution of trifluoracetic acid optionally containing scavengers; and oxidizing the obtained peptide solution using a suitable oxidizing agent selected from air, hydrogen peroxide, DMSO or iodine.
24. A process for preparing octreotide comprising:
treating the peptide resin conjugate of Claim 22 with a mild acid such as a solution of trifluoracetic acid containing iodine;
deprotecting, purifying and lyophilizing and obtaining octreotide, wherein the octreotide has a purity of >99%.
treating the peptide resin conjugate of Claim 22 with a mild acid such as a solution of trifluoracetic acid containing iodine;
deprotecting, purifying and lyophilizing and obtaining octreotide, wherein the octreotide has a purity of >99%.
25. A partially protected peptide of the formula E-His(A)-Gly-Glu(C)-Gly-Thr(C)-Phe-Thr(C)-Ser(C)-Asp(C)-X-Lys(E)-Gln(A)-Met-Glu(C)-Glu(C)-Glu(C)-Ala-Val-Arg(D)-Leu-Phe-Ile-Glu(C)-Trp(F)-Leu-Lys(E)-Asn(A)-Gly-Gly-Pro-Ser(C)-Ser(C)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 wherein:
X is Leu-Ser(tBu) or Leu-tPSer;
Date Recue/Date Received 2023-01-12 each A is independently H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group elected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
F is H or Boc; and Resin is an acid labile resin suitable for solid-phase peptide synthesis.
X is Leu-Ser(tBu) or Leu-tPSer;
Date Recue/Date Received 2023-01-12 each A is independently H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group elected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
F is H or Boc; and Resin is an acid labile resin suitable for solid-phase peptide synthesis.
26. The peptide of Claim 25, wherein the peptide is exenatide.
27. A partially protected peptide Y-Glu(C)-Glu(C)-Ala-Val-Arg(D)-Leu-Phe-Ile-Glu(C)-Trp(F)-Leu-Lys(E)-Asn(A)-Gly-Gly-Pro-Ser(C)-Ser(C)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 that is the exenatide fragments 12-39, 13-39, 14-39, wherein:
Y is H, E-Glu(C), E-Met-Glu(C), E-Gln(A)-Met-Glu(C) or E-Lys(E')-Gln(A)-Met-Glu(C); wherein:
each A is independently H or a carboxamido protecting group selected from Trt, Mtt and Mmt;
E and E' are each independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
F is H or Boc;
each C is independently a hydroxy or carboxyl protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Y is H, E-Glu(C), E-Met-Glu(C), E-Gln(A)-Met-Glu(C) or E-Lys(E')-Gln(A)-Met-Glu(C); wherein:
each A is independently H or a carboxamido protecting group selected from Trt, Mtt and Mmt;
E and E' are each independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
F is H or Boc;
each C is independently a hydroxy or carboxyl protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
28. A partially protected peptides that is E-His(A)-Gly-Glu(C)-Gly-Thr(C)-Phe-Thr(C)-Ser(C)-Asp(C)-X-Y in particular sequences corresponding to the exenatide fragments 1-11, 1-13, 1-14 and 1-15, wherein:
X is Leu-Ser(tBu) or Leu-tPSer;
E is an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt and Clt;
A is H, a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
Date Recue/Date Received 2023-01-12 each C is independently a hydroxy or carboxyl protecting group selected from tBu, Trt or Clt; and Y is OZ, Lys(E)-G1n(A)-0Z, Lys(E)-Gln(A)-Met-OZ, Lys(E)-G1n(A)-Met-G1u(C)-0Z, wherein each Z is independently H or a group which activate electrophilically carboxyl groups selected from Bt, Su, Pfp, Tcp or Pnp.
X is Leu-Ser(tBu) or Leu-tPSer;
E is an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt and Clt;
A is H, a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
Date Recue/Date Received 2023-01-12 each C is independently a hydroxy or carboxyl protecting group selected from tBu, Trt or Clt; and Y is OZ, Lys(E)-G1n(A)-0Z, Lys(E)-Gln(A)-Met-OZ, Lys(E)-G1n(A)-Met-G1u(C)-0Z, wherein each Z is independently H or a group which activate electrophilically carboxyl groups selected from Bt, Su, Pfp, Tcp or Pnp.
29. A process for the production of exenatide comprising:
condensing of one fragment of the Claim 27 with one fragment of Claim 28 to form the partially or resin-bound protected exenatide sequence;
deprotecting or cleaving from the resin; and deprotecting, chromatographic purification and lyophilization of exenatide.
condensing of one fragment of the Claim 27 with one fragment of Claim 28 to form the partially or resin-bound protected exenatide sequence;
deprotecting or cleaving from the resin; and deprotecting, chromatographic purification and lyophilization of exenatide.
30. A partially protected peptide of the formula E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-Gln(A)-Arg(D)-Leu-Ala-Asn(A)-Phe-Leu-Val-His(A)-X-Asn(A)-Asn(A)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(C)-Asn(A)-Val-Gly-Ser(C)-Asn(A)-Thr(C)-Tyr(Resin)-wherein X = Ser(tBu)-Ser(tBu) or Ser(tBu)-'PSer, wherein:
Y is Ala-Thr(tBu) or Ala-tPThr;
each A is independently H, a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or StBu;
each C is independently hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Y is Ala-Thr(tBu) or Ala-tPThr;
each A is independently H, a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or StBu;
each C is independently hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
31. The peptide of Claim 30 that is protected or partially protected pramlintide.
32. A protected or partially protected peptide with the sequence Z-Asn(A)-Phe-Leu-Val-His(A)-X-Asn(A)-Asn(A)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(C)-Asn(A)-Val-Gly-Ser(C)-Asn(A)-Thr(C)-Tyr(Resin)-NH2 in particular the protected or partially protected 10-38, 11-38, 12-38 and 14-38 partially protected pramlintide fragments wherein:
Z is H or E-Gln(A)-Arg(D)-Leu-Ala, E-Arg(D)-Leu-Ala, E-Leu-Ala or E-Ala;
Date Recue/Date Received 2023-01-12 X is Ser(tBu)-Ser(tBu) or Ser(tBu)-'PSer;
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
C is a hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt and Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Z is H or E-Gln(A)-Arg(D)-Leu-Ala, E-Arg(D)-Leu-Ala, E-Leu-Ala or E-Ala;
Date Recue/Date Received 2023-01-12 X is Ser(tBu)-Ser(tBu) or Ser(tBu)-'PSer;
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
C is a hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt and Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
33. A process for the preparation of a partially protected and oxidized pramlintide fragments, comprising:
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-Arg(D)-Leu-Ala-OZ
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-Arg(D)-0Z
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-0Z
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-OZ
assembling protected peptides on an acid sensitive resin, such as a 2-CTC-resin;
cleaving the protected peptide from the resin; and oxidizing the resin-bound peptides by contacting the resin-bound peptides with mild acid, such as dilute trifluoroacetic acid in an organic solvent, such as dichloromethane, wherein the solvent comprises a 2-200 molar excess of iodine over the fragment.
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-Arg(D)-Leu-Ala-OZ
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-Arg(D)-0Z
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-0Z
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-OZ
assembling protected peptides on an acid sensitive resin, such as a 2-CTC-resin;
cleaving the protected peptide from the resin; and oxidizing the resin-bound peptides by contacting the resin-bound peptides with mild acid, such as dilute trifluoroacetic acid in an organic solvent, such as dichloromethane, wherein the solvent comprises a 2-200 molar excess of iodine over the fragment.
34. A process for the production of pramlintide, where a fragment of Claim 33 is condensed in solution or on solid phase with one of the fragments of Claim 32, Claim 33 or one of the fragments of the formula E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-Gln(A)-Arg(D)-Leu-Ala-OZ, E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-G1n(A)-Arg(D)-0Z, E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-G1n(A)-0Z, E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-OZ, wherein:
each Y is independently Ala-Thr(tBu) or Ala-tPThr;
Date Recue/Date Received 2023-01-12 Z is H or a group selected from Bt, Su, Pfp, Tcp or Pnp;
each A is independently H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
each D is independently H or a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or StBu and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
each Y is independently Ala-Thr(tBu) or Ala-tPThr;
Date Recue/Date Received 2023-01-12 Z is H or a group selected from Bt, Su, Pfp, Tcp or Pnp;
each A is independently H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
each D is independently H or a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or StBu and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
35. A protected or partially protected peptide of the formula E-D-Phe-Pro-Arg(D)-Pro-Gly-Gly-Gly-Gly-Asn(A)-Gly-Asp(C)-Phe-Glu(C)-Glu(C)-Ile-Pro-Glu(C)-Glu(C)-Tyr(Resin)-Leu-O-C, wherein:
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each C is independently a hydroxyl or carboxyl protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each C is independently a hydroxyl or carboxyl protecting group selected from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
36. The protected peptide of Claim 36, wherein the protected peptide is bivalirudin.
37. A protected or partially protected fragment of the formula E-X-Tyr(Resin)-Leu-O-C
wherein:
X is a bivalirudin peptide sequence;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
C is a hydroxyl carboxyl protecting group selected from tBu, Trt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
wherein:
X is a bivalirudin peptide sequence;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
C is a hydroxyl carboxyl protecting group selected from tBu, Trt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
38. A process for the preparation of bivalirudin comprising:
Date Recue/Date Received 2023-01-12 condensing a bivalirudin fragment of the formula E-X-Tyr(Resin)-Leu-O-C of Claim 37 in solution or on solid phase with a bivalirudin fragment of the formula E-D-Phe-Y-OZ to give E-D-Phe-Y-X-Tyr(Resin)-Leu-O-C;
deprotection or cleavage from the resin and deprotection provides bivalirudin;
purification of bivalirudin by chromatography;
lyophilizing to provide bivalirudin in >99% purity; wherein;
X and Y are independently bivalirudin peptide sequences;
D- designate the chirality of the amino acid that follows as being a D-amino acid;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps;
and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Date Recue/Date Received 2023-01-12 condensing a bivalirudin fragment of the formula E-X-Tyr(Resin)-Leu-O-C of Claim 37 in solution or on solid phase with a bivalirudin fragment of the formula E-D-Phe-Y-OZ to give E-D-Phe-Y-X-Tyr(Resin)-Leu-O-C;
deprotection or cleavage from the resin and deprotection provides bivalirudin;
purification of bivalirudin by chromatography;
lyophilizing to provide bivalirudin in >99% purity; wherein;
X and Y are independently bivalirudin peptide sequences;
D- designate the chirality of the amino acid that follows as being a D-amino acid;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps;
and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
39. A protected or partially protected peptide with the formula E-Ser(C)-Tyr(C)-Ser(C)-Met-Glu(C)-His(A)-Phe-Arg(D)-Trp(F)-Gly-Lys(E)-Pro- Val-Gly-Lys(E)-Lys(E)-Arg(D)-Arg(D)-Pro-Val-Lys(E)-Val-Tyr(Resin)-Pro-O-C, wherein:
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
each D is independently H or a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
F is H or Boc and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt;
each D is independently H or a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
F is H or Boc and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
40. The protected or partially protected peptide of Claim 39 that is a ACTH(1-24) peptide.
41. A protected or partially protected fragment with the formula E-X-Tyr(Resin)-Pro-O-C
wherein:
X is a ACTH(1-24) sequence;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
C is a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Date Recue/Date Received 2023-01-12
wherein:
X is a ACTH(1-24) sequence;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt or Clt;
C is a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or Clt; and Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Date Recue/Date Received 2023-01-12
42. A process for the production of ACTH(1-24) comprising:
condensing a ACTH fragment of the formula E-X-Tyr(Resin)-Pro-O-C of Claim 37 in solution or in solid phase with a ACTH fragment of the formula E-Y-OZ to give E-Y-X-Tyr(Resin)-Pro-O-C;
deprotecting or cleaving from the resin and deprotection provides ACTH(1-24) which is purified by chromatography and lyophilized to provide ACTH(1-24) of >9 9%
purity, wherein;
X and Y are each independently ACTH sequences;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps;
and Resin represents H or an acid labile resin suitable for solid-phase peptide synthesis.
Date Recue/Date Received 2023-01-12
condensing a ACTH fragment of the formula E-X-Tyr(Resin)-Pro-O-C of Claim 37 in solution or in solid phase with a ACTH fragment of the formula E-Y-OZ to give E-Y-X-Tyr(Resin)-Pro-O-C;
deprotecting or cleaving from the resin and deprotection provides ACTH(1-24) which is purified by chromatography and lyophilized to provide ACTH(1-24) of >9 9%
purity, wherein;
X and Y are each independently ACTH sequences;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps;
and Resin represents H or an acid labile resin suitable for solid-phase peptide synthesis.
Date Recue/Date Received 2023-01-12
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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CA3186364A CA3186364A1 (en) | 2013-02-21 | 2013-02-21 | Solid phase peptide synthesis via side chain attachment |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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CA3186364A CA3186364A1 (en) | 2013-02-21 | 2013-02-21 | Solid phase peptide synthesis via side chain attachment |
CA2807162A CA2807162C (en) | 2013-02-21 | 2013-02-21 | Solid phase peptide synthesis via side chain attachment |
Related Parent Applications (1)
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CA2807162A Division CA2807162C (en) | 2013-02-21 | 2013-02-21 | Solid phase peptide synthesis via side chain attachment |
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