CN112194708A - Preparation method of procatide - Google Patents
Preparation method of procatide Download PDFInfo
- Publication number
- CN112194708A CN112194708A CN202011168301.7A CN202011168301A CN112194708A CN 112194708 A CN112194708 A CN 112194708A CN 202011168301 A CN202011168301 A CN 202011168301A CN 112194708 A CN112194708 A CN 112194708A
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- fmoc
- resin
- peptide
- solution
- 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
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 239000011347 resin Substances 0.000 claims abstract description 122
- 229920005989 resin Polymers 0.000 claims abstract description 121
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 114
- 238000000746 purification Methods 0.000 claims abstract description 56
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 44
- 230000003647 oxidation Effects 0.000 claims abstract description 43
- 241001208007 Procas Species 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 29
- 238000004440 column chromatography Methods 0.000 claims abstract description 19
- 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 claims abstract description 13
- 230000008569 process Effects 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 109
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 81
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 66
- 239000007864 aqueous solution Substances 0.000 claims description 51
- 239000007788 liquid Substances 0.000 claims description 44
- 239000003153 chemical reaction reagent Substances 0.000 claims description 41
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- 238000001514 detection method Methods 0.000 claims description 34
- 239000007800 oxidant agent Substances 0.000 claims description 33
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 31
- 238000010828 elution Methods 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 26
- 238000002156 mixing Methods 0.000 claims description 26
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 230000001590 oxidative effect Effects 0.000 claims description 22
- 239000002904 solvent Substances 0.000 claims description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000003638 chemical reducing agent Substances 0.000 claims description 19
- 238000005859 coupling reaction Methods 0.000 claims description 19
- 238000009833 condensation Methods 0.000 claims description 18
- 230000005494 condensation Effects 0.000 claims description 18
- 238000005336 cracking Methods 0.000 claims description 18
- KJYAFJQCGPUXJY-UMSFTDKQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-(tritylamino)butanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KJYAFJQCGPUXJY-UMSFTDKQSA-N 0.000 claims description 17
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 claims description 17
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 claims description 17
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 claims description 16
- CSMYOORPUGPKAP-IBGZPJMESA-N (2r)-3-(acetamidomethylsulfanyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CSCNC(=O)C)C(O)=O)C3=CC=CC=C3C2=C1 CSMYOORPUGPKAP-IBGZPJMESA-N 0.000 claims description 16
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 claims description 16
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 16
- 125000006239 protecting group Chemical group 0.000 claims description 16
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 15
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 12
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 11
- 239000005695 Ammonium acetate Substances 0.000 claims description 11
- 235000019257 ammonium acetate Nutrition 0.000 claims description 11
- 229940043376 ammonium acetate Drugs 0.000 claims description 11
- 238000004237 preparative chromatography Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 238000006722 reduction reaction Methods 0.000 claims description 9
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 claims description 8
- 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 claims description 8
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 8
- 238000006482 condensation reaction Methods 0.000 claims description 8
- 238000010511 deprotection reaction Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 230000002378 acidificating effect Effects 0.000 claims description 7
- 230000009089 cytolysis Effects 0.000 claims description 7
- 150000002730 mercury Chemical class 0.000 claims description 7
- GGCZERPQGJTIQP-UHFFFAOYSA-N sodium;9,10-dioxoanthracene-2-sulfonic acid Chemical compound [Na+].C1=CC=C2C(=O)C3=CC(S(=O)(=O)O)=CC=C3C(=O)C2=C1 GGCZERPQGJTIQP-UHFFFAOYSA-N 0.000 claims description 7
- 150000003608 titanium Chemical class 0.000 claims description 7
- 239000002262 Schiff base Substances 0.000 claims description 6
- 150000004753 Schiff bases Chemical class 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000012298 atmosphere Substances 0.000 claims description 6
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims description 6
- 238000012856 packing Methods 0.000 claims description 6
- 230000001681 protective effect Effects 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 239000012279 sodium borohydride Substances 0.000 claims description 5
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 5
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 238000011097 chromatography purification Methods 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 239000006166 lysate Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims 1
- 239000012266 salt solution Substances 0.000 claims 1
- 150000001413 amino acids Chemical class 0.000 abstract description 11
- 239000012535 impurity Substances 0.000 abstract description 8
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 238000003786 synthesis reaction Methods 0.000 abstract description 6
- 229920001184 polypeptide Polymers 0.000 abstract description 4
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000006340 racemization Effects 0.000 abstract description 2
- 230000006334 disulfide bridging Effects 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 114
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 29
- 238000005406 washing Methods 0.000 description 28
- 108090000565 Capsid Proteins Proteins 0.000 description 18
- 239000003960 organic solvent Substances 0.000 description 17
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- 230000008961 swelling Effects 0.000 description 15
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 14
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 238000001035 drying Methods 0.000 description 12
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 10
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 9
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 9
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 8
- DIZZIOFQEYSTPV-UHFFFAOYSA-N [I].CO Chemical compound [I].CO DIZZIOFQEYSTPV-UHFFFAOYSA-N 0.000 description 8
- 229910052740 iodine Inorganic materials 0.000 description 8
- 239000011630 iodine Substances 0.000 description 8
- 238000000926 separation method Methods 0.000 description 8
- 239000012043 crude product Substances 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 6
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 6
- 229930003268 Vitamin C Natural products 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000000967 suction filtration Methods 0.000 description 6
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 6
- 235000019154 vitamin C Nutrition 0.000 description 6
- 239000011718 vitamin C Substances 0.000 description 6
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 5
- 150000001408 amides Chemical class 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 4
- 239000001099 ammonium carbonate Substances 0.000 description 4
- 238000006664 bond formation reaction Methods 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005882 aldol condensation reaction Methods 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 239000007975 buffered saline Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- -1 alcohol amine Chemical class 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 2
- 238000005562 fading Methods 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 2
- 229960004919 procaine Drugs 0.000 description 2
- SJMPVWVIVWEWJK-AXEIBBKLSA-N uroguanylin Chemical class SC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(N)=O SJMPVWVIVWEWJK-AXEIBBKLSA-N 0.000 description 2
- NSPHQWLKCGGCQR-DLJDZFDSSA-N (2s)-2-[[(1r,4s,7s,10s,13s,16r,21r,27s,34r,37s,40s)-10-(2-amino-2-oxoethyl)-34-[[(2s)-4-carboxy-2-[[(2s)-3-carboxy-2-[[(2s)-2,4-diamino-4-oxobutanoyl]amino]propanoyl]amino]butanoyl]amino]-37-(2-carboxyethyl)-27-[(1r)-1-hydroxyethyl]-4-methyl-40-(2-methylp Chemical compound N1C(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(N)=O)CSSC[C@@H]2NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CSSC[C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)CNC(=O)[C@H]([C@@H](C)O)NC2=O NSPHQWLKCGGCQR-DLJDZFDSSA-N 0.000 description 1
- RSWGJHLUYNHPMX-UHFFFAOYSA-N 1,4a-dimethyl-7-propan-2-yl-2,3,4,4b,5,6,10,10a-octahydrophenanthrene-1-carboxylic acid Chemical compound C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-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
- 108010078321 Guanylate Cyclase Proteins 0.000 description 1
- 102000014469 Guanylate cyclase Human genes 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 102400000230 Uroguanylin Human genes 0.000 description 1
- 101800000255 Uroguanylin Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000008855 peristalsis Effects 0.000 description 1
- 108010018859 plecanatide Proteins 0.000 description 1
- 229950008515 plecanatide Drugs 0.000 description 1
- 238000006068 polycondensation reaction Methods 0.000 description 1
- 230000012495 positive regulation of renal sodium excretion Effects 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229960000244 procainamide Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
The invention provides a preparation method of procatide, and relates to the technical field of polypeptide synthesis. The AM resin or the MBHA resin is modified through FMPB, H-Leu-OtBu is used as a first amino acid to be coupled to the modified resin, amino acids at positions which are difficult to be connected are modified and then are coupled to the modified resin step by step according to a sequence of a proca peptide main chain peptide, so that the synthetic difficulty of the proca linear peptide is greatly reduced, and the problems of more impurities, low purity, low yield, racemization of amino acids and the like in the conventional synthesis are solved; performing first oxidation bonding on Cys at the 4 th site and Cys at the 12 th site in the sequence, and performing column chromatography coarse purification, thereby improving the purity of the obtained procapsipeptide; the purification solution is diluted and then subjected to secondary oxidation bonding of Cys at the 7 th site and Cys at the 15 th site, so that the concentration of the pre-oxidized peptide of the proca peptide can be reduced, intermolecular disulfide bonding in the process of secondary oxidation bonding is reduced, the yield of the proca peptide is improved, and the method is simple and convenient to operate and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of procainatide.
Background
Plecanatide was developed by Synergy pharmaceutical corporation of the united states,is an analogue of uroguanylin (uroguanylin) and contains a cyclic polypeptide with 16 amino acids, and the sequence of the cyclic polypeptide is as follows: asn (n)1-Asp2-Glu3-Cys4-Glu5-Leu6-Cys7-Val8-Asn9-Val10-Ala11-Cys12-Thr13-Gly14-Cys15-Leu16(two disulfide bond: Cys)4&Cys12And Cys7&Cys15) The medicine has the function of guanylate cyclase receptor stimulant for promoting natriuresis, can regulate acid and alkali ions in gastrointestinal tracts, induces liquid to be transported into the gastrointestinal tracts, increases the peristalsis of the gastrointestinal tracts and accelerates defecation.
Chinese patent CN107383170A discloses a preparation method of procatide: (1) coupling a resin solid phase carrier and Fmoc-Leu-OH in the presence of an activator system to obtain Fmoc-Leu-resin; (2) sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the peptide sequence of the main chain of the procatide by a solid-phase synthesis method; (3) cracking and liquid phase cyclization; (4) purifying, freezing and drying to obtain the procatide. However, the sequence of the proca peptide is easy to form resin polycondensation in the coupling process, and amino acid on the main chain is not modified, so that the coupling efficiency of the proca peptide is influenced, and the linear peptide is high in synthesis difficulty, low in purity and low in yield.
Disclosure of Invention
In view of this, the present invention aims to provide a preparation method of procainatide, which has the advantages of low synthesis difficulty, high yield and high purity of procainatide.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of procatide, which comprises the following steps:
(1) mixing the initial resin, FMPB and a condensation reagent, and modifying to obtain modified resin; the starting resin comprises an MBHA resin or an AM resin;
(2) under the protective atmosphere, mixing the modified resin, H-Leu-OtBu and furan solvents, carrying out an amino-aldehyde condensation reaction, mixing the obtained Schiff base with a reducing agent, and carrying out a reduction reaction to obtain H-Leu-OtBu-FMPB MBHA/AM resin;
(3) according to the sequence of the backbone peptide of the procapsipeptide, Fmoc-Cys (Acm) -OH and R are connected to the H-Leu-OtBu-FMPB MBHA/AM resin in sequence1-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Acm)-OH、R2-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn (Trt) -OH to obtain a fully protected procapside peptide resin; the R is1And R2Independently comprise Fmoc and/or Fmoc-Hmb, and the R1And R2Not being Fmoc at the same time; the connection comprises a coupling reaction and a Fomc protecting group removing reaction which are sequentially carried out;
(4) cracking the fully-protected procapsipeptide resin in a cracking solution to obtain procapsipeptide linear peptide;
(5) dissolving the procainatide linear peptide into a reagent, adjusting the pH value of the obtained procainatide linear peptide solution to 7-10, mixing the solution with a first oxidizing agent, and carrying out first oxidation bonding on disulfide bonds in Cys at positions 4 and 12 in the procainatide linear peptide sequence to obtain pre-oxidized procainatide linear peptide;
the first oxidizing agent comprises air, hydrogen peroxide or dimethyl sulfoxide;
(6) carrying out column chromatography coarse purification on the pre-oxidized proca peptide, diluting the obtained purified liquid, mixing the diluted purified liquid with a second oxidant, carrying out second oxidation bonding on disulfide bonds in Cys at the 7 th site and the 15 th site in the pre-oxidized proca peptide sequence, and carrying out reversed-phase preparative chromatography purification on the obtained second oxidation product to obtain proca peptide;
the mobile phase adopted by the column chromatography crude purification comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an acetic acid aqueous solution, a trifluoroacetic acid aqueous solution, a triethylamine phosphate aqueous solution or an ammonium acetate aqueous solution, and the mobile phase B comprises acetonitrile, methanol or ethanol;
the second oxidant comprises iodine, a divalent mercury salt, a silver salt, or a trivalent titanium salt.
Preferably, in the step (1), the substitution degree of the initial resin is 0.3-1.6 mmol/g;
the condensation reagent comprises HOBT-DIC, HBTU-NMM, HATU-NMM or PyBOP-HOBT-NMM;
the molar ratio of the starting resin, FMPB and condensing agent is 1: (1.5-4): (1.5-4).
Preferably, in the step (2), the molar ratio of the modified resin to H-Leu-OtBu is 1: (1-6);
the ammonia-aldehyde condensation reaction is carried out under acidic or alkaline conditions, the pH value of the acidic conditions is 2-6, and the pH value of the alkaline conditions is 7.5-11.
Preferably, in the step (2), the reducing agent includes one or more of sodium borohydride, sodium cyanoborohydride and sodium acetate borohydride;
the mol ratio of the modified resin to the reducing agent is 1: (2-6).
Preferably, in the step (3), the condensation reagent adopted in the coupling reaction comprises HOBT-DIC, HBTU-NMM, HATU-NMM or PyBOP-HOBT-NMM;
the deprotection reagent adopted in the reaction for removing the Fomc protecting group is piperidine, 1, 8-diazabicycloundecen-7-ene or triethylamine.
Preferably, in step (4), the composition of the lysis solution comprises TFA, EDT, TIS and H2O, said TFA, EDT, TIS and H2The volume ratio of O is (85-100): (0-5): (0-5): (0-5);
the ratio of the mass of the fully-protected procatide resin to the volume of the lysis solution is 1 g: (3-15) mL.
Preferably, the cracking temperature is 15-35 ℃, and the time is 2-6 h.
Preferably, in step (5), the reagent comprises water, an aqueous acetonitrile solution or an aqueous buffered saline solution;
the concentration of the procatide linear peptide solution is 0.2-10 mg/mL;
preferably, in the step (6), the molar ratio of the second oxidant to the procapsipeptide is (0.6-3): 1.
Preferably, in the step (6), the dilution ratio is 2-20.
Preferably, in the step (6), the reverse phase preparative chromatography purification comprises a first purification and a second purification which are sequentially performed;
the conditions of the first purification include: the chromatographic column packing is C18Alkyl bonded silica gel; the column temperature was 35 ℃; the mobile phase A is 0.5-2 v/v% triethylamine phosphate aqueous solution, 2-32 g/L ammonium acetate aqueous solution or 10-50 mol/L dihydric phosphate aqueous solution; the mobile phase B is acetonitrile; the elution mode is gradient elution, the gradient elution procedure is 0-6 min, the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B is 20%; 6-66 min, wherein the volume fraction of the mobile phase A is 60%, and the volume fraction of the mobile phase B is 40%; the detection wavelength is 214 nm;
the conditions of the second purification include: the chromatographic column packing is C18Alkyl bonded silica gel; the column temperature was 35 ℃; the mobile phase A is 0.05-5 v/v% acetic acid aqueous solution; the mobile phase B is acetonitrile; the elution mode is gradient elution, the gradient elution procedure is 0-6 min, the volume fraction of the mobile phase A is 75%, and the volume fraction of the mobile phase B is 25%; 6-66 min, wherein the volume fraction of the mobile phase A is 55%, and the volume fraction of the mobile phase B is 45%; the detection wavelength was 214 nm.
The invention provides a preparation method of procatide, which comprises the following steps: (1) mixing the initial resin, FMPB and a condensation reagent, and modifying to obtain modified resin; the starting resin comprises an MBHA resin or an AM resin; (2) under the protective atmosphere, mixing the modified resin, H-Leu-OtBu and furan solvents, carrying out an amino-aldehyde condensation reaction, mixing the obtained Schiff base with a reducing agent, and carrying out a reduction reaction to obtain H-Leu-OtBu-FMPB MBHA/AM resin; (3) according to the sequence of the backbone peptide of the procapsipeptide, Fmoc-Cys (Acm) -OH and R are connected to the H-Leu-OtBu-FMPB MBHA/AM resin in sequence1-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Acm)-OH、R2-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn (Trt) -OH to obtain a fully protected procapside peptide resin; the R is1And R2Independently comprise Fmoc and/or Fmoc-Hmb, and the R1And R2Not being Fmoc at the same time; the connection comprises a coupling reaction and a Fomc protecting group removing reaction which are sequentially carried out; (4) cracking the fully-protected procapsipeptide resin in a cracking solution to obtain procapsipeptide linear peptide; (5) dissolving the procainatide linear peptide into a reagent, adjusting the pH value of the obtained procainatide linear peptide solution to 7-10, mixing the solution with a first oxidizing agent, and carrying out first oxidation bonding on disulfide bonds in Cys at positions 4 and 12 in the procainatide linear peptide sequence to obtain pre-oxidized procainatide linear peptide; the first oxidizing agent comprises air, hydrogen peroxide or dimethyl sulfoxide; (6) carrying out column chromatography coarse purification on the pre-oxidized proca peptide, diluting the obtained purified liquid, mixing the diluted purified liquid with a second oxidant, carrying out second oxidation bonding on disulfide bonds in Cys at the 7 th site and the 15 th site in the pre-oxidized proca peptide sequence, and carrying out reversed-phase preparative chromatography purification on the obtained second oxidation product to obtain proca peptide; the mobile phase adopted by the column chromatography crude purification comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an acetic acid aqueous solution, a trifluoroacetic acid aqueous solution, a triethylamine phosphate aqueous solution or an ammonium acetate aqueous solution, and the mobile phase B comprises acetonitrile, methanol or ethanol; the second oxidant comprises iodine, a divalent mercury salt, a silver salt, or a trivalent titanium salt. According to the preparation method provided by the invention, AM resin or MBHA resin is modified through FMPB, the main chain of amino acid at a position difficult to be connected is modified, beta folding in a space structure is broken through steric hindrance, the difficulty in synthesizing the proca linear peptide is greatly reduced, and the problems of more impurities, low purity, low yield and racemization of amino acid in the conventional synthesis are solved.
Performing first oxidation bonding on disulfide bonds in Cys at positions 4 and 12 in the sequence, and performing column chromatography coarse purification, so that the purity of the finally obtained procapsipeptide is improved; the purified solution is diluted and then subjected to subsequent second oxidation bonding, so that the concentration of the pre-oxidized peptide of the procainatide can be reduced, intermolecular disulfide bond formation in the second oxidation bonding process is reduced, the yield of the procainatide is improved, the operation is simple and convenient, and the method is suitable for industrial production.
Detailed Description
The invention provides a preparation method of procatide, which comprises the following steps:
(1) mixing the initial resin, FMPB and a condensation reagent, and modifying to obtain modified resin; the starting resin comprises an MBHA resin or an AM resin;
(2) under the protective atmosphere, mixing the modified resin, H-Leu-OtBu and furan solvents, carrying out an amino-aldehyde condensation reaction, mixing the obtained Schiff base with a reducing agent, and carrying out a reduction reaction to obtain H-Leu-OtBu-FMPB MBHA/AM resin;
(3) according to the sequence of the backbone peptide of the procapsipeptide, Fmoc-Cys (Acm) -OH and R are connected to the H-Leu-OtBu-FMPB MBHA/AM resin in sequence1-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Acm)-OH、R2-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn (Trt) -OH to obtain a fully protected procapside peptide resin; the R is1And R2Independently comprise Fmoc and/or Fmoc-Hmb, and the R1And R2Not being Fmoc at the same time; the connection comprises a coupling reaction and a Fomc protecting group removing reaction which are sequentially carried out;
(4) cracking the fully-protected procapsipeptide resin in a cracking solution to obtain procapsipeptide linear peptide;
(5) dissolving the procainatide linear peptide into a reagent, adjusting the pH value of the obtained procainatide linear peptide solution to 7-10, mixing the solution with a first oxidizing agent, and carrying out first oxidation bonding on disulfide bonds in Cys at positions 4 and 12 in the procainatide linear peptide sequence to obtain pre-oxidized procainatide linear peptide;
the first oxidizing agent comprises air, hydrogen peroxide or dimethyl sulfoxide;
(6) carrying out column chromatography coarse purification on the pre-oxidized proca peptide, diluting the obtained purified liquid, mixing the diluted purified liquid with a second oxidant, carrying out second oxidation bonding on disulfide bonds in Cys at the 7 th site and the 15 th site in the pre-oxidized proca peptide sequence, and carrying out reversed-phase preparative chromatography purification on the obtained second oxidation product to obtain proca peptide;
the mobile phase adopted by the column chromatography crude purification comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an acetic acid aqueous solution, a trifluoroacetic acid aqueous solution, a triethylamine phosphate aqueous solution or an ammonium acetate aqueous solution, and the mobile phase B comprises acetonitrile, methanol or ethanol;
the second oxidant comprises iodine, a divalent mercury salt, a silver salt, or a trivalent titanium salt.
In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.
In the present invention, abbreviations represent materials as shown in table 1:
TABLE 1 abbreviations corresponding names of substances
The method comprises the steps of mixing initial resin, FMPB and a condensation reagent, and modifying to obtain modified resin; the starting resin comprises an MBHA resin or an AM resin.
In the present invention, the structural formula of the MBHA resin is shown in formula I:
wherein,
is a resin carrier;
the structural formula of the AM resin is shown as a formula II:
wherein,
is a resin carrier.
In the invention, the structural formula of FMPB is shown as formula III
In the present invention, the degree of substitution of the starting resin is preferably 0.3 to 1.6mmol/g, more preferably 0.5 to 1.5mmol/g, and most preferably 0.8 to 1 mmol/g. In the present invention, the starting resin preferably further comprises, before use, a pretreatment, the pretreatment preferably comprising: mixing the starting resin and a swelling agent for swelling; then removing the swelling agent, washing with an organic solvent and removing the organic solvent. In the invention, the swelling agent is preferably a solution of a basic reagent, and the basic reagent preferably comprises one or more of preferably N, N' -diisopropylethylamine, N-methylmorpholine and triethylamine; the volume percentage concentration of the alkaline reagent solution is preferably 2-5%, and more preferably 3-4%; the solvent in the alkaline reagent solution preferably comprises dichloromethane, tetrahydrofuran or pyrrolidone. In the present invention, the temperature of the swelling is preferably room temperature; the time is preferably 30 to 120min, and more preferably 50 to 100 min. In the present invention, the swelling is to neutralize the excess acid in the initial resin, and the swelling can expand the resin, so that the reaction sites are spread, which is beneficial to FMPB modification of the initial resin and amino acid coupling. In the present invention, the manner of removing the swelling agent is preferably suction filtration. In the present invention, the organic solvent is preferably N, N-dimethylformamide or N, N-dimethylacetamide; the number of times of washing with the organic solvent is preferably 2-3 times; the purpose of the organic solvent washing is to remove impurities as well as unreacted starting materials. In the present invention, the organic solvent is preferably removed by suction filtration.
In the present invention, the condensation reagent preferably comprises HOBT-DIC, HBTU-NMM, HATU-NMM or PyBOP-HOBT-NMM; the preferable molar ratio of HOBT to DIC in the HOBT-DIC is (1.5-6): (1.5-6), more preferably (2-5): (2-5), most preferably (3-4): (3-4); the molar ratio of HBTU to NMM in the HBTU-NMM is preferably (1.425-5.7): (3-12), more preferably (2-5): (5-10), most preferably (3-4): (6-8); the molar ratio of HATU to NMM in the HATU-NMM is preferably (1.425-5.7): (3-12), more preferably (2-5): (5-10), most preferably (3-4): (6-8); the mole ratio of PyBOP, HOBT and NMM in the PyBOP-HOBT-NMM is preferably (1.425-5.7): (1.5-6): (3-12), more preferably (2-5): (2-5): (5-10), most preferably (3-4): (6-8).
In the present invention, the molar ratio of the starting resin and the condensing agent is preferably 1: (1.5-6), more preferably 1: (2-3).
In the present invention, the molar ratio of the starting resin and FMPB is preferably 1: (1.5-4), more preferably 1: (2 to 3.5), most preferably 1: (2.5-3). In the present invention, the FMPB is preferably used in the form of an FMPB solution, and the solvent in the FMPB solution is preferably N, N-dimethylformamide or N, N-dimethylacetamide.
In the invention, the modification temperature is preferably 15-40 ℃, and more preferably 25-35 ℃; the modification time is not particularly limited, and the negative detection result can be detected by ninhydrin detection solution. In the invention, in the modification reaction process, under the action of a condensation reagent, FMPB and amino on the initial resin are subjected to amido bond condensation and are connected to AM or MBHA resin.
The operation of the ninhydrin detection solution is not particularly limited, and the ninhydrin detection solution known to those skilled in the art can be used.
After the modification, the invention preferably further comprises washing the modified system with an organic solvent and then removing the organic solvent to obtain the modified resin. In the present invention, the organic solvent washing includes DMF washing and THF washing, which are sequentially performed; the number of times of DMF washing is preferably 3-5 times; the THF washing frequency is preferably 2-3 times; the purpose of the DMF wash and THF wash was to remove unreacted FMPB to avoid FMPB affecting subsequent reactions. In the present invention, the organic solvent is preferably removed by suction filtration.
After obtaining the modified resin, mixing the modified resin, H-Leu-OtBu and a furan solvent under a protective atmosphere, carrying out an amino-aldehyde condensation reaction, mixing the obtained Schiff base with a reducing agent, and carrying out a reduction reaction to obtain H-Leu-OtBu-FMPB MBHA/AM resin; the structural formula of the H-Leu-OtBu-FMPB MBHA/AM resin is shown as the formula IV:
the protective atmosphere in the present invention is not particularly limited, and those known to those skilled in the art may be used, specifically, nitrogen or argon.
In the present invention, the molar ratio of the modified resin to H-Leu-OtBu is preferably 1: (1 to 6), more preferably 1: (1-3), most preferably 1: (1.5-2). In the present invention, the H-Leu-OtBu is preferably used in the form of H-Leu-OtBu. HCl.
In the present invention, the furan-based solvent preferably includes tetrahydrofuran. In the present invention, the volume ratio of the modified resin to the furan-based solvent is preferably 1: (3-6), more preferably 1: (4-5) mL.
In the present invention, the aldol condensation reaction is preferably carried out under acidic or basic conditions. In the invention, the pH value of the acidic condition is preferably 2-6, and more preferably 3-5; the reagent for providing the acidic condition is not particularly limited in the present invention, and an acid known to those skilled in the art, specifically acetic acid, may be used. In the invention, the pH value of the alkaline condition is preferably 7.5-11, and more preferably 8-10; the reagent for providing the alkaline condition is not particularly limited in the present invention, and a base known to those skilled in the art may be used, specifically, sodium hydroxide or potassium hydroxide.
In the present invention, the temperature of the aldol condensation reaction is preferably room temperature; the time is preferably 10-30 min, and more preferably 20 min; in the ammonia-aldehyde condensation reaction process, the aldehyde in the modified resin and the amino in the H-Leu-OtBu are subjected to ammonia-aldehyde condensation to generate Schiff base.
In the present invention, the reducing agent preferably includes one or more of sodium borohydride, sodium cyanoborohydride and sodium acetate borohydride, and more preferably includes sodium borohydride, sodium cyanoborohydride or sodium acetate borohydride. In the present invention, the reducing agent is preferably used in the form of a reducing agent solution, and the solvent in the reducing agent solution preferably includes methanol or ethanol; the concentration of the reducing agent solution is preferably 10-20 mol/L, and more preferably 13-15 mol/L. In the present invention, the molar ratio of the modified resin to the reducing agent is preferably 1: (2-6), more preferably 1: (3-5), and most preferably 1: 4. In the present invention, the temperature of the reduction reaction is preferably room temperature; the time is preferably 2 to 24 hours, more preferably 5 to 20 hours, and most preferably 10 to 15 hours.
After the reduction reaction, the invention preferably also comprises washing and drying the organic solvent of the reduction reaction system to obtain H-Leu-OtBu-FMPB MBHA/AM resin. In the invention, the organic solvent washing comprises amide solvent washing and alcohol solvent washing which are sequentially carried out; the amide-based solvent preferably includes N, N-dimethylformamide or N, N-dimethylacetamide; the washing times of the amide solvent are preferably 3-4 times; the alcohol solvent preferably comprises methanol or ethanol; the number of times of washing with the alcohol amine solvent is preferably 3-4. In the present invention, the drying is preferably performed by vacuum pumping.
After H-Leu-OtBu-FMPB MBHA/AM resin is obtained, according to the sequence of the procapside backbone peptide, Fmoc-Cys (Acm) -OH and R are sequentially connected to the H-Leu-OtBu-FMPB MBHA/AM resin1-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Acm)-OH、R2-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn (Trt) -OH to obtain a fully protected procapside peptide resin; the R is1And R2Independently comprise Fmoc and/or Fmoc-Hmb, and the R1And R2Not being Fmoc at the same time; the connection comprises a coupling reaction and a Fomc protecting group removing reaction which are sequentially carried out. In the invention, the structural formula of the fully-protected procapsipeptide resin is as follows:
asn (Trt) -Asp (OtBu) -Glu (OtBu) -Cys (Trt) -Glu (OtBu) -Leu-Cys (Acm) -Val-A sn (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) -Gly-Cys (Acm) -Leu-OtBu-FMPB AM resin.
In the present invention, the H-Leu-OtBu-FMPB MBHA/AM resin is preferably swollen before use. In the present invention, the organic solvent used for swelling preferably includes N, N-dimethylformamide, N-dimethylacetamide, or dichloromethane; the volume ratio of the H-Leu-OtBu-FMPB MBHA/AM resin to the swelling organic solvent is preferably 1: (1-3), more preferably 1: 2; the temperature of the swelling is preferably room temperature; the swelling time is preferably 30-120 min, and more preferably 50-100 min.
In the invention, the Fmoc-Cys (Acm) -OH and R1-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Acm)-OH、R2The molar ratio of-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn (Trt) -OH to H-Leu-OtBu-FMPB MBHA/AM resin is independently preferably (1.5 to 6): 1, more preferably (2-5): 1, most preferably (3-4): 1.
in the present invention, the condensation reagent is preferably the same as the optional species of the condensation reagent used in the preparation process of the modified resin, and is not described in detail herein.
In the invention, the molar ratio of the condensation reagent to H-Leu-OtBu-FMPB MBHA/AM resin is preferably (1.5-6): 1, more preferably (2-3): 1.
in the present invention, the deprotection reagent used in the Fomc protecting group removing reaction preferably comprises piperidine, 1, 8-diazabicycloundecen-7-ene or triethylamine, more preferably piperidine; the deprotection reagent is preferably used in the form of a deprotection reagent solution, and the volume concentration of the deprotection reagent solution is preferably 20-30%, and more preferably 20-25%. In the present invention, the solvent in the deprotection reagent solution is preferably an amide-based solvent, and the amide-based solvent preferably includes N, N-dimethylformamide or N, N-dimethylacetamide. In the present invention, the volume ratio of the H-Leu-OtBu-FMPB MBHA/AM resin to the deprotection reagent is preferably 1: (1-3), more preferably 1: 2.
In the present invention, the temperature for removing the protecting group is preferably room temperature; the time is preferably 5min to 1h, and more preferably 20min to 30 min. In the invention, the end point of the reaction is preferably determined by ninhydrin detection, and when the detection result is positive, the reaction for removing the Fomc protecting group is finished.
In the invention, the temperature of the coupling reaction is preferably 20-40 ℃, and more preferably room temperature; the time is preferably 0.5 to 4 hours, and more preferably 1 to 3 hours. In the present invention, the end point of the coupling reaction is preferably determined by ninhydrin detection, and when the detection result is negative, the second coupling reaction is completed. In the present invention, the reaction occurring during the coupling reaction is represented by the formula (1):
r in the formula (1)1Is absent or is Hmb, R2Is absent or is Hmb, R1And R2At least one is Hmb.
After the full-protection procapsipeptide resin is obtained, the full-protection procapsipeptide resin is cracked in a cracking solution to obtain the procapsipeptide wire peptide.
In the present invention, the structural formula of the procapsipeptide is:
Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH。
in the present invention, the composition of the lysis solution preferably comprises TFA, EDT, TIS and H2O, said TFA, EDT, TIS and H2The volume ratio of O is preferably (85-100): (0-5): (0-5): (0-5), more preferably (88-98): (1-4): (1-4): (1-4), more preferably (90-95): (2-3): (2-3): (2-3). In the present invention, the ratio of the mass of the fully protected proca peptide resin to the volume of the lysis solution is preferably 1 g: (3-15) mL, more preferably 1 g: (5-12) mL, most preferably 1 g: (5-10) mL.
In the invention, the cracking temperature is preferably 15-35 ℃, more preferably 20-30 ℃, and most preferably 25 ℃; the time is preferably 2 to 6 hours, more preferably 3 to 5 hours, and most preferably 4 hours. In the present invention, the reaction occurring during the cracking process is represented by the formula (2):
after the cracking, the method preferably further comprises the steps of carrying out first solid-liquid separation on the cracking system to obtain a solid component and a liquid component; washing the obtained solid component with TFA to obtain washing liquid; and combining the washing liquid and the liquid component, adding a settling agent for precipitation, performing second solid-liquid separation, washing the obtained solid component with an organic solvent, and drying to obtain the peptide-procainamide. In the invention, the first solid-liquid separation mode is preferably filtration or suction filtration; the purpose of the first solid-liquid separation is to remove the modified resin. In the present invention, the settling agent preferably comprises methyl tert-butyl ether; the volume ratio of the settling agent to the liquid component is preferably (3-10): 1, more preferably (5-8): 1. in the invention, the sedimentation temperature is preferably-20-15 ℃, and more preferably 0-10 ℃; the time is preferably 0.5 to 4 hours, and more preferably 1 to 2.5 hours. In the present invention, the second solid-liquid separation method is preferably centrifugal separation, and the conditions for centrifugal separation in the present invention are not particularly limited, and may be centrifugal separation conditions well known to those skilled in the art. In the present invention, the organic solvent washing is preferably methyl tert-butyl ether washing; the number of washing with the organic solvent is preferably 2 to 3. In the present invention, the drying is preferably performed by vacuum drying; the drying temperature is preferably 15-40 ℃, and more preferably 25-35 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.
After the peptide line of the procainatide is obtained, the method comprises the steps of dissolving the peptide line of the procainatide in a reagent, adjusting the pH value of the obtained solution of the procainatide to 7-10, mixing the solution of the procainatide with a first oxidizing agent, and carrying out first oxidation bonding on disulfide bonds in Cys at the 4 th site and the 12 th site in a sequence of the procainatide line of the procainatide to obtain pre-oxidized peptide line of the procainatide;
the first oxidizing agent comprises air, hydrogen peroxide or dimethyl sulfoxide.
In the present invention, the reagent preferably comprises water, an aqueous acetonitrile solution or a buffered saline solution; the volume fraction of acetonitrile in the acetonitrile water solution is preferably 0-50%, and more preferably 10-20%; the buffer saline solution preferably comprises an ammonium bicarbonate aqueous solution, ammonia water or a triethylamine phosphate aqueous solution; the concentration of the buffered saline solution is not particularly limited, and the pH value can be adjusted to 7-10; in an embodiment of the present invention, the aqueous ammonium bicarbonate solution is preferably a saturated aqueous ammonium bicarbonate solution.
In the invention, the concentration of the procapsipeptide wire peptide solution is preferably 0.2-10 mg/mL, more preferably 0.5-5 mg/mL, and most preferably 2-5 mg/mL.
In the present invention, the pH is 7 to 10, preferably 8 to 9. The reagent used for adjusting the pH value is not particularly limited, and an alkaline reagent well known to those skilled in the art can be used, specifically, ammonia water, sodium bicarbonate, potassium bicarbonate or ammonium bicarbonate; the mass percentage concentration of the ammonia water is preferably 1-25%, and more preferably 5-10%.
In the present invention, the first oxidizing agent includes air, hydrogen peroxide or dimethyl sulfoxide. In the invention, the air is preferably introduced; the air is preferably introduced in an amount of 100-2000 mL/min, more preferably 500-1000 mL/min. In the invention, the hydrogen peroxide is preferably used in the form of aqueous hydrogen peroxide, and the mass percentage concentration of the aqueous hydrogen peroxide is preferably 0.5-30%, and more preferably 1-10%; the molar ratio of the procapsipeptide wire peptide to the hydrogen peroxide is preferably 1: (0.8-2), more preferably 1: (1-1.5). In the present invention, the volume ratio of the procatide linear peptide solution to the dimethyl sulfoxide is preferably 1: (0.05 to 0.3), more preferably 1: (0.1-0.2). In the invention, the adding mode of the hydrogen peroxide and the dimethyl sulfoxide is preferably dropwise, the dropwise adding speed is not particularly limited, and the hydrogen peroxide and the dimethyl sulfoxide can be added dropwise at a constant speed.
In the present invention, it is preferable to stop the first oxidation bond when the remaining amount of the peptide of procapsipeptide is 2.5% or less of the total amount of the procapsipeptide. In the present invention, the temperature of the first oxidation bond is preferably room temperature; the time is preferably 2 to 72 hours, more preferably 10 to 60 hours, and still more preferably 30 to 50 hours. In the invention, in the first oxidation bonding process, because the side chain sulfydryl of two Cys at the 7-position and the 15-position in the peptide line peptide of the proca is protected by Acm, the protecting group is stable in the presence of air, hydrogen peroxide and a dimethyl sulfoxide oxidant, the two Cys at the 7-position and the 15-position cannot react; the reaction occurring during the first oxidation bonding process is as shown in formula (3):
after the pre-oxidized procapsid peptide is obtained, the pre-oxidized procapsid peptide is subjected to column chromatography coarse purification, the obtained purification solution is diluted and then is mixed with a second oxidant, the disulfide bonds in Cys at the 7 th site and the 15 th site in the sequence of the pre-oxidized procapsid peptide are subjected to second oxidation bonding, and the obtained second oxidation product is subjected to reversed phase preparative chromatography purification to obtain the procapsid peptide;
the mobile phase adopted by the column chromatography crude purification comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises acetonitrile, methanol or ethanol, and the mobile phase B comprises an acetic acid aqueous solution, a trifluoroacetic acid aqueous solution, a triethylamine phosphate aqueous solution or an ammonium acetate aqueous solution;
the second oxidant comprises iodine, a divalent mercury salt, a silver salt, or a trivalent titanium salt.
In the invention, the mobile phase adopted by the column chromatography crude purification comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an acetic acid aqueous solution, a trifluoroacetic acid aqueous solution, a triethylamine phosphate aqueous solution or an ammonium acetate aqueous solution, and the mobile phase B comprises acetonitrile, methanol or ethanol. In the invention, the mass percentage concentration of the acetic acid aqueous solution is preferably 0.1-5%, and more preferably 0.5-2%; the mass percentage concentration of the trifluoroacetic acid aqueous solution is preferably 0.05-0.5%, and more preferably 0.08-0.2%; the mass percentage concentration of the triethylamine phosphate aqueous solution is preferably 0.5-5%, and more preferably 0.5-2%; the mass percentage concentration of the ammonium acetate aqueous solution is preferably 0.1-2%, and more preferably 0.5-1%. Other conditions for the column chromatography crude purification are not particularly limited in the present invention, and impurities can be removed by using column chromatography crude purification conditions well known to those skilled in the art. In the invention, after the crude purification by the column chromatography, the impurities such as residual solvent in the pre-oxidized proca peptide, alkaline amino acid salt generated in the cracking process, lysate, removed side chain protecting group and the like can be removed, and the subsequent second oxidation is carried out to form bonds, thereby improving the purity of the final proca peptide.
In the present invention, the diluent used for the dilution preferably includes water or an aqueous acetonitrile solution; the volume percentage of acetonitrile in the aqueous acetonitrile solution is preferably < 30%; the dilution factor is preferably 2-20 times, more preferably 5-15 times, and most preferably 10-15 times. In the invention, the concentration of the pre-oxidized pracapelin peptide in the purified solution is obviously reduced by dilution, so that the formation of disulfide bonds among the pre-oxidized pracapelin peptide molecules in the subsequent second oxidation bonding process is reduced.
After the dilution, the invention preferably also comprises the step of adjusting the pH value of the diluted system to 3-6. In the invention, the pH value is preferably 4-5; the reagent used for the pH adjustment in the present invention is not particularly limited, and an acid known to those skilled in the art, specifically, acetic acid or phosphoric acid, may be used.
In the present invention, the second oxidant includes iodine, a divalent mercury salt, a silver salt, or a trivalent titanium salt; the second oxidant is preferably used in the form of a second oxidant solution, the solvent in the second oxidant solution preferably comprising methanol, acetic acid or acetonitrile; the concentration of the second oxidant solution is preferably 0.01-1 mol/L, more preferably 0.1-0.8 mol/L, and most preferably 0.3-0.5 mol/L. The divalent mercury salt, silver salt or trivalent titanium salt is not particularly limited in kind, and may be dissolved in methanol, acetic acid or acetonitrile. In the invention, the adding mode of the second oxidant solution is preferably dropwise, the dropwise adding speed is not particularly limited, and the second oxidant solution can be dropwise added at a constant speed. In the present invention, the second oxidant is preferably added in 2 to 3 times. In the invention, the molar ratio of the second oxidant to the procapsipeptide is preferably (0.6-3): 1, more preferably (1-2): 1, and most preferably 1.5: 1.
In the invention, the temperature of the second oxidation bonding is preferably 10-40 ℃, and more preferably room temperature; the time is preferably 5 to 24 hours, and more preferably 5 to 12 hours. In the present invention, the reaction occurring during the second oxidation reaction is represented by formula (4):
after the second oxidation bonding, the present invention preferably further comprises adding a reducing agent to the second oxidation bonding system to remove excess iodine. In the present invention, the reducing agent preferably includes vitamin C or sodium bisulfite. The dosage of the reducing agent is not particularly limited, and the reducing agent is added until the faint yellow (the color of iodine) of a system for forming bonds by second oxidation fades.
In the present invention, the reverse phase preparative chromatographic purification comprises a first purification and a second purification performed in sequence. In the present invention, the conditions of the first purification include: the chromatographic column packing is preferably C18Alkyl bonded silica gel; the column temperature is preferably 35 ℃; the detection wavelength is preferably 214 nm; the mobile phase A is preferably 0.5-2 v/v% triethylamine phosphate aqueous solution, 2-32 g/L ammonium acetate aqueous solution or 10-50 mol/L dihydrogen phosphate aqueous solution, the concentration of the triethylamine phosphate aqueous solution is more preferably 1-1.5 v/v%, the concentration of the ammonium acetate aqueous solution is more preferably 10-16 g/L, and the concentration of the dihydrogen phosphate aqueous solution is more preferably 20-30 mol/L; the mobile phase B is preferably acetonitrile; the flow rates of the mobile phase A and the mobile phase B are preferably 5-15 mL/min independently, and more preferably 10 mL/min; the elution mode is gradient elution, the gradient elution procedure is 0-6 min, the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B isThe volume fraction is 20%; and 6-66 min, wherein the volume fraction of the mobile phase A is 60%, and the volume fraction of the mobile phase B is 40%.
In the present invention, the conditions of the second purification include: the chromatographic column packing is preferably C18Alkyl bonded silica gel; the column temperature is preferably 35 ℃; the detection wavelength is preferably 214 nm; the mobile phase A is preferably 0.05-5 v/v% acetic acid aqueous solution, the concentration of the acetic acid aqueous solution is more preferably 1-4%, and the most preferably 2-3%; the mobile phase B is preferably acetonitrile; the flow rates of the mobile phase A and the mobile phase B are preferably 5-15 mL/min independently, and more preferably 10 mL/min; the elution mode is gradient elution, the gradient elution procedure is 0-6 min, the volume fraction of the mobile phase A is 75%, and the volume fraction of the mobile phase B is 25%; and 6-66 min, wherein the volume fraction of the mobile phase A is 55%, and the volume fraction of the mobile phase B is 45%.
In the present invention, after the purification by reverse phase preparative chromatography, the present invention preferably further comprises concentrating the purified solution obtained by the purification by reverse phase preparative chromatography and drying the concentrated solution to obtain the procainatide. The concentration method of the present invention is not particularly limited, and a concentration method known to those skilled in the art may be used, specifically, distillation under reduced pressure. In the invention, the drying temperature is preferably 10-40 ℃, and more preferably 20-30 ℃; the drying time is not particularly limited, and the drying is carried out until the weight is constant; the drying is preferably carried out in a hanging bottle lyophilizer.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
(1) After swelling 10g of AM resin with a degree of substitution of 1.0mmol/g, 200mL of 5 v/v% DIEA/DCM resin for 1h, DMF was washed 2 times, the solution was removed by suction filtration, 3.33g of FMPB (15mmol) was added to the reactor, DMF was added to dissolve it, 2.36mL of DIC (15mmol) and 2.03g of HoBt (15mmol) were added to mix, and after modification for 1.5h, ninhydrin was detected as negative, washed 3 times with DMF, THF was washed 2 times, and the solvent was removed by suction filtration to give a modified resin (13.56 g).
(2) Under the protection of nitrogen, 1.36g of the modified resin obtained in the step (1), 4.47g H-Leu-OtBu & HCl (20mmol) and 200mL of THF are mixed, 2.3mL (40mmol) of acetic acid is added and mixed uniformly, then, the mixture is subjected to an aldol condensation reaction for 30min, 3mL of a sodium cyanoborohydride/methanol solution with the concentration of 13.3mol/L is added and mixed, the reduction reaction is carried out for 16H, the mixture is washed with DMF for 3 times, methanol for 3 times and vacuum-pumped to dryness, and H-Leu-OtBu-FMPBAM resin (1.49g, the yield is 96.3%) is obtained.
(3) Placing 1.49g of H-Leu-OtBu-FMPB AM resin (1mmol) obtained in the step (2) in 25mL of DCM for swelling for 30min, taking 3mmol of DIC and 3mmol of HoBt as condensation reagents, carrying out coupling reaction on the swollen H-Leu-OtBu-FMPB AM resin and Fmoc-Cys (Acm) -OH at room temperature, determining the reaction end point by using ninhydrin detection liquid, wherein the detection result is negative, then removing the Fmoc protecting group in the presence of 30mL of 20 v/v% piperidine DMF solution, determining the reaction end point by using ninhydrin detection liquid, wherein the detection result is positive, completing the Fmoc protecting group removing reaction, and obtaining Cys (Cys) (Acm) -Leu-OtBu-FMPB AM resin; the coupling reaction and the Fmoc-protecting group removing reaction are repeated, and Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Cys (Acm) -OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn (Trt) -OH are sequentially connected to the Prokana main chain peptide sequence of Prokana (Cys) (Acm) -Leu-Cys-Glu- (OtBu) -Glu) (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn- (Trt) -OH to obtain the fully-protected Prokana -Val-Ala-Cys (Trt) -Thr (tBu) -Gly-Cys (Acm) -Leu-OtBu-FMPB AM resin (4.18 g); wherein the dosage of each amino acid is 3 mmol.
(4) 4.18g of the fully protected procaine resin obtained in step (3) and 40mL of a lysis solution (TFA: EDT: TIS: H)2O volume ratio 90:5:2.5:2.5), splitting at room temperature for 3h, filtering to obtain resin and collecting liquid components; washing the resin with 3mL of TFA for 1 time to obtain a washing solution; combining said wash solution and said liquid componentThen, the solid was washed 2 times with 100mL of methyl t-butyl ether per washing, and vacuum dried to obtain the peptide line peptide Asn-Asp-Glu-Cys-Glu-Leu-Cys (Acm) -Val-Asn-Val-Ala-Cys-Thr-Gly-Cys (Acm) -Leu-OH (1.77g, 97.0% yield, 68.6% purity).
(5) Putting 200mg of the procapsid peptide obtained in the step (4) into pure water, adding a saturated ammonium bicarbonate solution to adjust the pH value to 7.5, magnetically stirring until the procapsid peptide crude product is completely dissolved to obtain a 1mg/mL procapsid peptide solution, dropwise adding 50 mu L of 7.5 wt% hydrogen peroxide solution for 60min in two times (wherein the dropwise adding amount is 25 mu L each time, and the first oxidation bond is 30min after each dropwise adding) to obtain a preoxidized procapsid peptide solution, and performing HPLC (high performance liquid chromatography) detection to obtain the purity of 70.2%;
(6) and (3) carrying out column chromatography coarse purification on the pre-oxidized proca peptide liquid obtained in the step (5) to obtain a purified liquid, adding 20mL of pure water into the purified liquid for dilution, dropwise adding 0.25mL of 0.50mol/L iodine methanol solution until the system color is light yellow brown, stopping adding the solution when the system color is light yellow brown, continuously dropwise adding the iodine methanol solution until the system color is not changed within 30min after the solution is faded, oxidizing the solution into a bond for 12h for the second time, adding vitamin C to reduce excessive iodine to be light yellow and fading away to obtain a proca peptide crude product liquid, and detecting by HPLC (high performance liquid chromatography) to obtain the purity of 56.8%.
(7) And (3) filtering the crude prucalotide solution obtained in the step (6) by a 0.45-micron filter membrane, performing first purification and second purification by using reverse phase preparative chromatography, and drying for 48 hours at the temperature of 30 ℃ by using a bottle hanging freeze dryer to obtain prucalotide (31.2mg, the purification yield is 15.6%, the purity is 99.74%, and the maximum single impurity content is 0.08%).
Wherein the first purification conditions are: the chromatographic column is Luna-C18 (20X 250mm, 10 μm); the column temperature was 35 ℃; the detection wavelength is 214 nm; mobile phase a was a 1 v/v% TEAP aqueous solution (pH 3); the mobile phase B is acetonitrile; the flow rate of the mobile phase A and the mobile phase B is 10 mL/min; the elution mode is gradient elution: 0-6 min, wherein the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B is 20%; and 6-66 min, wherein the volume fraction of the mobile phase A is 60%, and the volume fraction of the mobile phase B is 40%.
The conditions for the second purification were: the chromatographic column is Luna-C18 (20X 250mm, 10 μm); the column temperature was 35 ℃; the detection wavelength is 214 nm; mobile phase a was 0.3 v/v% aqueous acetic acid (pH 3); the mobile phase B is acetonitrile; the flow rate of the mobile phase A and the mobile phase B is 10 mL/min; the elution mode is gradient elution: 0-6 min, wherein the volume fraction of the mobile phase A is 75%, and the volume fraction of the mobile phase B is 25%; and 6-66 min, wherein the volume fraction of the mobile phase A is 55%, and the volume fraction of the mobile phase B is 45%.
Example 2
(1) Placing 6.30g of H-Leu-OtBu-FMPBAM resin (1mmol) obtained in the step (2) in the example 1 in 25mL of DCM for swelling for 30min, taking 3mmol of DIC and 3mmol of HoBt as condensation reagents, carrying out coupling reaction on the swollen H-Leu-OtBu-FMPB AM resin and Fmoc-Cys (Acm) -OH at room temperature, judging the reaction end point by ninhydrin detection liquid, wherein the detection result is negative, then removing the Fmoc protecting group in 140mL of 20 v/v% piperidine DMF solution, judging the reaction end point by ninhydrin detection liquid, wherein the detection result is positive, and completing the Fmoc protecting group removing reaction to obtain Cys Acm-Leu-OtBu-FMPB AM resin; the coupling reaction and the Fmoc-protecting group removing reaction are repeated, and Fmoc-Hmb-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Cys (Acm) -OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn (Trt) -OH are sequentially connected to Cys (Acm) -Leu-Hmb-Gly-FMO-Cys (Trt) -OH, Fmoc-Leu-Asn-Glu (OtBu) (Tru) -OH, and Fmoc-Glu (OtBu) -OH to obtain the fully (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) -Gly-Cys (Acm) -Leu-OtBu-FMPB AM resin (21.27 g); wherein the dosage of each amino acid is 3 mmol.
(2) 21.27g of the fully protected procaine resin prepared in step (1) and 250mL of a lysis solution (TFA: EDT: TIS: H)2O volume ratio 90:5:2.5:2.5), splitting at room temperature for 3h, filtering to obtain resin and a collection liquid; washing the resin with 10mL TFA for 1 time to obtain a washing solution; mixing said wash solution with said collectorThe collected liquids were combined and added to 1100mL of methyl tert-butyl ether, and the mixture was allowed to settle at-10 ℃ for 30min, centrifuged, and the resulting solid fraction was washed 2 times with 100mL of methyl tert-butyl ether per washing, and dried under vacuum to give the peptide of praena peptide (9.02g, yield 96.7%, purity 81.2%).
(3) Putting 200mg of the peptide-peptide of the proca obtained in the step (2) into pure water, adding a saturated ammonium bicarbonate solution to adjust the pH value to 7.5, magnetically stirring until the crude product of the proca is completely dissolved to obtain a 1mg/mL solution of the proca, introducing air at the flow rate of 200mL/min, dropwise adding 50 mu L of a 7.5 wt% hydrogen peroxide solution for 60min in two times, wherein the addition amount of each dropwise adding is 25 mu L, and the first oxidation bond is 30min after each dropwise adding to obtain a preoxidized proca peptide solution, and detecting by HPLC to obtain the purity of 83.4%.
(4) And (3) carrying out column chromatography coarse purification on the pre-oxidized procapsid peptide liquid obtained in the step (3) by taking 1 v/v% TEAP aqueous solution as a mobile phase to obtain 18mL of purified liquid, adding 102mL of pure water into the purified liquid for dilution to obtain a diluted liquid, adjusting the pH value of the diluted liquid to 3 by using acetic acid, dropwise adding 0.25mL of a 0.50mol/L iodine methanol solution until the system color is light yellow brown, stopping adding the solution when the system color is not changed after the solution fades, continuously dropwise adding the iodine methanol solution until the system color is not changed within 30min, carrying out secondary oxidation for 8h, adding vitamin C to reduce excessive iodine until the light yellow color is faded, and obtaining a procapsid peptide crude product liquid with the purity of 89.4% by HPLC detection.
(5) And (3) filtering the crude procapsipeptide obtained in the step (4) through a 0.45-micron filter membrane, performing first purification and second purification by using reverse phase preparative chromatography, and performing freeze-drying on a bottle hanging freeze-drying machine at room temperature for 24 hours to obtain the procapsipeptide (65.2mg, the purification yield is 32.6%, the purity is 99.76%, and the maximum single impurity content is 0.06%).
Wherein the first purification conditions are: the chromatographic column is Luna-C18 (20X 250mm, 10 μm); the column temperature was 35 ℃; the detection wavelength is 214 nm; mobile phase a was a 1 v/v% TEAP aqueous solution (pH 3); the mobile phase B is acetonitrile; the flow rate of the mobile phase A and the mobile phase B is 10 mL/min; the elution mode is gradient elution: 0-6 min, wherein the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B is 20%; and 6-66 min, wherein the volume fraction of the mobile phase A is 60%, and the volume fraction of the mobile phase B is 40%.
The conditions for the second purification were: the chromatographic column is Luna-C18 (20X 250mm, 10 μm); the column temperature was 35 ℃; the detection wavelength is 214 nm; mobile phase a was 0.3 v/v% aqueous acetic acid (pH 3); the mobile phase B is acetonitrile; the flow rate of the mobile phase A and the mobile phase B is 10 mL/min; the elution mode is gradient elution: 0-6 min, wherein the volume fraction of the mobile phase A is 75%, and the volume fraction of the mobile phase B is 25%; and 6-66 min, wherein the volume fraction of the mobile phase A is 55%, and the volume fraction of the mobile phase B is 45%.
Example 3
(1) Putting 8.02g of the procapsipeptide wire peptide prepared according to the step (1) in the example 2 into pure water, adding a saturated ammonium bicarbonate solution to adjust the pH value to 7.5, magnetically stirring until the crude procapsipeptide wire peptide product is completely dissolved to obtain a procapsipeptide wire peptide solution with the concentration of 1mg/mL, introducing air at the flow rate of 600mL/min, dropwise adding 2.1mL of 7.5 wt% hydrogen peroxide solution for 90min in three times for the first oxidation bond formation (wherein the dropwise adding amount is 700 mu L each time, and the first oxidation bond formation is 30min after each dropwise adding) to obtain a pre-oxidized procapsipeptide wire peptide solution, and detecting by HPLC (high performance liquid chromatography) to obtain the purity of 81.6%.
(2) Carrying out column chromatography coarse purification on the pre-oxidized procapsid peptide liquid obtained in the step (1) by taking 1 v/v% TEAP aqueous solution as a mobile phase to obtain 850mL of purified liquid, adding 3650mL of pure water into the purified liquid for dilution to obtain a diluted liquid, wherein the pH value of the diluted solution is about 3.5, dropwise adding 2.35mL of a 0.50mol/L iodine methanol solution until the system color is light yellow brown, stopping dropwise adding until the iodine methanol solution is not discolored within 30min after the solution fades, carrying out secondary oxidation for 12h, adding vitamin C to reduce excessive iodine to light yellow, and fading away, carrying out HPLC detection to obtain a procapsid peptide crude liquid, wherein the purity of the procapsid peptide liquid is 90.2% after the HPLC detection.
(3) Enabling the crude prucalotide solution obtained in the step (1) to pass through a 0.45-micron filter membrane, performing first purification and second purification by using a reverse phase preparative chromatography, and performing freeze-drying for about 32 hours by using a bottle hanging freeze-drying machine at room temperature to obtain 2.55g of prucalotide, wherein the purification yield is 39.6%; purity 99.80%, maximum single impurity content 0.04%).
Wherein the first purification conditions are: the chromatographic column is Luna-C18 (20X 250mm, 10 μm); the column temperature was 35 ℃; the detection wavelength is 214 nm; mobile phase a was a 1 v/v% TEAP aqueous solution (pH 3); the mobile phase B is acetonitrile; the flow rate of the mobile phase A and the mobile phase B is 10 mL/min; the elution mode is gradient elution: 0-6 min, wherein the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B is 20%; and 6-66 min, wherein the volume fraction of the mobile phase A is 60%, and the volume fraction of the mobile phase B is 40%.
The conditions for the second purification were: the chromatographic column is Luna-C18 (20X 250mm, 10 μm); the column temperature was 35 ℃; the detection wavelength is 214 nm; mobile phase a was 0.3 v/v% aqueous acetic acid (pH 3); the mobile phase B is acetonitrile; the flow rate of the mobile phase A and the mobile phase B is 10 mL/min; the elution mode is gradient elution: 0-6 min, wherein the volume fraction of the mobile phase A is 75%, and the volume fraction of the mobile phase B is 25%; and 6-66 min, wherein the volume fraction of the mobile phase A is 55%, and the volume fraction of the mobile phase B is 45%.
Comparative example 1
200mg of the procapsipeptide wire peptide obtained in the step (4) in the example 1 is placed in pure water, saturated ammonium bicarbonate solution is added to adjust the pH value to 7.5, the mixture is magnetically stirred until the crude procapsipeptide wire peptide is completely dissolved to obtain 1mg/mL of procapsipeptide wire peptide solution, 50 mu L of 7.5 wt% hydrogen peroxide solution is dropwise added to form a first oxidation bond for 40min to obtain pre-oxidized procapsipeptide wire peptide solution, and the purity of the pre-oxidized procapsipeptide wire peptide solution is 67.2% by HPLC detection.
And (3) regulating the pH value of the pre-oxidized procapsid peptide solution to 5 by using acetic acid, dropwise adding 0.3mL of 0.50mol/L iodine methanol solution for secondary oxidation for 3 hours, adding vitamin C to reduce excessive iodine to be faint yellow and remove to obtain a procapsid peptide crude product solution, wherein the purity of the procapsid peptide crude product solution is 55.4% by HPLC (high performance liquid chromatography).
Comparative example 2
200mg of the procapsipeptide wire peptide obtained in the step (4) in the example 1 is placed in 20 v/v% acetonitrile water solution, saturated ammonium bicarbonate solution is added to adjust the pH value to 7.5, the mixture is magnetically stirred until the crude procapsipeptide wire peptide is completely dissolved to obtain 1mg/mL of procapsipeptide wire peptide solution, 50 mu L of 7.5 wt% hydrogen peroxide solution is dropwise added to form a first oxidation bond for 40min to obtain pre-oxidized procapsipeptide wire peptide solution, and the purity of the pre-oxidized procapsipeptide wire peptide solution is 69.4% by HPLC detection.
Adjusting the pH value of the pre-oxidized procapsid peptide solution to 5 by using acetic acid, dropwise adding 0.3mL of 0.50mol/L iodine methanol solution for secondary oxidation for 3 hours, adding vitamin C to reduce excessive iodine to be faint yellow and remove to obtain a procapsid peptide crude product solution, and detecting by HPLC (high performance liquid chromatography) to obtain the purity of 46.6%.
As can be seen from comparative examples 1-2, after the first oxidation bond is completed, the pH value is directly adjusted without chromatographic purification, and then the iodine/methanol solution is added for the second oxidation bond formation, so that the purity of the crude pracapecitabine liquid is obviously reduced.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of procatide is characterized by comprising the following steps:
(1) mixing the initial resin, FMPB and a condensation reagent, and modifying to obtain modified resin; the starting resin comprises an MBHA resin or an AM resin;
(2) under the protective atmosphere, mixing the modified resin, H-Leu-OtBu and furan solvents, carrying out an amino-aldehyde condensation reaction, mixing the obtained Schiff base with a reducing agent, and carrying out a reduction reaction to obtain H-Leu-OtBu-FMPB MBHA/AM resin;
(3) according to the sequence of the backbone peptide of the procapsipeptide, Fmoc-Cys (Acm) -OH and R are connected to the H-Leu-OtBu-FMPB MBHA/AM resin in sequence1-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Acm)-OH、R2-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Asn (Trt) -OH to obtain a fully protected procapside peptide resin; the R is1And R2Independent bagIncluding Fmoc and/or Fmoc-Hmb, said R1And R2Not being Fmoc at the same time; the connection comprises a coupling reaction and a Fomc protecting group removing reaction which are sequentially carried out;
(4) cracking the fully-protected procapsipeptide resin in a cracking solution to obtain procapsipeptide linear peptide;
(5) dissolving the procainatide linear peptide into a reagent, adjusting the pH value of the obtained procainatide linear peptide solution to 7-10, mixing the solution with a first oxidizing agent, and carrying out first oxidation bonding on disulfide bonds in Cys at positions 4 and 12 in the procainatide linear peptide sequence to obtain pre-oxidized procainatide linear peptide;
the first oxidizing agent comprises air, hydrogen peroxide or dimethyl sulfoxide;
(6) carrying out column chromatography coarse purification on the pre-oxidized proca peptide, diluting the obtained purified liquid, mixing the diluted purified liquid with a second oxidant, carrying out second oxidation bonding on disulfide bonds in Cys at the 7 th site and the 15 th site in the pre-oxidized proca peptide sequence, and carrying out reversed-phase preparative chromatography purification on the obtained second oxidation product to obtain proca peptide;
the mobile phase adopted by the column chromatography crude purification comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an acetic acid aqueous solution, a trifluoroacetic acid aqueous solution, a triethylamine phosphate aqueous solution or an ammonium acetate aqueous solution, and the mobile phase B comprises acetonitrile, methanol or ethanol;
the second oxidant comprises iodine, a divalent mercury salt, a silver salt, or a trivalent titanium salt.
2. The method according to claim 1, wherein in the step (1), the degree of substitution of the starting resin is 0.3 to 1.6 mmol/g;
the condensation reagent comprises HOBT-DIC, HBTU-NMM, HATU-NMM or PyBOP-HOBT-NMM;
the molar ratio of the starting resin, FMPB and condensing agent is 1: (1.5-4): (1.5-4).
3. The production method according to claim 1, wherein in the step (2), the molar ratio of the modified resin to H-Leu-OtBu is 1: (1-6);
the ammonia-aldehyde condensation reaction is carried out under acidic or alkaline conditions, the pH value of the acidic conditions is 2-6, and the pH value of the alkaline conditions is 7.5-11.
4. The preparation method according to claim 1 or 3, wherein in the step (2), the reducing agent comprises one or more of sodium borohydride, sodium cyanoborohydride and sodium borohydride acetate;
the mol ratio of the modified resin to the reducing agent is 1: (2-6).
5. The process according to claim 1, wherein in step (3), the condensation reagent used in the coupling reaction comprises HOBT-DIC, HBTU-NMM, HATU-NMM or PyBOP-HOBT-NMM;
the deprotection reagent adopted in the reaction for removing the Fomc protecting group comprises piperidine, 1, 8-diazabicycloundecen-7-ene or triethylamine.
6. The method according to claim 1, wherein in the step (4), the composition of the lysate comprises TFA, EDT, TIS and H2O, said TFA, EDT, TIS and H2The volume ratio of O is (85-100): (0-5): (0-5): (0-5);
the ratio of the mass of the fully-protected procatide resin to the volume of the lysis solution is 1 g: (3-15) mL;
the cracking temperature is 15-35 ℃, and the cracking time is 2-6 h.
7. The method according to claim 1, wherein in the step (5), the reagent comprises water, an aqueous acetonitrile solution or an aqueous buffered salt solution;
the concentration of the procapsipeptide solution is 0.2-10 mg/mL.
8. The method according to claim 1, wherein in the step (6), the molar ratio of the second oxidizing agent to the procapsipeptide is (0.6-3): 1.
9. The method according to claim 1, wherein in the step (6), the dilution ratio is 2 to 20.
10. The production method according to claim 1, wherein in the step (6), the reverse phase preparative chromatographic purification comprises a first purification and a second purification which are carried out in this order;
the conditions of the first purification include: the chromatographic column packing is C18Alkyl bonded silica gel; the column temperature was 35 ℃; the mobile phase A is 0.5-2 v/v% triethylamine phosphate aqueous solution, 2-32 g/L ammonium acetate aqueous solution or 10-50 mol/L dihydric phosphate aqueous solution; the mobile phase B is acetonitrile; the flow rates of the mobile phase A and the mobile phase B are independently 5-15 mL/min; the elution mode is gradient elution, the gradient elution procedure is 0-6 min, the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B is 20%; 6-66 min, wherein the volume fraction of the mobile phase A is 60%, and the volume fraction of the mobile phase B is 40%; the detection wavelength is 214 nm;
the conditions of the second purification include: the chromatographic column packing is C18Alkyl bonded silica gel; the column temperature was 35 ℃; the mobile phase A is 0.05-5 v/v% acetic acid aqueous solution; the mobile phase B is acetonitrile; the flow rates of the mobile phase A and the mobile phase B are independently 5-15 mL/min; the elution mode is gradient elution, the gradient elution procedure is 0-6 min, the volume fraction of the mobile phase A is 75%, and the volume fraction of the mobile phase B is 25%; 6-66 min, wherein the volume fraction of the mobile phase A is 55%, and the volume fraction of the mobile phase B is 45%; the detection wavelength was 214 nm.
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| CN114315973A (en) * | 2021-12-30 | 2022-04-12 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying procatide |
| CN114573662A (en) * | 2021-09-30 | 2022-06-03 | 深圳翰宇药业股份有限公司 | Preparation method of procainatide ammonium salt |
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