CN111018962A - Method for preparing teduglutide based on solid phase step-by-step method - Google Patents
Method for preparing teduglutide based on solid phase step-by-step method Download PDFInfo
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- CN111018962A CN111018962A CN201911375592.4A CN201911375592A CN111018962A CN 111018962 A CN111018962 A CN 111018962A CN 201911375592 A CN201911375592 A CN 201911375592A CN 111018962 A CN111018962 A CN 111018962A
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- 238000000034 method Methods 0.000 title claims abstract description 126
- CILIXQOJUNDIDU-ASQIGDHWSA-N teduglutide Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O)[C@@H](C)CC)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)CC)C1=CC=CC=C1 CILIXQOJUNDIDU-ASQIGDHWSA-N 0.000 title claims abstract description 111
- 239000007790 solid phase Substances 0.000 title claims abstract description 55
- 108010073046 teduglutide Proteins 0.000 title claims abstract description 43
- 229960002444 teduglutide Drugs 0.000 title claims abstract description 39
- 239000011347 resin Substances 0.000 claims abstract description 392
- 229920005989 resin Polymers 0.000 claims abstract description 392
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 83
- 239000012634 fragment Substances 0.000 claims abstract description 43
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 12
- 239000012043 crude product Substances 0.000 claims abstract description 10
- 238000004108 freeze drying Methods 0.000 claims abstract description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 267
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 178
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 115
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 68
- 238000005859 coupling reaction Methods 0.000 claims description 64
- 238000010168 coupling process Methods 0.000 claims description 49
- 230000008878 coupling Effects 0.000 claims description 48
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 36
- 150000001413 amino acids Chemical class 0.000 claims description 28
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 claims description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims description 27
- 125000006239 protecting group Chemical group 0.000 claims description 26
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 claims description 25
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 25
- 239000000203 mixture Substances 0.000 claims description 24
- 238000006467 substitution reaction Methods 0.000 claims description 24
- XXMYDXUIZKNHDT-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(N=C1)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXMYDXUIZKNHDT-QNGWXLTQSA-N 0.000 claims description 19
- AUBMZAMQCOYSIC-MNXVOIDGSA-N Leu-Ile-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O AUBMZAMQCOYSIC-MNXVOIDGSA-N 0.000 claims description 18
- 150000007530 organic bases Chemical class 0.000 claims description 16
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 16
- 239000007821 HATU Substances 0.000 claims description 13
- 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 12
- 230000002194 synthesizing effect Effects 0.000 claims description 11
- TYPIQBXHJBDEBC-HNNXBMFYSA-N tert-butyl (3s)-3-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxobutanoate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C=O)C3=CC=CC=C3C2=C1 TYPIQBXHJBDEBC-HNNXBMFYSA-N 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- JMEWFDUAFKVAAT-WDSKDSINSA-N Met-Asn Chemical compound CSCC[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CC(N)=O JMEWFDUAFKVAAT-WDSKDSINSA-N 0.000 claims description 10
- WKTSCAXSYITIJJ-PCBIJLKTSA-N Phe-Ile-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O WKTSCAXSYITIJJ-PCBIJLKTSA-N 0.000 claims description 10
- 230000015572 biosynthetic process Effects 0.000 claims description 10
- 238000003786 synthesis reaction Methods 0.000 claims description 10
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 claims description 9
- OUUCIIJSBIBCHB-ZPFDUUQYSA-N Ile-Leu-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O OUUCIIJSBIBCHB-ZPFDUUQYSA-N 0.000 claims description 9
- 239000003875 Wang resin Substances 0.000 claims description 9
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 claims description 8
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 6
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical group C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 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 6
- KPFBUSLHFFWMAI-HYRPPVSQSA-N [(8r,9s,10r,13s,14s,17r)-17-acetyl-6-formyl-3-methoxy-10,13-dimethyl-1,2,7,8,9,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-17-yl] acetate Chemical compound C1C[C@@H]2[C@](CCC(OC)=C3)(C)C3=C(C=O)C[C@H]2[C@@H]2CC[C@](OC(C)=O)(C(C)=O)[C@]21C KPFBUSLHFFWMAI-HYRPPVSQSA-N 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 108010016626 Dipeptides Proteins 0.000 claims description 4
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- GVIXTVCDNCXXSH-AWEZNQCLSA-N (2s)-2-amino-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]pentanoic acid Chemical compound OC(=O)[C@@H](N)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C GVIXTVCDNCXXSH-AWEZNQCLSA-N 0.000 claims description 3
- SCCPDJAQCXWPTF-VKHMYHEASA-N Gly-Asp Chemical compound NCC(=O)N[C@H](C(O)=O)CC(O)=O SCCPDJAQCXWPTF-VKHMYHEASA-N 0.000 claims description 3
- ROHDXJUFQVRDAV-UWVGGRQHSA-N Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 ROHDXJUFQVRDAV-UWVGGRQHSA-N 0.000 claims description 3
- 108010051242 phenylalanylserine Proteins 0.000 claims description 3
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 3
- -1 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl Chemical group 0.000 claims description 2
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 2
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 5
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 982
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 96
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- 230000004913 activation Effects 0.000 description 17
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 14
- 238000001035 drying Methods 0.000 description 13
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- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 12
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- JBLIDPPHFGWTKU-UHFFFAOYSA-N 2,6-dichlorobenzoyl chloride Chemical compound ClC(=O)C1=C(Cl)C=CC=C1Cl JBLIDPPHFGWTKU-UHFFFAOYSA-N 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
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- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- WVZWEMOFSIEEMU-UHFFFAOYSA-N indene-1,2,3-trione Chemical compound C1=CC=C2C(=O)C(=O)C(=O)C2=C1 WVZWEMOFSIEEMU-UHFFFAOYSA-N 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 235000016236 parenteral nutrition Nutrition 0.000 description 3
- VVQIIIAZJXTLRE-QMMMGPOBSA-N (2s)-2-amino-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound CC(C)(C)OC(=O)NCCCC[C@H](N)C(O)=O VVQIIIAZJXTLRE-QMMMGPOBSA-N 0.000 description 2
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- WBGMQHNUPJENDC-MERQFXBCSA-N dicyclohexylazanium;(2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-[1-[(2-methylpropan-2-yl)oxycarbonyl]imidazol-4-yl]propanoate Chemical compound C1CCCCC1NC1CCCCC1.CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CN(C(=O)OC(C)(C)C)C=N1 WBGMQHNUPJENDC-MERQFXBCSA-N 0.000 description 1
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Classifications
-
- 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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Abstract
The invention relates to a method for preparing teduglutide based on a solid phase step-by-step method. According to the sequence, a full-protection 20-33 fragment sequence is synthesized by a solid phase method, a 19-33 fragment sequence is synthesized in a solid phase together with Fmoc-HmbAla-OH or Fmoc-DmbAla-OH, a full-protection 4-18 fragment sequence is synthesized by a solid phase method according to the sequence, the 4-18 fragment sequence is connected with a sequence 2 and/or a sequence 3, then two long sequences are connected, and finally the first sequence is connected to form a crude product of the teduglutide, and the crude product of the teduglutide is obtained after purification and freeze-drying. The method has the advantages that the resin can be prevented from polycondensation during connection, and the purity and the total yield of the obtained crude peptide of the teduglutide have high levels and less impurities.
Description
Technical Field
The invention belongs to the field of preparation of polypeptide medicines, and particularly relates to a method for preparing teduglutide based on a solid-phase stepwise method.
Background
The teduglutide is GLP-2 (glucagon-like peptide-2) recombinantAnalogs having the chemical structure H-His1-Gly2-Asp3-Gly4-Ser5-Phe6-Ser7-Asp8-Glu9-Met10-Asn11-Thr12-Ile13-Leu14-Asp15-Asn16-Leu17-Ala18-Ala19-Arg20-Asp21-Phe22-Ile23-Asn24-Trp25-Leu26-Ile27-Gln28-Thr29-Lys30-Ile31-Thr32-Asp33-OH
Glucagon-like peptide-2 (GLP-2) analogue teduglutide (teduglutide) produced by NPS pharmaceutical company was first approved for the market in the european union at 8 months 2012 (trade name: Revestive); in the same year, 12 months, approved by the U.S. FDA for marketing in the united states (trade name Gattex). The composition is in form of injection, and can be used for treating Short Bowel Syndrome (SBS) of adult dependent on parenteral nutrition.
Short Bowel Syndrome (SBS) is caused by partial or complete surgical resection of the small or large intestine. Extensive small bowel loss results in life-sustaining fluids and nutrient malabsorption, and thus, adult patients with short bowel syndrome often require parenteral nutrition. Clinical studies prove that: subcutaneous injection of teduglutide 1 time a day helps to improve absorption of intestinal fluids and nutrients and may reduce the number and amount of parenteral nutrition administered.
At present, in the existing preparation method of the teduglutide, a solid-phase fragment method and a step-by-step coupling method are mainly adopted. In the fragment method synthesis strategy, for example, patent CN103314010A discloses a method for synthesizing teduglutide, which comprises preparing 1-4 position peptide fragment and 5-33 position peptide resin by solid phase method, then connecting 1-4 position peptide fragment to 5-33 position peptide resin by solid phase method, reducing Asp3-Gly4 rearrangement reaction; the patent CN104418949A uses a solid phase to synthesize a tedulpit peptide resin fragment 4-33 of the tedulpit peptide sequence and a tedulpit peptide resin fragment 1-3 of the tedulpit peptide sequence, then uses a suitable resin as a carrier, couples the tedulpit peptide resin fragment 4-33 of the tedulpit peptide sequence and the tedulpit peptide resin fragment 1-3 of the tedulpit peptide sequence to obtain the tedulpit peptide resin, finally performs cracking to obtain crude tedulpit peptide, and performs purification to obtain a purified tedulpit peptide product; the patent CN104072605A discloses another preparation method of teduglutide, which comprises the steps of dividing the teduglutide into 3-5 segments through reasonable division of peptide sequences, and then sequentially connecting the segments by a solid phase method to obtain the teduglutide; in the patent CN104817638A, a liquid phase method is used for synthesizing a 1-2-bit polypeptide fragment and a 3-4-bit polypeptide fragment, a solid phase step-by-step method is used for synthesizing a 5-33-bit peptide resin, and finally, the solid phase method is used for connecting the 1-2-bit polypeptide fragment and the 3-4-bit polypeptide fragment to the 5-33-bit peptide resin in sequence to obtain a tedulpit peptide sequence. Compared with a fragment method, the solid-phase stepwise method for synthesizing the teduglutide has only one patent with the patent number of CN104072603A, the patent utilizes Wang resin or CTC resin, adopts a method of coupling amino acids one by one to synthesize 2-33-bit polypeptide fragment resin, and then uses Trt-His (Trt) -OH, Boc-His (Boc) -OH or Boc-His (Boc) -OH DCHA to connect His, so as to reduce the content of [ D-His1] -teduglutide impurity in the polypeptide synthesis process.
The synthetic methods described in the above patents fall into two categories. The first type is that fragments are synthesized first and then are connected to synthesize the teduglutide. The first four patents mentioned above are all in this manner. These methods require the synthesis of two or more polypeptide fragments for ligation. First, the fragments need to be ligated one by one on a solid support, then the fragments are cleaved off with full protection, and the fragments are ligated one by one. The biggest problem is that some side chain protecting groups may be stripped off when the fragments are subjected to full-protection cleavage and fragment impurities are introduced when the next liquid phase ligation is performed. Also, if the purity of the fully protected fragments is low, these fragments may need to be purified, further increasing the complexity of the process. Secondly, in the process of segment condensation, in order to ensure the reaction is complete, multiple times of the amount of the segments are generally required to be fed, and a large amount of raw materials are wasted. Furthermore, racemization of amino acids during fragment ligation is often unavoidable, which also presents a great challenge for subsequent purification.
The second type is the stepwise synthesis of teduglutide using a solid phase method. Because the teduglutide is a hydrophobic sequence with medium length, the problems of resin sphere polycondensation, difficult connection and the like can occur when amino acids are gradually connected, so that impurities such as missing peptide exist in a crude product, and the missing peptide impurities are difficult to remove in the purification process. The second type of patent of the stepwise method only examines the racemization problem of [ D-His1], but does not research the problems of resin polycondensation, difficult connection, low crude product purity and the like in the specific synthesis of the sequence of the teduolutide.
Disclosure of Invention
In view of this, the present invention aims to provide a method for synthesizing teduolutide, which can simultaneously ensure that a resin is not subjected to condensation polymerization during connection, the purity and the total yield of the obtained crude teduolutide peptide are high, and the contents of common impurities of [ D-His1] -teduolutide and [ Asu3] -teduolutide in the synthesis of teduolutide are further reduced. In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for preparing teduglutide, comprising:
step 1) synthesizing a fully protected sequence I according to the sequence by a solid phase method: arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin.
Step 2) synthesizing the sequence I with Fmoc-HmbAla-OH or Fmoc-DmbAla-OH in a solid phase to obtain a sequence II: XmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin.
Step 3), synthesizing a sequence III in a solid phase according to the sequence of the teduglutide: Gly-Asp (OtBu) - (Xmb) Gly-Ser (tBu) - (Phe-Ser (tBu) - (OtBu) - (Glu), (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) - (Trt) - (Leu-Ala-XmbAla-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) - (Boc) - (Trp) (Leu-Ile-Gln (Trt) - (Thr) - (tBu) - (Boc) -Ile-Thr- (tBu) -Asp (OtBu) -Resin. Wherein the Asp3-Gly4 sequence employs the dipeptide Fmoc-Asp (OtBu) -XmbGly-OH; stepwise ligation of Fmoc-XmbGly-OH and Fmoc-Asp- (OtBu) -OH can also be used.
And 4) carrying out coupling reaction on the sequence III and Fmoc-His (Trt) -OH, and removing the Fmoc protecting group to obtain the teduglutide peptide resin.
And 5) removing side chain protecting groups by acidolysis to obtain a crude product of the teduolutide, and purifying and freeze-drying the crude product to obtain a pure product of the teduolutide.
Preferably, the Asp3-Gly4 sequence in step 3) is selected from the group consisting of the dipeptide Fmoc-Asp (OtBu) -XmbGly-OH; or step-wise linked using Fmoc-XmbGly-OH and Fmoc-Asp (OtBu) -OH. Wherein Xmb is Hmb or Dmb.
Preferably, the Resin used in the step 1) for synthesizing the sequence I is Wang Resin or CTC Resin, wherein the initial peptide Resin has a degree of substitution of Fmoc-Asp (OtBu) -Wang Resin of 0.22-0.48 mmol/g; the initial peptide Resin has a degree of substitution of Fmoc-Asp (OtBu) -CTC Resin of 0.25-0.50 mmol/g.
Preferably, in the steps 1), 3) and 4), the side chain protecting group adopted by His is trityl, the side chain protecting group adopted by Asp is tert-butoxy, the side chain protecting group adopted by Ser is tert-butyl, the side chain protecting group adopted by Glu is tert-butoxy, the side chain protecting group adopted by Asn is trityl, the side chain protecting group adopted by Thr is tert-butyl, the side chain protecting group adopted by Arg is 2,2,4,6, 7-pentamethyl dihydrobenzofuran-5-sulfonyl, the side chain protecting group adopted by Trp is tert-butoxycarbonyl, the side chain protecting group adopted by Gln is trityl, and the side chain protecting group adopted by Lys is tert-butoxycarbonyl.
Preferably, the amino acid at the second 19 th position of the sequence in the step 2) adopts Fmoc-HmbAla-OH or Fmoc-DmbAla-OH.
Preferably, the coupling reagent for coupling in step 1), 2), 3) is DIC/HOBt, DIC/HOAt, or a mixture of PyAop, PyBop, HBTU, HATU and an organic base. Wherein the organic base is one of DIPEA and NMM. More preferably a combination of DIC and HOBt. The ratio of coupling reagent to base was 1: 2.
Preferably, the reaction system used in step 4) is DIC/HOBt, DIC/HOOBt, DIC/Oxyma or HBTU, a mixture of TDBTU and an organic base. Wherein the organic base is one of DIPEA and NMM. More preferably, the condensing agent is selected from the group consisting of DIC/HOOBt in combination. The ratio of coupling reagent to base was 1: 2.
Preferably, the Fmoc removal method used in steps 1), 2), 3), 4) is a piperidine method or a diethylamine method, and more preferably, a 20% piperidine/DMF solution is used.
Preferably, the cleavage reagent used in step 5) comprises trifluoroacetic acid, thioanisole, phenol, 1, 2-ethanedithiol, and water. More preferably, the ratio of lysis reagent is trifluoroacetic acid: 1, 2-ethanedithiol: triisopropylsilane: water 90:5:2.5: 2.5.
Detailed Description
The meanings of abbreviations used in the specification and claims are listed in the following table:
the embodiment of the invention discloses a preparation method of teduglutide. The contents of this document can be referred to by those skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
step 1) Synthesis of the fully protected sequence-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin according to the sequence by solid phase method.
Specifically, the method for preparing the full protection sequence one in this embodiment is as follows: Fmoc-Asp (OtBu) -OH was coupled to Wang Resin to give Fmoc-Asp (OtBu) -Wang Resin. Wherein the coupling reagent is pyridine and 2, 6-dichlorobenzoyl chloride. Or coupling Fmoc-Asp (OtBu) -OH with CTC Resin to obtain Fmoc-Asp (OtBu) -CTC Resin. Wherein the coupling reagent is DIPEA.
More specifically, Fmoc-Asp (OtBu) -OH and Wang Resin are mixed and dissolved in DCM, pyridine is added, 2, 6-dichlorobenzoyl chloride is added under stirring, and the mixture is stirred and reacts to obtain Fmoc-Asp (OtBu) -Wang Resin; or dissolving Fmoc-Asp (OtBu) -OH and CTC Resin in DCM, and slowly dropwise adding DIPEA to obtain Fmoc-Asp (OtBu) -CTC Resin.
The molar ratio of Wang resin to Fmoc-Asp (OtBu) -OH, pyridine and 2, 6-dichlorobenzoyl chloride is 1: 2: 8: 4. the molar ratio of CTC resin to Fmoc-Asp (OtBu) -OH and DIPEA is 1: 2: 6.
the coupling reaction is carried out at room temperature for 2.5-3.0 hours.
After the coupling reaction is finished, adding acetic anhydride into Wang Resin for end capping, then washing the Wang Resin with DMF (dimethyl formamide), methanol, DCM (DCM) and methanol in sequence, and drying the obtained product in vacuum to obtain Fmoc-Asp (OtBu) -Wang Resin; and (3) adding methanol into the CTC Resin for end capping, washing with methanol, DCM and methanol in sequence, and drying in vacuum to obtain Fmoc-Asp (OtBu) -CTC Resin. According to the sequence of the teduu peptide, the sequence of complete protection, Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin, is obtained by successive coupling.
Wherein, the coupling reagent for the coupling reaction is a mixture of HOBt and DIC, or a mixture of PyAop, PyBop, HBTU, HATU and organic base. Wherein the organic base is one of DIPEA and NMM. If the combination of HOBt and DIC is adopted, the effect is better.
The molar ratio of the amino acid to the HOBt and the DIC is 1:1:1, and the coupling reaction in the step is detected and judged by an indantrione method to obtain the end point of each coupling reaction. If the resin is colorless and transparent, the reaction is complete; if the resin is developed, the reaction is not complete and recoupling is required.
Step 2) Synthesis of the sequence one with Fmoc-HmbAla-OH or Fmoc-DmbAla-OH in solid phase the sequence bis XmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin.
Wherein, the coupling reagent for the coupling reaction is a mixture of HOBt and DIC, or a mixture of PyAop, PyBop, HBTU, HATU and organic base. Wherein the organic base is one of DIPEA and NMM. If the combination of HOBt and DIC is adopted, the effect is better.
The molar ratio of the amino acid to the HOBt and the DIC is 1:1:1, and the coupling reaction in the step is detected and judged by an indantrione method to obtain the end point of each coupling reaction. If the resin is colorless and transparent, the reaction is complete; if the resin is developed, the reaction is not complete and recoupling is required.
Step 3) connecting the sequence II to Gly according to the sequence of the teduglutide peptide2Obtaining the sequence of tri Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) - (Phe-Ser (tBu) - (OtBu) - (Glu) (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) - (Trt) - (Leu-Ala-XmbAla-Arg (Pbf) - (OtBu) - (Phe-Ile-Asn (Trt) - (Trp) (Boc) -Leu-Ile-Gln (Trt) - (Thr tBu) - (Boc) -Ile-Thr) - (tBu) -Asp (OtBu) -Resin.
Wherein, the peptide sequence Asp3-Gly4 is formed by dipeptide Fmoc-Asp (OtBu)) - (Xmb) Gly-OH, and Fmoc-XmbGly-OH and Fmoc-Asp (OtBu)) -OH can be gradually connected.
Furthermore, the coupling reagent for the above coupling reaction is a mixture of HOBt and DIC, or a mixture of PyAop, PyBop, HBTU, HATU and an organic base. Wherein the organic base is one of DIPEA and NMM. If the combination of HOBt and DIC is adopted, the effect is better.
The molar ratio of the amino acid to the HOBt and the DIC is 1:1:1, and the coupling reaction in the step is detected and judged by an indantrione method to obtain the end point of each coupling reaction. If the resin is colorless and transparent, the reaction is complete; if the resin is developed, the reaction is not complete and recoupling is required.
Step 4) Synthesis of the sequence III with Fmoc-His (Trt) -OH in solid phase of the teduglutide peptide Resin His (Trt) -Gly-Asp (OtBu) - (Xmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-XmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr) -Asp (OtBu) -Resin.
The coupling reagent for the coupling reaction is DIC/HOBt, DIC/HOOBt, DIC/Oxyma or a mixture of HBTU, TDBTU and an organic base. Wherein the organic base is one of DIPEA and NMM. The condensation reagent is preferably selected from DIC/HOOBt.
The molar ratio of the amino acid to the HOOBt to the DIC is 1:1:1, and the coupling reaction in the steps is detected by an indetrione method to judge the end point of each coupling reaction. If the resin is colorless and transparent, the reaction is complete; if the resin is developed, the reaction is not complete and recoupling is required.
And 5) removing side chain protecting groups by acidolysis to obtain a crude product of the teduolutide, and purifying and freeze-drying the crude product to obtain a pure product of the teduolutide.
Specifically, adding fully protected teduolutide into a prepared cutting solution for reaction, slowly pouring the reaction solution into pre-cooled ether after cutting, stirring, standing to separate out the crude teduolutide peptide, centrifuging to remove the ether solution, washing the obtained solid with ether again, centrifuging, repeating twice, and finally vacuum drying to obtain the crude teduolutide.
The cracking reagent used in the step 5) comprises trifluoroacetic acid, thioanisole, phenol, 1, 2-ethanedithiol and water. More preferably, the ratio of lysis reagent is trifluoroacetic acid: 1, 2-ethanedithiol: triisopropylsilane: water 90:5:2.5: 2.5.
The amount of the cutting fluid used in the step 5) is 10 to 20 times by volume based on the weight of the resin, preferably 15 times.
The reaction temperature used in step 5) is preferably from 0 to 40 ℃ and in general 25 ℃.
The reaction time used in step 5) is 2 to 5 hours, and 2.5 hours is generally preferred.
The purification method used in step 5) is reversed phase high performance liquid chromatography.
The invention is further illustrated below with reference to examples.
Example 1: preparation of Fmoc-Asp (OtBu) -Wang Resin with a degree of substitution of 0.35mmol/g
Weighing 70.00g of blank Wang resin with a substitution degree of 0.79mmol/g and 16.28g of Fmoc-Asp (OtBu) -OH16, mixing, adding DCM for dissolving, adding 12.86mL of pyridine with 4 equivalents to amino acid, finally slowly adding 11.34mL of 2, 6-dichlorobenzoyl chloride, stirring for reaction for 2.5 hours, transferring to a reaction injection, removing reaction liquid, washing with DCM for 2 times, washing with DMF for 2 times, and then adding 1L of end-capping solution (Ac)2O NMM: DMF: 10:5:85), capping reaction for 30 min. The blocking solution was aspirated off, washed 3 times with DMF, 1 time with methanol, 3 times with DCM and then with methanolWashing for 3 times, draining the solvent, and drying in vacuum to obtain Fmoc-Asp (OtBu) -Wang Resin, wherein the detection substitution degree is 0.33 mmol/g.
Example 2: preparation of Fmoc-Asp (OtBu) -Wang Resin with a degree of substitution of 0.20mmol/g
Weighing 70.00g of blank Wang resin with a substitution degree of 0.79mmol/g and 8.14g of Fmoc-Asp (OtBu) -OH8.14g, mixing, adding DCM for dissolution, adding 6.43mL of pyridine with 4 equivalents to amino acid, finally slowly adding 5.67mL of 2, 6-dichlorobenzoyl chloride, stirring for reaction for 2.5 hours, transferring to a reaction injection, pumping out the reaction solution, washing with DCM for 2 times, washing with DMF for 2 times, and then adding 0.5L of end-capping solution (Ac)2O NMM: DMF: 10:6:84), capping reaction for 30 min. And (3) pumping out the blocking solution, washing with DMF for 3 times, washing with methanol for 1 time, washing with DCM for 3 times, then washing with methanol for 3 times, pumping out the solvent, and drying in vacuum to obtain Fmoc-Asp (OtBu) -Wang Resin, wherein the detection substitution degree is 0.22 mmol/g.
Example 3: preparation of Fmoc-Asp (OtBu) -Wang Resin with a degree of substitution of 0.50mmol/g
Weighing 70.00g of blank Wang resin with a substitution degree of 0.79mmol/g and 45.51g of Fmoc-Asp (OtBu) -OH45, mixing, adding DCM for dissolution, adding 35.60mL of pyridine with 4 equivalents to amino acid, finally slowly adding 31.95mL of 2, 6-dichlorobenzoyl chloride, stirring for reaction for 2.5 hours, transferring to a reaction injection, removing reaction liquid, washing with DCM for 2 times, washing with DMF for 2 times, and then adding 1L of end-capping solution (Ac)2O NMM: DMF: 10:6:84), capping reaction for 30 min. And (3) pumping out the blocking solution, washing with DMF for 3 times, washing with methanol for 1 time, washing with DCM for 3 times, then washing with methanol for 3 times, pumping out the solvent, and drying in vacuum to obtain Fmoc-Asp (OtBu) -Wang Resin, wherein the detection substitution degree is 0.48 mmol/g.
Example 4: preparation of Fmoc-Asp (OtBu) -CTC Resin with a degree of substitution of 0.55mmol/g
143g of CTC resin with a degree of substitution of 0.7mmol/g was weighed into a solid phase reaction column, washed 2 times with DMF and the resin was swollen with DMF for 30 minutes. 82.3g of Fmoc-Asp (OtBu) -OH (200mmol) is weighed and dissolved in DMF solution, and is added with 104.3mL of DIPEA (400mmol) under ice water bath for activation for 3min, and then is added into a solid phase reaction column for reaction at room temperature for 60 min. Washed 3 times with DMF and blocked for 8 hours by adding 81mL of blocking solution (methanol: 2000mmol) (DMF was added as solvent if the resin did not diffuse completely). Washing with DMF for 6 times, shrinking and draining methanol to obtain Fmoc-Asp (OtBu) -CTC resin. The degree of substitution was determined to be 0.50 mmol/g.
Example 5: preparation of Fmoc-Asp (OtBu) -CTC Resin with a degree of substitution of 0.20mmol/g
285g of CTC resin with a substitution degree of 0.35mmol/g was weighed into a solid phase reaction column, washed 2 times with DMF, and the resin was swollen with DMF for 30 minutes. 82.3g of Fmoc-Asp (OtBu) -OH (200mmol) is weighed and dissolved in DMF solution, and is added with 104.3mL of DIPEA (400mmol) under ice water bath for activation for 3min, and then is added into a solid phase reaction column for reaction at room temperature for 60 min. Washed 3 times with DMF and blocked for 8 hours by adding 81mL of blocking solution (methanol: 2000mmol) (DMF was added as solvent if the resin did not diffuse completely). Washing with DMF for 6 times, shrinking and draining methanol to obtain Fmoc-Asp (OtBu) -CTC resin. The degree of substitution was determined to be 0.25 mmol/g.
Example 6: preparation of Fmoc-Asp (OtBu) -CTC Resin with a degree of substitution of 0.35mmol/g
222g of CTC resin with a substitution degree of 0.45mmol/g was weighed into a solid phase reaction column, washed 2 times with DMF, and the resin was swollen with DMF for 30 minutes. 82.3g of Fmoc-Asp (OtBu) -OH (200mmol) is weighed and dissolved in DMF solution, and is added with 104.3mL of DIPEA (400mmol) under ice water bath for activation for 3min, and then is added into a solid phase reaction column for reaction at room temperature for 60 min. Washed 3 times with DMF and blocked for 8 hours by adding 81mL of blocking solution (methanol: 2000mmol) (DMF was added as solvent if the resin did not diffuse completely). Washing with DMF for 6 times, shrinking and draining methanol to obtain Fmoc-Asp (OtBu) -CTC resin. The detection substitution degree was 0.36 mmol/g.
Example 7 Fmoc-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Asn
Preparation of Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Fmoc-Asp (OtBu) -Wang resin60.61g (20mmol) with a degree of substitution of 0.33mmol/g was weighed into the solid phase reaction mixture, washed 2 times with DMF and the resin was swollen with DMF for 1.5 h. Adding 20% piperidine solution with a volume being three times that of the resin into DMF for reaction for 30min to remove Fmoc protection, pumping out the deprotection solution, and then adding DMF to wash the resin for 5 times. 23.86g of Fmoc-Thr (tBu) -OH (60mmol) and 8.12g of HOBt (60mmol) were weighed out and dissolved in 400mL of DMF, and 9.30mL of DIC (60mmol) were added to the mixture in an ice-water bath for activation for 10min, followed by addition to the solid phase reaction column and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, sequentially completing coupling of 20-33 fragments from the C end to the N end according to the backbone peptide sequence of the teduotide, shrinking by using methanol after the reaction is finished, and drying the resin in vacuum overnight to obtain the teduotide fragment Fmoc- [20-33] -WangResin 122.82g.
Example 8 Fmoc-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Asn
Preparation of Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Fmoc-Asp (OtBu) -CTC Resin55.56g (20mmol) with a degree of substitution of 0.36mmol/g was weighed into the solid phase reaction mixture, washed 2 times with DMF and the resin was swollen with DMF for 1.5 h. Adding 20% piperidine solution with a volume being three times that of the resin into DMF for reaction for 30min to remove Fmoc protection, pumping out the deprotection solution, and then adding DMF to wash the resin for 5 times. 23.86g of Fmoc-Thr (tBu) -OH (60mmol) and 8.12g of HOBt (60mmol) were weighed out and dissolved in 400mL of DMF, and 9.30mL of DIC (60mmol) were added to the mixture in an ice-water bath for activation for 10min, followed by addition to the solid phase reaction column and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, sequentially completing coupling of 20-33 fragments from the C end to the N end according to the backbone peptide sequence of the teduotide, shrinking by using methanol after the reaction is finished, and drying the resin in vacuum overnight to obtain the teduotide fragment Fmoc- [20-33] -CTCResin 112.57g.
Example 9: Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Asn
Preparation of Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 4.06g of HOBt (30mmol) were dissolved in 200mL of DMF, and 4.70mL of DIC (30mmol) were added to the resulting solution in an ice-water bath to activate the solution for 10min, followed by addition to the solid phase reaction column and reaction at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -WangResin was obtained.
Example 10: Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc)
Preparation of-Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 4.06g of HOBt (30mmol) were dissolved in 200mL of DMF, and 4.70mL of DIC (30mmol) were added to the resulting solution in an ice-water bath to activate the solution for 10min, followed by addition to the solid phase reaction column and reaction at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTCResin was obtained.
Example 11: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 4.08g of HOAt (30mmol) were weighed out and dissolved in 200mL of DMF, and activated by adding 4.70mL of DIC (30mmol) in an ice-water bath for 10min, and then introduced into a solid-phase reaction column and reacted at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -WangResin was obtained.
Example 12: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 4.08g of HOAt (30mmol) were weighed out and dissolved in 200mL of DMF, and activated by adding 4.70mL of DIC (30mmol) in an ice-water bath for 10min, and then introduced into a solid-phase reaction column and reacted at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTCResin was obtained.
Example 13: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 15.64g of PyAOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 14: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 15.64g of PyAOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 15: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 15.61g of PyBOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 16: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 15.61g of PyBOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 17: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 11.38g of HBTU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 18: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 11.38g of HBTU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 19: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 11.41g of HATU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 20: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 11.41g of HATU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 21: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 15.64g of PyAOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 22: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 15.64g of PyAOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 23: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 15.61g of PyBOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 24: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 15.61g of PyBOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 25: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 11.38g of HBTU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 26: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 11.38g of HBTU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 27: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 11.41g of HATU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 28: preparation of Fmoc-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-HmbAla-OH (30mmol) and 11.41g of HATU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 29: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
The Fmoc- [20-33] -Wang Resin10mmol obtained in example 4 was weighed and added to a solid phase reactor, three Resin volumes of 20% piperidine in DMF was added for reaction for 30min to remove the Fmoc protection, and DMF was added again to wash the Resin5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 4.06g of HOBt (30mmol) were dissolved in DMF, activated by adding 4.7mL of DIC (30mmol) in an ice water bath for 10min, and then introduced into a solid phase reaction column to react at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 30: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
The Fmoc- [20-33] -Wang Resin10mmol obtained in example 4 was weighed and added to a solid phase reactor, three Resin volumes of 20% piperidine in DMF was added for reaction for 30min to remove the Fmoc protection, and DMF was added again to wash the Resin5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 4.06g of HOBt (30mmol) were dissolved in DMF, activated by adding 4.7mL of DIC (30mmol) in an ice water bath for 10min, and then introduced into a solid phase reaction column to react at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 31: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
The Fmoc- [20-33] -CTC Resin10mmol obtained in example 4 was weighed into a solid phase reactor, added with 20% piperidine solution in DMF three times the Resin volume for reaction for 30min to remove the Fmoc protection, and added with DMF again to wash the Resin5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 4.06g of HOBt (30mmol) were dissolved in DMF, activated by adding 4.7mL of DIC (30mmol) in an ice water bath for 10min, and then introduced into a solid phase reaction column to react at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 32: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
The Fmoc- [20-33] -Wang Resin10mmol obtained in example 4 was weighed and added to a solid phase reactor, three Resin volumes of 20% piperidine in DMF was added for reaction for 30min to remove the Fmoc protection, and DMF was added again to wash the Resin5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 4.08g of HOAt (30mmol) were dissolved in DMF, and after activation by 4.7mL of DIC (30mmol) in an ice-water bath for 10min, the mixture was loaded onto a solid-phase reaction column and reacted at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 33: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
The Fmoc- [20-33] -CTC Resin10mmol obtained in example 4 was weighed into a solid phase reactor, added with 20% piperidine solution in DMF three times the Resin volume for reaction for 30min to remove the Fmoc protection, and added with DMF again to wash the Resin5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 4.08g of HOAt (30mmol) were dissolved in DMF, and after activation by 4.7mL of DIC (30mmol) in an ice-water bath for 10min, the mixture was loaded onto a solid-phase reaction column and reacted at room temperature for 6 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 34: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 15.64g of PyAOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 35: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 15.64g of PyAOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 36: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 15.61g of PyBOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 37: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 15.61g of PyBOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 38: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 11.38g of HBTU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 39: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 11.38g of HBTU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 40: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 11.41g of HATU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 41: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 11.41g of HATU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 9.92mL of DIPEA (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 42: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 15.64g of PyAOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 43: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 15.64g of PyAOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 44: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 15.61g of PyBOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 45: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 15.61g of PyBOP (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 46: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 11.38g of HBTU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 47: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 11.38g of HBTU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 48: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -Wang Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 11.41g of HATU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 3.30mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -Wang Resin was obtained.
Example 49: preparation of Fmoc-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -CTC Resin
Weighing 10mmol of teduglutide fragment Fmoc- [20-33] -CTC Resin, adding the teduglutide fragment into a solid phase reactor, adding a DMF solution of 20 percent piperidine with the volume being three times that of the Resin, reacting for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.82g of Fmoc-DmbAla-OH (30mmol) and 11.41g of HATU (30mmol) were added to the resin, 200mL of DMF was added to dissolve the solids, and after stirring for 5min, 3.30mL of NMM (60mmol) were added dropwise and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Fmoc- [19-33] -CTC Resin was obtained.
Example 50: Fmoc-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmbal-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr-tBu) -Asp (OtBu) -Wang Resin preparation of amino acid
Weighing 10mmol of Fmoc- [19-33] -Wang Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with three Resin volumes into the solid phase reaction column for reaction for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 9.34g of Fmoc-Ala-OH (30mmol), 4.05g of HOBt (30mmol) were weighed out and dissolved in 200mL of DMF, and after activation by 4.7mL of DIC (30mmol) in an ice-water bath for 10min, the mixture was reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 5-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [5-33] -Wang Resin.
Example 51: Fmoc-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmbal-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Lys) (Ile-Thr tBu) -Asp (OtBu) -CTC Resin preparation of
Weighing 10mmol of Fmoc- [19-33] -CTC Resin, adding the Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column to react for 30min, removing Fmoc protection, and adding DMF again to wash the Resin for 5 times. 9.34g of Fmoc-Ala-OH (30mmol), 4.05g of HOBt (30mmol) were weighed out and dissolved in 200mL of DMF, and after activation by 4.7mL of DIC (30mmol) in an ice-water bath for 10min, the mixture was reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 5-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [5-33] -CTC Resin.
Example 52: Fmoc-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Lys) (Ile-Thr-tBu) -Asp (OtBu) -Wang Resin preparation of amino acid
Weighing 10mmol of Fmoc- [19-33] -Wang Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with three Resin volumes into the solid phase reaction column for reaction for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 9.34g of Fmoc-Ala-OH (30mmol), 4.05g of HOBt (30mmol) were weighed out and dissolved in 200mL of DMF, and after activation by 4.7mL of DIC (30mmol) in an ice-water bath for 10min, the mixture was reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 5-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [5-33] -Wang Resin.
Example 53: Fmoc-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-DmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Lys) (Ile-Thr tBu) -Asp (OtBu) -CTC Resin preparation of
Weighing 10mmol of Fmoc- [19-33] -CTC Resin, adding the Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column to react for 30min, removing Fmoc protection, and adding DMF again to wash the Resin for 5 times. 9.34g of Fmoc-Ala-OH (30mmol), 4.05g of HOBt (30mmol) were weighed out and dissolved in 200mL of DMF, and after activation by 4.7mL of DIC (30mmol) in an ice-water bath for 10min, the mixture was reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 5-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [5-33] -CTC Resin.
Example 54: Fmoc-Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) - (OtBu) - (Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Waring-restoring in preparation of
Weighing 10mmol of Fmoc- [5-33] -Wang Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with three Resin volumes into the solid phase reaction column for reaction for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.13g of Fmoc-Asp (OtBu) -HmbGly-OH (30mmol) and 4.05g of HOBt (30mmol) were weighed out and dissolved in 200mL of DMF, and 4.7mL of DIC (30mmol) were added under ice-water bath for activation for 10min, followed by solid phase reaction and reaction at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 2-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [2-33] -Wang Resin.
Example 55: Fmoc-Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) - (OtBu) - (Glu) (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) - (Trt) -Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) - (Trp) (Boc) -Leu-Ile-Gln (Trt) - (Thr tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC Resin preparation of
Weighing 10mmol of Fmoc- [5-33] -CTC Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with the volume being three times that of the Resin into the solid phase reaction column to react for 30min, removing the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.13g of Fmoc-Asp (OtBu) -HmbGly-OH (30mmol) and 4.05g of HOBt (30mmol) were weighed out and dissolved in 200mL of DMF, and 4.7mL of DIC (30mmol) were added under ice-water bath for activation for 10min, followed by solid phase reaction and reaction at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 2-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [2-33] -CTC Resin.
Example 56: Fmoc-Gly-Asp (OtBu) - (Dmb) Gly-Ser (tBu) -Phe-Ser (tBu) - (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) -Asn (Trt) - (Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) - (Trp) (Boc) -Leu-Ile-Gln (Trt) - (Thr tBu) - (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of Fmoc- [5-33] -Wang Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with three Resin volumes into the solid phase reaction column for reaction for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.55g of Fmoc-Asp (OtBu) -DmbGly-OH (30mmol) and 4.05g of HOBt (30mmol) were weighed out and dissolved in 200mL of DMF, and 4.7mL of DIC (30mmol) were added under ice-water bath for activation for 10min, followed by solid phase reaction and reaction at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 2-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [2-33] -Wang Resin.
Example 57: Fmoc-Gly-Asp (OtBu) - (Dmb) Gly-Ser (tBu) -Phe-Ser (tBu) - (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) -Asn (Trt) - (Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) - (Trp) (Boc) -Leu-Ile-Gln (Trt) - (Thr tBu) - (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC Resin preparation of
Weighing 10mmol of Fmoc- [5-33] -CTC Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with the volume being three times that of the Resin into the solid phase reaction column to react for 30min, removing the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.55g of Fmoc-Asp (OtBu) -DmbGly-OH (30mmol) and 4.05g of HOBt (30mmol) were weighed out and dissolved in 200mL of DMF, and 4.7mL of DIC (30mmol) were added under ice-water bath for activation for 10min, followed by solid phase reaction and reaction at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 2-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [2-33] -CTC Resin.
Example 58: Fmoc-Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) - (Glu) (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of Fmoc- [5-33] -Wang Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with three Resin volumes into the solid phase reaction column for reaction for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.01g of Fmoc-HmbGly-OH (30mmol) and 4.05g of HOBt (30mmol) are weighed and dissolved in 200mL of DMF, and after activation by adding 4.7mL of LDIC (30mmol) in an ice water bath for 10min, the mixture is added into a solid phase reaction column and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 2-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [2-33] -Wang Resin.
Example 59: Fmoc-Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) - (OtBu) - (Glu) (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) - (Trt) -Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) - (Trp) (Boc) -Leu-Ile-Gln (Trt) - (Thr tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC Resin preparation of
Weighing 10mmol of Fmoc- [5-33] -CTC Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with the volume being three times that of the Resin into the solid phase reaction column to react for 30min, removing the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.01g of Fmoc-HmbGly-OH (30mmol) and 4.05g of HOBt (30mmol) are weighed and dissolved in 200mL of DMF, and after activation by adding 4.7mL of LDIC (30mmol) in an ice water bath for 10min, the mixture is added into a solid phase reaction column and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 2-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [2-33] -CTC Resin.
Example 60: Fmoc-Gly-Asp (OtBu) - (Dmb) Gly-Ser (tBu) -Phe-Ser (tBu) - (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) -Asn (Trt) - (Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) - (Trp) (Boc) -Leu-Ile-Gln (Trt) - (Thr tBu) - (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin
Weighing 10mmol of Fmoc- [5-33] -Wang Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with three Resin volumes into the solid phase reaction column for reaction for 30min to remove the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-DmbGly-OH (30mmol) and 4.05g of HOBt (30mmol) are weighed, dissolved in 200mL of DMF, activated for 10min by adding 4.7mL of LDIC (30mmol) in an ice-water bath, added into a solid phase reaction column and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. And repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 2-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [2-33] -Wang Resin.
Example 61: Fmoc-Gly-Asp (OtBu) - (Dmb) Gly-Ser (tBu) -Phe-Ser (tBu) - (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) - (Trt) -Leu-Ala-DmbAla-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) - (Trp) (Boc) -Leu-Ile-Gln (Trt) - (Thr) (tBu) - (Lys (Boc) -Ile-T hr (tBu) -Asp OtBu) -CTC Resin
Weighing 10mmol of Fmoc- [5-33] -CTC Resin, adding the Fmoc Resin into a solid phase reaction column, adding 20 percent piperidine solution with the volume being three times that of the Resin into the solid phase reaction column to react for 30min, removing the Fmoc protection, and adding DMF again to wash the Resin for 5 times. 13.42g of Fmoc-DmbGly-OH (30mmol) and 4.05g of HOBt (30mmol) are weighed, dissolved in 200mL of DMF, activated for 10min by adding 4.7mL of LDIC (30mmol) in an ice-water bath, added into a solid phase reaction column and reacted at room temperature for 4 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing the coupling of 2-18 fragments from the C end to the N end according to the peptide sequence of the backbone of the teduotide to obtain Fmoc- [2-33] -CTC Resin.
Example 62: H-His (Trt) -Gly-Asp (OtBu) -HmbGly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn Trt-Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin preparation
Adding Fmoc- [2-33] -Wang Resin10mmol into a solid phase reaction column, adding 20% piperidine solution with three Resin volumes in DMF for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 4.05g of HOBt (30mmol) were weighed out and dissolved in 300mL of DMF, and activated for 10min by adding 4.7mL of LDIC (30mmol) in an ice-water bath, followed by addition to the solid phase reaction column and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times to obtain 93.12g of H- [1-33] -Wang Resin.
Example 63: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC-Resin preparation
Adding 10mmol of Fmoc- [2-33] -CTC Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 4.05g of HOBt (30mmol) were weighed out and dissolved in 300mL of DMF, and activated for 10min by adding 4.7mL of LDIC (30mmol) in an ice-water bath, followed by addition to the solid phase reaction column and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times to obtain 87.20g of H- [1-33] -CTC Resin (teduglutide peptide Resin).
Example 64: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin preparation of
Adding Fmoc- [2-33] -Wang Resin10mmol into a solid phase reaction column, adding 20% piperidine solution with three Resin volumes in DMF for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 4.89g of HOOBt (30mmol) were weighed out and dissolved in 300mL of DMF, and activated by adding 4.7mL of DIC (30mmol) in an ice-water bath for 10min, followed by addition to the solid phase reaction mixture and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, shrinking with methanol, and vacuum drying the peptide Resin overnight to obtain 94.02g of H- [1-33] -Wang Resin.
Example 65: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC-Resin preparation
Adding 10mmol of Fmoc- [2-33] -CTC Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 4.89g of HOOBt (30mmol) were weighed out and dissolved in 300mL of DMF, and activated by adding 4.7mL of DIC (30mmol) in an ice-water bath for 10min, followed by addition to the solid phase reaction mixture and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF three times the volume of the Resin for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, then shrinking with methanol, and drying the peptide Resin in vacuum overnight to obtain 88.51g of H- [1-33] -CTC Resin (teduolutide peptide Resin).
Example 66: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin preparation of
Adding Fmoc- [2-33] -Wang Resin10mmol into a solid phase reaction column, adding 20% piperidine solution with three Resin volumes in DMF for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 4.26g of Oxyma (30mmol) were weighed out and dissolved in 300mL of DMF, and activated for 10min by adding 4.7mL of LDIC (30mmol) in an ice-water bath, followed by addition to the solid phase reaction column and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, shrinking with methanol, and vacuum drying the peptide Resin overnight to obtain 95.68g of H- [1-33] -Wang Resin.
Example 67: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC-Resin preparation
Adding 10mmol of Fmoc- [2-33] -CTC Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 4.26g of Oxyma (30mmol) were weighed out and dissolved in 300mL of DMF, and activated for 10min by adding 4.7mL of LDIC (30mmol) in an ice-water bath, followed by addition to the solid phase reaction column and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF three times the volume of the Resin for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, then shrinking by methanol, and drying the peptide Resin in vacuum overnight to obtain 86.09g of H- [1-33] -CTC Resin (teduolutide peptide Resin).
Example 68: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin preparation of
Adding Fmoc- [2-33] -Wang Resin10mmol into a solid phase reaction column, adding 20% piperidine solution with three Resin volumes in DMF for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 11.38g of HBTU (30mmol) were weighed out and added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) was added dropwise and reacted at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, shrinking with methanol, and vacuum drying the peptide Resin overnight to obtain 90.05g of H- [1-33] -Wang Resin.
Example 69: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC-Resin preparation
Adding 10mmol of Fmoc- [2-33] -CTC Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 11.38g of HBTU (30mmol) were weighed out and added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) was added dropwise and reacted at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF three times the volume of the Resin for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, then shrinking with methanol, and drying the peptide Resin in vacuum overnight to obtain 84.70g of H- [1-33] -CTC Resin (teduolutide peptide Resin).
Example 70: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin preparation of
Adding Fmoc- [2-33] -Wang Resin10mmol into a solid phase reaction column, adding 20% piperidine solution with three Resin volumes in DMF for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol), 10.47g of TDBTU (30mmol) were weighed out and added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) was added dropwise and reacted at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, shrinking with methanol, and vacuum drying the peptide Resin overnight to obtain 98.63g of H- [1-33] -Wang Resin.
Example 71: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC-Resin preparation
Adding 10mmol of Fmoc- [2-33] -CTC Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol), 10.47g of TDBTU (30mmol) were weighed out and added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 9.92mL of DIPEA (60mmol) was added dropwise and reacted at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF three times the volume of the Resin for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, then shrinking with methanol, and drying the peptide Resin in vacuum overnight to obtain 78.43g of H- [1-33] -CTC Resin (teduolutide peptide Resin).
Example 72: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin preparation of
Adding Fmoc- [2-33] -Wang Resin10mmol into a solid phase reaction column, adding 20% piperidine solution with three Resin volumes in DMF for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 11.38g of HBTU (30mmol) were weighed out and added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) was added dropwise and reacted at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, shrinking with methanol, and vacuum drying the peptide Resin overnight to obtain 91.21g of H- [1-33] -Wang Resin.
Example 73: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC-Resin preparation
Adding 10mmol of Fmoc- [2-33] -CTC Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 11.38g of HBTU (30mmol) were weighed out and added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) was added dropwise and reacted at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF three times the volume of the Resin for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, then shrinking with methanol, and drying the peptide Resin in vacuum overnight to obtain 87.55g of H- [1-33] -CTC Resin (teduolutide peptide Resin).
Example 74: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin preparation of
Adding Fmoc- [2-33] -Wang Resin10mmol into a solid phase reaction column, adding 20% piperidine solution with three Resin volumes in DMF for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 10.47g of TDBTU (30mmol) were weighed out and added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) was added dropwise and reacted at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, shrinking with methanol, and vacuum drying the peptide Resin overnight to obtain 89.98g of H- [1-33] -Wang Resin.
Example 75: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Hmba a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -CTC-Resin preparation
Adding 10mmol of Fmoc- [2-33] -CTC Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 10.47g of TDBTU (30mmol) were weighed out and added to the resin, 200mL of DMF was added to dissolve the solid, and after stirring for 5min, 6.60mL of NMM (60mmol) was added dropwise and reacted at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, shrinking with methanol, and vacuum drying the peptide Resin overnight to obtain 75.67g of H- [1-33] -CTC Resin.
Example 76: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Wang Resin preparation of
Adding Fmoc- [2-33] -Wang Resin10mmol into a solid phase reaction column, adding 20% piperidine solution with three Resin volumes in DMF for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 4.89g of HOOBt (30mmol) were weighed out and dissolved in 300mL of DMF, and activated by adding 4.7mL of DIC (30mmol) in an ice-water bath for 10min, followed by addition to the solid phase reaction mixture and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF of three Resin volumes for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, shrinking with methanol, and vacuum drying the peptide Resin overnight to obtain 93.77g of H- [1-33] -Wang Resin.
Example 77: H-His (Trt) -Gly-Asp (OtBu) - (Hmb) Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-DmbAl a-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Leu-Ile-Gln) (Trt) -Thr (tBu) -Lys (Boc) -Ile-T hr (tBu) -Asp (OtBu) -Boc-Resin preparation
Adding 10mmol of Fmoc- [2-33] -CTC Resin into a solid phase reaction column, adding 20 percent of piperidine solution with the volume being three times that of the Resin into the solid phase reaction column for reaction for 30min to remove Fmoc protection, and adding DMF again to wash the Resin for 5 times. 18.59g of Fmoc-His (Trt) -OH (30mmol) and 4.89g of HOOBt (30mmol) were weighed out and dissolved in 300mL of DMF, and activated by adding 4.7mL of DIC (30mmol) in an ice-water bath for 10min, followed by addition to the solid phase reaction mixture and reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method. After the reaction, the reaction solution was drained and DMF was added to wash the resin 3 times. Then adding 20% piperidine solution in DMF three times the volume of the Resin for reaction for 30min to remove Fmoc protection, adding DMF again to wash the Resin for 5 times, then shrinking by methanol, and drying the peptide Resin in vacuum overnight to obtain 89.82g of H- [1-33] -CTC Resin (teduolutide peptide Resin).
Example 78: preparation of crude peptide of teduglutide
94.02g H-His (Trt) -Gly-Asp (OtBu) -HmbGly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-HmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr) (tBu) -Asp (OtBu) -Wang Resin prepared in example 64 was placed in a cleavage reactor, and cleavage reagent (trifluoroacetic acid: 1, 2-ethanedithiol: triisopropylsilane: water: 5:2.5: V/5) V/V (V/V) was added at a ratio of 15mL/g Resin, stir at rt for 2.5 h. The reaction was filtered through a sand funnel, the filtrate was collected, the resin was washed 3 times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. Adding frozen anhydrous ether for precipitation, collecting precipitate, washing with anhydrous ether for 3 times, and vacuum drying to obtain white powder solid, i.e. crude peptide of teduglutide 41.82g with HPLC purity of 73.58%.
Example 79: preparation of crude peptide of teduglutide
88.51g H-His (Trt) -Gly-Asp (OtBu) -HmbGly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln-Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin prepared in example 65 was placed in a cleavage reactor, cleavage reagent (trifluoroacetic acid: 1, 2-ethanedithiol: triisopropylsilane: water: (5: 2.5: V/5) was added to the cleavage reactor at a ratio of 15mL/g Resin, stir at rt for 2.5 h. The reaction was filtered through a sand funnel, the filtrate was collected, the resin was washed 3 times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. Adding frozen anhydrous ether for precipitation, collecting precipitate, washing with anhydrous ether for 3 times, and vacuum drying to obtain white powder solid, i.e. crude peptide 37.42g of teduglutide with HPLC purity of 74.82%.
Example 80: preparation of crude peptide of teduglutide
93.77g H-His (Trt) -Gly-Asp (OtBu) -HmbGly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-DbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr) (tBu) -Asp (OtBu) -Wang Resin the case of preparation in example 76, a cleavage reactor was charged with a cleavage reagent (trifluoroacetic acid: 1, 2-ethanedithiol: triisopropylsilane: water: 5:2.5: V/5, V/V) (V5, V/g Resin, 5, V, 5, V, and 5, and the cleavage reagent was added to the resulting mixture in, stir at rt for 2.5 h. The reaction was filtered through a sand funnel, the filtrate was collected, the resin was washed 3 times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. Adding frozen anhydrous ether for precipitation, collecting precipitate, washing with anhydrous ether for 3 times, and vacuum drying to obtain white powder solid, i.e. crude peptide 40.98g of teduglutide with HPLC purity of 70.43%.
Example 81: preparation of crude peptide of teduglutide
89.82g H-His (Trt) -Gly-Asp (OtBu) -HmbGly-Ser (tBu) -Phe-Ser (tBu) -Asp (OtBu) -Glu (OtBu) -Met-Asn (Trt) -Thr (tBu) -Ile-Leu-Asp (OtBu) -Asn (Trt) -Leu-Ala-DbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr) (tBu) -Asp (OtBu) -Asn (Trt) -Trp (Boc) -Leu-Ile-Gln) (Trt) -Thr (Thr) and Lys (Boc) -Ile-Thr) (OtBu) -Asp (Trt-Ile) -Tr) prepared in example 77 was placed in a cleavage reactor CTC, and cleavage reagent (trifluoroacetic acid: 1, 2-dithiol: triisopropylsilane: water, stir at rt for 2.5 h. The reaction was filtered through a sand funnel, the filtrate was collected, the resin was washed 3 times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. Adding frozen anhydrous ether for precipitation, collecting precipitate, washing with anhydrous ether for 3 times, and vacuum drying to obtain white powder solid, i.e. crude peptide of teduglutide 36.85g with HPLC purity of 73.89%.
Example 82: preparation of teduglutide fine peptide acetate
Weighing any 20.0g of the crude teduglutide peptide in the example 78-81, dissolving the crude teduglutide peptide in 2000mL of water, purifying by a Waters2545RP-HPLC system with the wavelength of 230nm and a chromatographic column of 50 multiplied by 250mm reversed phase C18 through a conventional 0.2% TFA/acetonitrile mobile phase, and collecting target peak fractions to obtain the refined peptide with the purity of more than 98.5%. And (3) adopting a Waters2545RP-HPLC system to obtain a refined peptide solution, wherein a chromatographic column is a 50 multiplied by 250mm reversed phase C18 column, 0.1% acetic acid solution/acetonitrile mobile phase is used for converting salt, a target peak fraction is collected, and the refined peptide solution is lyophilized to obtain more than or equal to 7.0g of the teduglutide acetate refined peptide, the RP-HPLC purity is more than or equal to 98.50%, and Asp degradation impurities are less than 0.16%.
The above examples are given solely to aid in the understanding of the methods of the present invention and the core concepts thereof. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (10)
1. A method for preparing teduglutide based on a solid phase step-by-step method is characterized by comprising the following steps:
step 1), under the action of a coupling reagent, connecting 20-33 fragments one by using a solid-phase synthesis method according to a teduglutide sequence to obtain a synthesized fully-protected sequence I: arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin;
and 2) synthesizing the sequence I and Fmoc-HmbAla-OH or Fmoc-DmbAla-OH by a solid-phase synthesis method under the action of a coupling reagent to obtain a sequence II: XmbAla-Arg (Pbf) -Asp (OtBu) -Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-Ile-Gln (Trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin;
and 3) synthesizing a third sequence by a solid-phase synthesis method according to the sequence of the teduglutide under the action of a coupling reagent: Gly-Asp (OtBu) - (Xmb) Gly-Ser (tBu) - (Phe-Ser (tBu) - (OtBu) - (Glu), (OtBu) - (Met-Asn (Trt) - (tBu) - (Ile-Leu-Asp (OtBu) - (Trt) - (Leu-Ala-XmbAla-Arg (Pbf) -Asp (OtBu) - (Phe-Ile-Asn (Trt) - (Boc) - (Trp) (Leu-Ile-Gln (Trt) - (Thr) - (tBu) - (Boc) -Ile-Thr (tBu) -Asp (OtBu) -Resin;
step 4), carrying out coupling reaction on the sequence III and Fmoc-His (Trt) -OH, and removing an Fmoc protecting group to obtain the teduglutide peptide resin;
step 5), removing side chain protecting groups by acidolysis to obtain a crude product of the teduglutide;
and 6) purifying and freeze-drying the crude product of the teduolutide to obtain a pure product of the teduolutide, and completing the synthesis of the teduolutide.
2. The method as claimed in claim 1, wherein the Asp3-Gly4 sequence in step 3) is selected from the group consisting of dipeptide Fmoc-Asp (OtBu) -XmbGly-OH; or step-wise linked using Fmoc-XmbGly-OH and Fmoc-Asp (OtBu) -OH. Wherein Xmb is Hmb or Dmb.
3. The method of claim 1, wherein the resin used in step 1) to synthesize sequence one is Wang resin or CTC resin; wherein the degree of substitution of the initial peptide Resin Fmoc-Asp (OtBu) -Wang Resin is 0.22-0.48 mmol/g; the initial peptide Resin has a degree of substitution of Fmoc-Asp (OtBu) -CTC Resin of 0.25-0.50 mmol/g.
4. The method according to claim 1, wherein the side chain protecting group used in His in the steps 1), 3) or 4) is trityl, the side chain protecting group used in Asp is tert-butoxy, the side chain protecting group used in Ser is tert-butyl, the side chain protecting group used in Glu is tert-butoxy, the side chain protecting group used in Asn is trityl, the side chain protecting group used in Thr is tert-butyl, the side chain protecting group used in Arg is 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl, the side chain protecting group used in Trp is tert-butoxycarbonyl, the side chain protecting group used in Gln is trityl, and the side chain protecting group used in Lys is tert-butoxycarbonyl.
5. The method of claim 1, wherein the second 19 th amino acid in the sequence of step 2) is Fmoc-HmbAla-OH or Fmoc-DmbAla-OH.
6. The process of claim 1, wherein the coupling reagent for coupling in steps 1), 2), 3) is DIC/HOBt, DIC/HOAt, or a mixture of one of PyAop, PyBop, HBTU, and HATU with an organic base; wherein the organic base is one of DIPEA and NMM.
7. The process of claim 1, wherein the coupling reagent used in step 4) is DIC/HOBt, DIC/HOOBt, DIC/Oxyma or a mixture of HBTU and TDBTU with an organic base; wherein the organic base is one of DIPEA and NMM.
8. The method as claimed in claim 1, wherein the acidolysis reagent used in step 5) comprises trifluoroacetic acid, triisopropylsilane, thioanisole, 1, 2-ethanedithiol, and water.
9. The method of claim 6 or 7, wherein the ratio of the coupling reagent to the base is 1: 2.
10. The process of claim 1, wherein the ratio of the acidolysis reagents used in step 5) is trifluoroacetic acid: 1, 2-ethanedithiol: triisopropylsilane: water 90:5:2.5: 2.5; the Fmoc removal method used in the steps 1), 2), 3) and 4) is a piperidine method or a diethylamine method, and a 20% piperidine/DMF solution is used.
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