CN105753964A - Preparation method of semaglutide and intermediate of semaglutide - Google Patents
Preparation method of semaglutide and intermediate of semaglutide Download PDFInfo
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- CN105753964A CN105753964A CN201410784161.4A CN201410784161A CN105753964A CN 105753964 A CN105753964 A CN 105753964A CN 201410784161 A CN201410784161 A CN 201410784161A CN 105753964 A CN105753964 A CN 105753964A
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- WSTNFGAKGUERTC-UHFFFAOYSA-N CCCCCCNCC Chemical compound CCCCCCNCC WSTNFGAKGUERTC-UHFFFAOYSA-N 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention belongs to the technical field of pharmaceutical chemistry, and discloses a preparation method of semaglutide and an intermediate of semaglutide. The preparation method comprises: obtaining a compound having a structure represented as the formula I by taking a resin, activating the resin, and coupling L-glycine, L-arginine, L-glycine, L-arginine, L-valine, L- leucine, L-tryptophan, L-alanine, L-isoleucine, L-phenylalanine, and L-glutamic acid to the activated resin step by step so as to obtain a first peptide resin; taking the first peptide resin, and coupling amino acids or amino acid derivatives to the second peptide resin step by step; and taking the second peptide resin, and performing cracking and purifying to obtain semaglutide. According to the method, the compound having a structure represented as the formula I is taken as an intermediate and coupling of each amino acid or amino acid derivative step by step through solid-phase synthesis is carried out, thereby improving the yield of semaglutide. In the formula I, R1 is selected from Fmoc, Boc, and ivDde, and R2 is selected from tBu, Dmab, and Bzl.
Description
Technical field
The invention belongs to field of pharmaceutical chemistry technology, particularly to the Preparation Method And Their Intermediate of a kind of Sa Molu peptide.
Background technology
Diabetes are a kind of because of a series of clinical syndromes that internal insulin is absolute or relative deficiency causes, have very close associating with gene.The main clinical manifestation of diabetes is polydipsia, polyuria, polyphagia and weight loss, and containing glucose etc. in blood glucose height, urine.Diabetes are divided into four kinds of types: type Ⅰ diabetes mellitus, type Ⅱdiabetes mellitus, other types diabetes and gestational diabetes.Any kind of diabetes all can cause that the β cell in pancreas can not produce enough insulins to drop hypoglycemic concentration, causes the generation of hyperglycemia.World Health Organization's report of 2011 points out that the whole world has 3.46 hundred million people to suffer from diabetes, and estimation in 2004 has 3,400,000 people to die from the disease that hyperglycemia causes, and the Diabetes Death more than 80% occurs in low income and middle income country.
Type Ⅱdiabetes mellitus, it is once called as non-insulin-dependent diabetes mellitus (NIDDM) or Adult Onset's patients with type Ⅰ DM (adult-onsetdiabetes), it is a kind of metabolic disease, it is characterized by hyperglycemia, mainly relatively lacked by insulin resistant and insulin and cause.Wherein, patients with NIDDM accounts in diabetics about 90%, and all the other 10% are mainly type Ⅰ diabetes mellitus and gestational diabetes, so the research and development tool for the medicine of type Ⅱdiabetes mellitus is of great significance.
Antidiabetic drug kind for type Ⅱdiabetes mellitus is a lot, and the receptor agonism element of GLP-1 (GLP-1) is the focus of Recent study.Wherein, Sa Molu peptide (semaglutide) is one of receptor agonism element of GLP-1, and this medicine is developed by Novo Nordisk Co., Ltd of Denmark.Sa Molu peptide as one expendable subcutaneous injection formulation weekly, can play and good drop hypoglycemic effect, also have effect of fat-reducing simultaneously, and its chemistry is expressed as Nε26-{ 18-[N-(17-carboxyheptadecanoyl)-L-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaoctadecanoyl}-[8-(2-amino-2-propanoicacid), 34-L-arginine] humanglucagon-likepeptide1 (7-37), it has structure shown in Formula II, and the main chain of Sa Molu peptide has the aminoacid sequence as shown in SEQIDNO:1:
At present; the preparation method of disclosed Sa Molu peptide is solid-phase synthesis, mainly comprises the steps that acquisition the first amino-acid resin, and progressively coupling synthesizes main chain peptide resin afterwards; then the protection base of Lys side chain is removed, the more progressively aminoacid of coupling side chain and other group.But, when employing the method, on the one hand, side chain elimination is wayward, not exclusively or in this process, other protect coming off of bases to there is protection base elimination, produce various by-product, affect the purity of thick peptide；On the other hand, the coupling of side chain is equivalent to extend the coupling of peptide sequence, increases coupling difficulty, have impact on the purity of thick peptide, thus having had influence on last yield.
Summary of the invention
In view of this, the goal of the invention of the present invention there are provided the Preparation Method And Their Intermediate of a kind of Sa Molu peptide.The 1B that the preparation method of Sa Molu peptide provided by the invention is modified using side chain is as intermediate; adopt each aminoacid or the amino acid derivativges of solid-phase synthesis progressively coupling Sa Molu peptide main chain; avoid the coupling process of side chain amino acid after main chain synthesizes; and then avoid the elimination of 1B Side chain protective group in coupling process; effectively prevent the generation of by-product, improve the yield of Sa Molu peptide.
In order to realize the goal of the invention of the present invention, the present invention adopts the following technical scheme that:
The preparation method that the invention provides a kind of Sa Molu peptide, it comprises the following steps:
Step 1: obtain the compound with structure shown in Formulas I；
Step 2: take resin, after activation, progressively coupling L-glycine, L-arginine, L-glycine, L-arginine, Valine, L-Leu, L-Trp, ALANINE, ILE, L-phenylalanine, Pidolidone, gained have the compound of structure shown in Formulas I, obtain the first peptide resin；
Step 3: taking gained the first peptide resin, progressively coupling amino acid or amino acid derivativges obtain the second peptide resin；
Step 4: take gained the second peptide resin, through cracking, purification, to obtain final product；
Wherein, R1Selected from Fmoc, Boc, ivDde；
R2Selected from tBu, Dmab, Bzl.
In some embodiments of the invention, R1For Fmoc or ivDde.
In the other embodiment of the present invention, R2For tBu.
In the present invention, the 1B modified using side chain, as intermediate, namely has the compound of structure shown in Formulas I, on solid phase carrier progressively each aminoacid of coupling, there is the compound of structure shown in Formulas I, namely obtain the first peptide resin；Progressively other aminoacid of coupling or amino acid derivativges more on this basis, namely obtains the second peptide resin, on the second peptide resin with peptide sequence information consistent with the peptide sequence information of Sa Molu peptide；Eventually pass cracking, purification, namely obtain Sa Molu peptide.The present invention utilizes the compound with structure shown in Formulas I as intermediate; adopt each aminoacid or the amino acid derivativges (Sa Molu peptide main chain has the aminoacid sequence as shown in SEQIDNO:1) of solid-phase synthesis progressively coupling Sa Molu peptide main chain; namely Sa Molu peptide is obtained; avoid the coupling process of side chain amino acid after main chain synthesizes; and then avoid the elimination of 1B Side chain protective group in coupling process; effectively prevent the generation of by-product; improve the yield of Sa Molu peptide, be more suitable for the industrialized great production of Sa Molu peptide.
Preferably, in preparation method provided by the invention, in the coupling in step 3, the Ala-Ala in the peptide sequence of Sa Molu peptide is with the form coupling of dipeptide fragment.In some embodiments of the invention, the dipeptide fragment used by coupling Ala-Ala is specially Fmoc-Ala-Ala-OH.
In the preparation process of Sa Molu peptide, when each aminoacid or amino acid derivativges in the mode coupling peptide sequence main chain adopting progressively coupling, Ala-Ala in peptide sequence is bigger with the form progressively coupling difficulty of single amino acids, it is easy to forms defect peptide, and then affects the yield of Sa Molu peptide.The present invention is preferably the form coupling with Ala-Ala dipeptide fragment, it is possible to effectively reduce the formation of defect peptide, improves the yield of Sa Molu peptide further.
Preferably, in preparation method provided by the invention, in the coupling in step 3, in the peptide sequence of Sa Molu peptide, Ser-Ser is with the form coupling of dipeptide fragment.In some embodiments of the invention, the dipeptide fragment used by coupling Ser-Ser is specially Fmoc-Ser (tBu)-Ser (tBu)-OH.
In the preparation process of Sa Molu peptide, when each aminoacid or amino acid derivativges in the mode coupling peptide sequence main chain adopting progressively coupling, Ser-Ser in peptide sequence is bigger with the form progressively coupling difficulty of single amino acids, it is easy to forms defect peptide, and then affects the yield of Sa Molu peptide.The present invention is preferably the form coupling with Ser-Ser dipeptide fragment, it is possible to effectively reduce the formation of defect peptide, improves the yield of Sa Molu peptide further.
Preferably, in preparation method provided by the invention, the resin in step 2 is king's resin or 2-CTC resin (2-trityl chloride resin).In some embodiments of the invention, in preparation method provided by the invention, the resin in step 2 is specially king's resin.
In some embodiments of the invention, in preparation method provided by the invention, when in step 2, coupling L-glycine prepares Fmoc-Gly-king's resin, coupling agent used is DIC/DMAP, HOBt/DIC/DMAP or PyBOP/HOBt/DIPEA.In the other embodiment of the present invention, in preparation method provided by the invention, when in step 2, coupling L-glycine prepares Fmoc-Gly-king's resin, coupling agent used is HOBt/DIC/DMAP.
Preferably, in preparation method provided by the invention, in step 4, cracking lytic reagent used is the mixture of TFA, thioanisole, methyl phenyl ethers anisole and EDT.
In some embodiments of the invention, in preparation method provided by the invention, cracking the ratio of the volume of TFA, thioanisole, methyl phenyl ethers anisole and EDT in agents useful for same in step 4 is (85～92): (4～6): (2～3): (2～6).In the other embodiment of the present invention, in preparation method provided by the invention, in step 4, in cracking lytic reagent used, the ratio of the volume of TFA, thioanisole, methyl phenyl ethers anisole and EDT is 90:5:3:2.
In the other embodiment of the present invention, in preparation method provided by the invention, in step 4, purification method therefor is chromatography.In the other embodiment of the present invention, in preparation method provided by the invention, in step 4, chromatographic column used by purification is C8 post.
Preferably, in preparation method provided by the invention, step 1 has the preparation method of the compound of structure shown in Formulas I particularly as follows:
Take resin, after activation, the 1B of coupling side chain protected, obtain amino-acid resin；
Take gained amino-acid resin; de-1B Side chain protective group; progressively coupling 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid, 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid, Pidolidone, octadecane diacid mono-methyl, obtain primary product；
Take gained primary product, through cracking, purification, must have the compound of structure shown in Formulas I；
Wherein, R1Selected from Fmoc, Boc, ivDde；
R2Selected from tBu, Dmab, Bzl.
There is the preparation method of compound of structure shown in Formulas I not by the restriction of preparation method provided by the invention, the method that those skilled in the art can select to synthesize the compound with structure shown in Formulas I according to practical situation.
In the other embodiment of the present invention, in preparation method provided by the invention, step 1 has in the preparation method of compound of structure shown in Formulas I, the preparation method of octadecane diacid mono-methyl particularly as follows:
Take octadecane diacid to react with methanol, to obtain final product.
Preferably, in preparation method provided by the invention, in step 1, resin used is 2-trityl chloride resin or HMPB-AM resin.In some embodiments of the invention, in preparation method provided by the invention, in step 1, resin used is 2-trityl chloride resin.
In some embodiments of the invention; in preparation method provided by the invention; the Side chain protective group of the 1B of the side chain protected in amino-acid resin in step 1 is allyloxycarbonyl or 1-(4,4-dimethyl-2,6-dioxocyclohexyl methylene)-3-methyl butyl.
In the other embodiment of the present invention, in preparation method provided by the invention, step 1 cracks the mixture that lytic reagent used is TFE and DCM.
In the other embodiment of the present invention, in preparation method provided by the invention, cracking the volume ratio of TFE and DCM in lytic reagent used in step 1 is (10～30): (70～90).In the other embodiment of the present invention, in preparation method provided by the invention, cracking the volume ratio of TFE and DCM in lytic reagent used in step 1 is 20:80.
In some embodiments of the invention, in preparation method provided by the invention, in step 1, the coupling reagent used by the 1B of coupling side chain protected is DIPEA or triethylamine.In the other embodiment of the present invention, in preparation method provided by the invention, in step 1, the coupling reagent used by the 1B of coupling side chain protected is DIPEA.
In some embodiments of the invention, in preparation method provided by the invention, having in the preparation method of compound of structure shown in Formulas I in step 1, the coupling reagent used by coupling 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid is PyBOP/HOBt/DIPEA, HOBt/DIC or DIC.In the other embodiment of the present invention, the coupling reagent used by coupling 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid is PyBOP/HOBt/DIPEA.
In the other embodiment of the present invention, in preparation method provided by the invention, the coupling reagent used by coupling Pidolidone is PyBOP/HOBt/DIPEA, HBTU/HOBt/DIPEA or HOBt/DIC.In the other embodiment of the present invention, in preparation method provided by the invention, the coupling reagent used by coupling Pidolidone is PyBOP/HOBt/DIPEA.
In the other embodiment of the present invention, in preparation method provided by the invention, the coupling reagent used by coupling octadecane diacid mono-methyl is DIC, HOBt/DIC or PyBOP/HOBt/DIPEA.In the other embodiment of the present invention, in preparation method provided by the invention, the coupling reagent used by coupling octadecane diacid mono-methyl is DIC.
Present invention also offers the intermediate of a kind of Sa Molu peptide, it is the compound with structure shown in Formulas I；
Wherein, R1Selected from Fmoc, Boc, ivDde；
R2Selected from tBu, Dmab, Bzl.
In some embodiments of the invention, in the intermediate of Sa Molu peptide provided by the invention, R1For Fmoc.
In the other embodiment of the present invention, in the intermediate of Sa Molu peptide provided by the invention, R2For tBu.
In some embodiments of the invention, the intermediate of Sa Molu peptide provided by the invention has structure shown in Formulas I-1, Formulas I-2:
The preparation method that present invention also offers the intermediate of a kind of Sa Molu peptide, including:
Take resin, after activation, the 1B of coupling side chain protected, obtain amino-acid resin；
Take gained amino-acid resin; de-1B Side chain protective group; progressively coupling 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid, 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid, Pidolidone, octadecane diacid mono-methyl, obtain primary product；
Take gained primary product, through cracking, purification, must have the compound of structure shown in Formulas I；
Wherein, R1Selected from Fmoc, Boc, ivDde；
R2Selected from tBu, Dmab, Bzl.
The intermediate of Sa Molu peptide, namely has the preparation method of compound of structure shown in Formulas I not by the restriction of preparation method provided by the invention, the method that those skilled in the art can select to synthesize the compound with structure shown in Formulas I according to practical situation.
In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the preparation method of octadecane diacid mono-methyl particularly as follows:
Take octadecane diacid to react with methanol, to obtain final product.
Preferably, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, resin used is 2-trityl chloride resin or HMPB-AM resin.In some embodiments of the invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, resin used is 2-trityl chloride resin.
In some embodiments of the invention; in the preparation method of the intermediate of Sa Molu peptide provided by the invention; the Side chain protective group of the 1B of the side chain protected in amino-acid resin is allyloxycarbonyl or 1-(4,4-dimethyl-2,6-dioxocyclohexyl methylene)-3-methyl butyl.
In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the mixture that lytic reagent is TFE and DCM that cracking is used.
In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, in the lytic reagent that cracking is used, the volume ratio of TFE and DCM is (10～30): (70～90).In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, in the lytic reagent that cracking is used, the volume ratio of TFE and DCM is 20:80.
In some embodiments of the invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the coupling reagent used by the 1B of coupling side chain protected is DIPEA or triethylamine.In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the coupling reagent used by the 1B of coupling side chain protected is DIPEA.
In some embodiments of the invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the coupling reagent used by coupling 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid is PyBOP/HOBt/DIPEA, HOBt/DIC or DIC.In the other embodiment of the present invention, the coupling reagent used by coupling 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid is PyBOP/HOBt/DIPEA.
In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the coupling reagent used by coupling Pidolidone is PyBOP/HOBt/DIPEA, HBTU/HOBt/DIPEA or HOBt/DIC.In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the coupling reagent used by coupling Pidolidone is PyBOP/HOBt/DIPEA.
In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the coupling reagent used by coupling octadecane diacid mono-methyl is DIC, HOBt/DIC or PyBOP/HOBt/DIPEA.In the other embodiment of the present invention, in the preparation method of the intermediate of Sa Molu peptide provided by the invention, the coupling reagent used by coupling octadecane diacid mono-methyl is DIC.
The invention provides the Preparation Method And Their Intermediate of a kind of Sa Molu peptide.The preparation method of Sa Molu peptide provided by the invention includes: obtain the compound with structure shown in Formulas I；Take resin, after activation, progressively coupling L-glycine, L-arginine, L-glycine, L-arginine, Valine, L-Leu, L-Trp, ALANINE, ILE, L-phenylalanine, Pidolidone, gained have the compound of structure shown in Formulas I, obtain the first peptide resin；Taking gained the first peptide resin, progressively coupling amino acid or amino acid derivativges obtain the second peptide resin；Take gained the second peptide resin, through cracking, purification, to obtain final product.The present invention utilizes the compound with structure shown in Formulas I as intermediate; adopt each aminoacid or the amino acid derivativges of solid-phase synthesis progressively coupling Sa Molu peptide main chain; namely Sa Molu peptide is obtained; avoid the coupling process of side chain amino acid after main chain synthesizes; and then avoid the elimination of 1B Side chain protective group in coupling process; effectively prevent the generation of by-product, improve the yield of Sa Molu peptide.Experimental result confirms, preparation method provided by the invention significantly improves the yield of Sa Molu peptide, is more suitable for the industrialized great production of Sa Molu peptide；
Wherein, R1Selected from Fmoc, Boc, ivDde；
R2Selected from tBu, Dmab, Bzl.
Accompanying drawing explanation
Fig. 1 shows the mass spectrogram of the product that embodiment 1 prepares；
Fig. 2 shows the Structural Identification result of the compound that embodiment 10 prepares；
Wherein, Fig. 2-A shows the mass spectrogram of this compound；Fig. 2-B shows this compound H PLC spectrogram；
Fig. 3 shows the HPLC spectrogram of the compound that embodiment 11 prepares；
Fig. 4 shows the HPLC spectrogram of the compound that embodiment 12 prepares；
Fig. 5 shows the HPLC spectrogram of the compound that embodiment 13 prepares；
Fig. 6 shows the structure detection qualification result of the purpose peak fraction of embodiment 23 gained；
Wherein, Fig. 6-A shows the mass spectrogram of the purpose peak fraction that the present embodiment obtains；Fig. 6-B shows the HPLC spectrogram of the purpose peak fraction that the present embodiment obtains；
Fig. 7 shows the structure detection qualification result of the purpose peak fraction of embodiment 24 gained；
Wherein, Fig. 7-A shows the mass spectrogram of the purpose peak fraction that the present embodiment obtains；Fig. 7-B shows the HPLC spectrogram of the purpose peak fraction that the present embodiment obtains；
Fig. 8 shows the structure detection qualification result of the purpose peak fraction of embodiment 25 gained；
Wherein, Fig. 8-A shows the mass spectrogram of the purpose peak fraction that the present embodiment obtains；Fig. 8-B shows the HPLC spectrogram of the purpose peak fraction that the present embodiment obtains；
Fig. 9 shows the structure detection qualification result of the purpose peak fraction of embodiment 26 gained；
Wherein, Fig. 9-A shows the mass spectrogram of the purpose peak fraction that the present embodiment obtains；Fig. 9-B shows the HPLC spectrogram of the purpose peak fraction that the present embodiment obtains.
Detailed description of the invention
The invention discloses the Preparation Method And Their Intermediate of a kind of Sa Molu peptide.Those skilled in the art are referred to present disclosure, it is thus achieved that Sa Molu peptide, it is accordingly required in particular to it is noted that all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention.Preparation method and the application of the present invention are described already by preferred embodiment, this paper preparation method and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, realizes and applies the technology of the present invention.
Reagent used in the Preparation Method And Their Intermediate of a kind of Sa Molu peptide provided by the invention and raw material all can be buied by market.
The bilingual of the english abbreviation related in the present invention is in Table 1.
Table 1 bilingual table
In order to make those skilled in the art better understood when technical scheme, below in conjunction with embodiment, the present invention is expanded on further:
Embodiment 1 octadecane diacid mono-methyl synthesizes
Weighing octadecane diacid 87g (0.5mol) and join in there-necked flask, add 500mLDCM, stirring and dissolving, add 16g (0.5mol) methanol, drip concentrated sulphuric acid 0.05g, stirring, to backflow, is reacted 4 hours.TLC monitoring reaction is complete, cools down reactant liquor, adds 2%Na2CO3(quality volume percentage) aqueous solution 100mL extracting twice, water layer 100mLDCM extracting twice, merge organic facies, dry with anhydrous sodium sulfate.Filtrate reduced in volume, obtains product 69.5g.
Adopting mass spectrum and proton nmr spectra detection products therefrom, gained mass spectrogram is shown in Fig. 1, it can be seen that MS, m/z329.19 (M+H)+；Gained hydrogen nuclear magnetic resonance modal data is:1H-NMR(CDCl3400MHz), 1.25～1.32 (m, 8H), 1.32～1.36 (m, 6H), 1.36～1.38 (m, 4H), 1.38～1.40 (m, 4H), 1.42～1.46 (m, 6H), 2.3 (t, 2H, J=7.2), 2.35 (t, 2H, J=7.2), 3.65 (s, 3H), 11.1 (s, 1H)；It is octadecane diacid mono-methyl through resolving this product.
Embodiment 2 substitution degree is the synthesis of Fmoc-Lys (the Alloc)-2CTC resin of 0.60mmol/g
Weigh 2-CTC resin (the being purchased from Tianjin Nankai Compositech Inc.) 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, after DMF swellable resins 30 minutes, take 9.05gFmoc-Lys (Alloc)-OH DMF to dissolve, after adding 6.65mLDIEA activation under ice-water bath, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 10mL absolute methanol and close 1 hour.Washing 3 times with DMF, DCM washes 3 times, closes 30 minutes with absolute methanol afterwards, and methanol shrinks to be drained, and obtains Fmoc-Lys (Alloc)-2CTC resin, and detection substitution degree is 0.605mmol/g.
Embodiment 3 substitution degree is the synthesis of Fmoc-Lys (the Alloc)-2CTC resin of 0.80mmol/g
Weigh 2-CTC resin (the being purchased from Tianjin Nankai Compositech Inc.) 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, after DMF swellable resins 30 minutes, take 13.75gFmoc-Lys (Alloc)-OH DMF to dissolve, after adding 9.98mLDIEA activation under ice-water bath, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 15mL absolute methanol and close 1 hour.Washing 3 times with DMF, DCM washes 3 times, closes 30 minutes with absolute methanol afterwards, and methanol shrinks to be drained, and obtains Fmoc-Lys (Alloc)-2CTC resin, and detection substitution degree is 0.810mmol/g.
Embodiment 4 substitution degree is the synthesis of Fmoc-Lys (the ivDde)-2CTC resin of 0.60mmol/g
Weigh 2-CTC resin (the being purchased from Tianjin Nankai Compositech Inc.) 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, after DMF swellable resins 30 minutes, take 11.48gFmoc-Lys (ivDde)-OH DMF to dissolve, after adding 6.65mLDIEA activation under ice-water bath, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 10mL absolute methanol and close 1 hour.Washing 3 times with DMF, DCM washes 3 times, closes 30 minutes with absolute methanol, and methanol shrinks to be drained, and obtains Fmoc-Lys (ivDde)-2CTC resin, and detection substitution degree is 0.612mmol/g.
Embodiment 5 substitution degree is the synthesis of Fmoc-Lys (the ivDde)-2CTC resin of 0.80mmol/g
Weigh 2-CTC resin (the being purchased from Tianjin Nankai Compositech Inc.) 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, after DMF swellable resins 30 minutes, take 17.22gFmoc-Lys (ivDde)-OH DMF to dissolve, after adding 9.98mLDIEA activation under ice-water bath, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 15mL absolute methanol and close 1 hour.Washing 3 times with DMF, DCM washes 3 times, closes 30 minutes with absolute methanol, and methanol shrinks to be drained, and obtains Fmoc-Lys (ivDde)-2CTC resin, and detection substitution degree is 0.794mmol/g.
The synthesis of Lys intermediate Fmoc-Lys (M)-resin that embodiment 6 side chain is modified
Weigh the Fmoc-Lys that substitution degree is 0.605mmol/g (Alloc)-2CTC resin 10.0g (6.0mmol) of embodiment 2 preparation, add in solid state reaction post, wash 2 times with DMF, after DMF swelling Fmoc-Lys (Alloc)-2CTC resin 30 minutes, add 30mL dichloromethane, add 8.8mL phenyl silane, react 3 minutes, add 1.86gPd (PPh3)4, room temperature reaction 45 minutes, to take out reactant liquor, detect color of resin with ninhydrin method, resin has color, represents that Alloc removes.
Weigh Fmoc-AEEA-OH11.56g (30mmol), PyBOP13.05g (30mmol), HOBt4.88g (36mmol), dissolve with 30mLDMF, add 10.80mLDIEA (60mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Same method is coupling Fmoc-AEEA-OH again.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Weigh Fmoc-Glu-OtBu12.72g (30mmol), PyBOP13.05g (30mmol), HOBt4.88g (36mmol), dissolve with 30mLDMF, add 10.80mLDIEA (60mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and elimination Fmoc, DMF wash 6 times.Weigh octadecane diacid mono-methyl 9.855g (30mmol) prepared according to the preparation method of embodiment 1 offer, dissolve with 30mLDMF, add 4.75mLDIC (30mmol) under ice-water bath to activate 3 minutes, add reaction column and react 2 hours.
Reaction is shunk with methanol after terminating, dried in vacuum overnight.Weigh and obtain the Lys intermediate resin 14.2g that side chain is modified.
The synthesis of Lys intermediate Fmoc-Lys (M)-resin that embodiment 7 side chain is modified
Weigh the Fmoc-Lys that substitution degree is 0.810mmol/g (Alloc)-2CTC resin 7.4g (6.0mmol) of embodiment 3 preparation, add in solid state reaction post, wash 2 times with DMF, after DMF swelling Fmoc-Lys (Alloc)-2CTC resin 30 minutes, add 30mL dichloromethane, add 8.8mL phenyl silane, react 3 minutes, add 1.86gPd (PPh3)4, room temperature reaction 45 minutes, to take out reactant liquor, detect color of resin with ninhydrin method, resin has color, represents that Alloc removes.
Weigh Fmoc-AEEA-OH11.56g (30mmol), PyBOP13.05g (30mmol), HOBt4.88g (36mmol), dissolve with 30mLDMF, add 10.80mLDIEA (60mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Same method is coupling Fmoc-AEEA-OH again.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Weigh Fmoc-Glu-OtBu12.72g (30mmol), PyBOP13.05g (30mmol), HOBt4.88g (36mmol), dissolve with 30mLDMF, add 10.80mLDIEA (60mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and elimination Fmoc, DMF wash 6 times.Weigh octadecane diacid mono-methyl 9.855g (30mmol) prepared according to the preparation method of embodiment 1 offer, dissolve with 30mLDMF, add 3.76mLDIC (24mmol) under ice-water bath to activate 3 minutes, add reaction column and react 2 hours.
Reaction is shunk with methanol after terminating, dried in vacuum overnight.Weigh and obtain the Lys intermediate resin 12.0g that side chain is modified.
The synthesis of Lys intermediate Fmoc-Lys (M)-resin that embodiment 8 side chain is modified
Weigh the Fmoc-Lys that substitution degree is 0.612mmol/g (ivDde)-2CTC resin 9.8g (6.0mmol) of embodiment 4 preparation, add in solid state reaction post, wash 2 times with DMF, after DMF swelling Fmoc-Lys (ivDde)-2CTC resin 30 minutes, add the NH of 30mL2%2NH2/ DMF solution, room temperature reaction 1 hour, to take out reactant liquor, detect color of resin with ninhydrin method, resin has color, represents that ivDde removes.
Weigh Fmoc-AEEA-OH11.56g (30mmol), PyBOP13.05g (30mmol), HOBt4.88g (36mmol), dissolve with 30mLDMF, add 10.80mLDIEA (60mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Same method is coupling Fmoc-AEEA-OH again.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Weigh Fmoc-Glu-OtBu12.72g (30mmol), PyBOP13.05g (30mmol), HOBt4.88g (36mmol), dissolve with 30mLDMF, add 10.80mLDIEA (60mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and elimination Fmoc, DMF wash 6 times.Weigh octadecane diacid mono-methyl 9.855g (30mmol) prepared according to the preparation method of embodiment 1 offer, dissolve with 30mLDMF, add 3.76mLDIC (24mmol) under ice-water bath to activate 3 minutes, add reaction column and react 2 hours.
Reaction is shunk with methanol after terminating, dried in vacuum overnight.Weigh and obtain the Lys intermediate resin 14.5g that side chain is modified.
The synthesis of Lys intermediate Fmoc-Lys (M)-resin that embodiment 9 side chain is modified
Weigh the Fmoc-Lys that substitution degree is 0.794mmol/g (ivDde)-2CTC resin 7.6g (6.0mmol) of embodiment 5 preparation, add in solid state reaction post, wash 2 times with DMF, after DMF swelling Fmoc-Ly (ivDde)-2CTC resin 30 minutes, add the NH of 30mL2%2NH2/ DMF solution, room temperature reaction 1 hour, to take out reactant liquor, detect color of resin with ninhydrin method, resin has color, represents that ivDde removes.
Weigh Fmoc-AEEA-OH11.56g (30mmol), PyBOP13.05g (30mmol), HOBt4.88g (36mmol), dissolve with 30mLDMF, add 10.80mLDIEA (60mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Same method is coupling Fmoc-AEEA-OH again.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Weigh Fmoc-Glu-OtBu12.72g (30mmol), PyBOP13.05g (30mmol), HOBt4.88g (36mmol), dissolve with 30mLDMF, add 10.80mLDIEA (60mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and elimination Fmoc, DMF wash 6 times.Weigh octadecane diacid mono-methyl 9.855g (30mmol) prepared according to the preparation method of embodiment 1 offer, dissolve with 30mLDMF, add 3.76mLDIC (24mmol) under ice-water bath to activate 3 minutes, add reaction column and react 2 hours.
Reaction is shunk with methanol after terminating, dried in vacuum overnight.Weigh and obtain the Lys intermediate resin 12.3g that side chain is modified.
Embodiment 10 has the preparation of the compound of structure shown in Formulas I
The Lys intermediate resin that 14.2g side chain embodiment 6 obtained is modified is transferred in round-bottomed flask, adds the 142mL lysate (with volume basis, TFE:DCM=20:80) prepared in advance, filters after being stirred at room temperature 2 hours, collects filtrate；Resin washs 2 times with the DCM of 10mL again, merging filtrate.Filtrate decompression is evaporated, and vacuum drying, obtains 6.05g compound as white solid.
Gained compound as white solid is utilized HPLC purification, and purification condition is: adopt with 8 alkyl linked silica gel for fixing phase, mobile phase: A phase: TFA；B phase: trifluoroacetic acid aqueous solution, gradient: B%:66% 86%, 50-70min.
Adopting Mass Spectrometer Method gained compound as white solid, result is shown in Fig. 2-A, Ms, m/z1154.3 (M+H)+；Its HPLC spectrogram is shown in Fig. 2-B.Through resolving, obtaining gained compound is the compound with structure shown in Formulas I-1, and its purity is 98.5%, and quality is 3.63g.
Embodiment 11 has the preparation of the compound of structure shown in Formulas I
The Lys intermediate resin that 12.0g side chain embodiment 7 obtained is modified is transferred in round-bottomed flask, adds the 120mL lysate (with volume basis, TFE:DCM=20:80) prepared in advance, filters after being stirred at room temperature 2 hours, collects filtrate；Resin washs 2 times with the DCM of 10mL again, merging filtrate.Filtrate decompression is evaporated, and vacuum drying, obtains 6.25g compound as white solid.
Utilizing HPLC to be purified gained compound as white solid, purification condition is: adopt with 8 alkyl linked silica gel for fixing phase, mobile phase: A phase: TFA；B phase: trifluoroacetic acid aqueous solution, gradient: B%:66% 86%, 50-70min.
Adopt Mass Spectrometer Method gained compound as white solid, its Ms, m/z1154.3 (M+H)+；Its HPLC spectrogram is shown in Fig. 3.Through resolving, obtaining gained compound is the compound with structure shown in Formulas I-1, and its purity is 98.5%, and quality is 3.54g.
Embodiment 12 has the preparation of the compound of structure shown in Formulas I
The Lys intermediate resin that 14.5g side chain embodiment 8 obtained is modified is transferred in round-bottomed flask, adds the 145mL lysate (with volume basis, TFE:DCM=20:80) prepared in advance, filters after being stirred at room temperature 2 hours, collects filtrate；Resin washs 2 times with the DCM of 10mL again, merging filtrate.Filtrate decompression is evaporated, and vacuum drying, obtains 6.10g compound as white solid.
Utilizing HPLC to be purified gained compound as white solid, purification condition is: adopt with 8 alkyl linked silica gel for fixing phase, mobile phase: A phase: TFA；B phase: trifluoroacetic acid aqueous solution, gradient: B%:66% 86%, 50-70min.
Adopt Mass Spectrometer Method gained compound as white solid, its Ms, m/z1154.3 (M+H)+；Its HPLC spectrogram is shown in Fig. 4.Through resolving, obtaining gained compound is the compound with structure shown in Formulas I-1, and its purity is 98.5%, and quality is 3.75g.
Embodiment 13 has the preparation of the compound of structure shown in Formulas I
The Lys intermediate resin that 12.3g side chain embodiment 9 obtained is modified is transferred in round-bottomed flask, adds the 120mL lysate (with volume basis, TFE:DCM=20:80) prepared in advance, filters after being stirred at room temperature 2 hours, collects filtrate；Resin washs 2 times with the DCM of 10mL again, merging filtrate.Filtrate decompression is evaporated, and vacuum drying, obtains 6.01g compound as white solid.
Utilizing HPLC to be purified gained compound as white solid, purification condition is: adopt with 8 alkyl linked silica gel for fixing phase, mobile phase: A phase: TFA；B phase: trifluoroacetic acid aqueous solution, gradient: B%:66% 86%, 50-70min.
Adopt Mass Spectrometer Method gained compound as white solid, its Ms, m/z1154.3 (M+H)+；Its HPLC spectrogram is shown in Fig. 5.Through resolving, obtaining gained compound is the compound with structure shown in Formulas I-1, and its purity is 98.5%, and quality is 3.60g.
Embodiment 14 substitution degree is the synthesis of Fmoc-Gly-king's resin of 0.15mmol/g
Weigh king's resin (the being purchased from Tianjin Nankai Compositech Inc.) 40g that substitution degree is 0.5mmol/g, join in solid state reaction post, wash 2 times with DMF, after DMF swellable resins 30 minutes, take 6.54gFmoc-Gly-OH, 3.56gHOBt DMF dissolves, after adding 4.12mLDIC activation 3min under ice-water bath, add above-mentioned equipped with in the reaction column of resin, 268mgDMAP is added after 5 minutes, after reacting 2 hours, DMF washs 3 times, DCM washes 3 times, with 120mL acetic anhydride/pyridine (with volume basis, acetic anhydride: pyridine=1:1) close overnight, methanol shrinks to be drained, obtain Fmoc-Gly-king's resin, detection substitution degree is 0.150mmol/g.
The preparation of embodiment 15 peptide resin
Weigh Fmoc-Gly-king's resin 10g (1.5mmol) that substitution degree is 0.15mmol/g of embodiment 14 preparation; add in solid state reaction post; wash 2 times with DMF; after the swelling Fmoc-Gly-king's resin of DMF 30 minutes, remove Fmoc protection with DBLK, then wash 4 times with DMF; DCM washes 2 times; detecting color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 4.87gFmoc-Arg (Pbf)-OH (7.5mmol), 1.22gHOBt (9mmol), 3.90gPyBOP (7.5mmol) is dissolved in DCM and the DMF mixed solution that volume ratio is 11, adding under ice-water bath after 1.92mLDIPEA (9mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that would react completely；Resin develops the color, then it represents that reaction not exclusively, needs coupling reaction 1 hour again, and this criterion judges reaction end with ninhydrin method detection suitable in subsequent content.
nullRepeat above-mentioned elimination Fmoc protection and add corresponding aminoacid and carry out the step of coupling，Peptide sequence according to Sa Molu peptide main chain，It is sequentially completed Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、There is the compound (preparation method that embodiment 10 provides prepares) of structure shown in Formulas I-1、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、The coupling of Fmoc-Aib-OH and Boc-His (Trt)-OH.Being washed 3 times by products therefrom DMF, DCM washes 5 times, shrinks with methanol, dried in vacuum overnight, obtains peptide resin 18.2g.
The preparation of embodiment 16 peptide resin
Weigh Fmoc-Gly-king's resin 10g (1.5mmol) that substitution degree is 0.15mmol/g of embodiment 14 preparation; add in solid state reaction post; wash 2 times with DMF; after the swelling Fmoc-Gly-king's resin of DMF 30 minutes, remove Fmoc protection with DBLK, then wash 4 times with DMF; DCM washes 2 times; detecting color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 4.87gFmoc-Arg (Pbf)-OH (7.5mmol), 1.22gHOBt (9mmol), 3.90gPyBOP (7.5mmol) is dissolved in DCM and the DMF mixed solution that volume ratio is 11, adding under ice-water bath after 1.92mLDIPEA (9mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that would react completely；Resin develops the color, then it represents that reaction not exclusively, needs coupling reaction 1 hour again, and this criterion judges reaction end with ninhydrin method detection suitable in subsequent content.
nullRepeat above-mentioned elimination Fmoc protection and add corresponding aminoacid and carry out the step of coupling，Peptide sequence according to Sa Molu peptide main chain，It is sequentially completed Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、There is the compound (preparation method that embodiment 11 provides prepares) of structure shown in Formulas I-1、Fmoc-Ala-Ala-OH (derives from gill biochemistry)、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、The coupling of Fmoc-Aib-OH and Boc-His (Trt)-OH.Being washed 3 times by products therefrom DMF, DCM washes 5 times, and coelonychia alcohol shrinks, and dried in vacuum overnight obtains peptide resin 18.3g.
The preparation of embodiment 17 peptide resin
Weigh Fmoc-Gly-king's resin 10g (1.5mmol) that substitution degree is 0.15mmol/g of embodiment 14 preparation; add in solid state reaction post; wash 2 times with DMF; after the swelling Fmoc-Gly-king's resin of DMF 30 minutes, remove Fmoc protection with DBLK, then wash 4 times with DMF; DCM washes 2 times; detecting color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 4.87gFmoc-Arg (Pbf)-OH (7.5mmol), 1.22gHOBt (9mmol), 3.90gPyBOP (7.5mmol) is dissolved in DCM and the DMF mixed solution that volume ratio is 11, adding under ice-water bath after 1.92mLDIPEA (9mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that would react completely；Resin develops the color, then it represents that reaction not exclusively, needs coupling reaction 1 hour again, and this criterion judges reaction end with ninhydrin method detection suitable in subsequent content.
nullRepeat above-mentioned elimination Fmoc protection and add corresponding aminoacid and carry out the step of coupling，Peptide sequence according to Sa Molu peptide main chain，It is sequentially completed Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、There is the compound (preparation method that embodiment 12 provides prepares) of structure shown in Formulas I-1、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser (tBu)-Ser (tBu)-OH (derives from gill biochemistry)、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、The coupling of Fmoc-Aib-OH and Boc-His (Trt)-OH.Being washed 3 times by products therefrom DMF, DCM washes 5 times, and methanol shrinks, and dried in vacuum overnight obtains peptide resin 18.2g.
The preparation of embodiment 18 peptide resin
Weigh Fmoc-Gly-king's resin 10g (1.5mmol) that substitution degree is 0.15mmol/g of embodiment 14 preparation; add in solid state reaction post; wash 2 times with DMF; after the swelling Fmoc-Gly-king's resin of DMF 30 minutes, remove Fmoc protection with DBLK, then wash 4 times with DMF; DCM washes 2 times; detecting color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 4.87gFmoc-Arg (Pbf)-OH (7.5mmol), 1.22gHOBt (9mmol), 3.90gPyBOP (7.5mmol) is dissolved in DCM and the DMF mixed solution that volume ratio is 11, adding under ice-water bath after 1.92mLDIPEA (9mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that would react completely；Resin develops the color, then it represents that reaction not exclusively, needs coupling reaction 1 hour again, and this criterion judges reaction end with ninhydrin method detection suitable in subsequent content.
nullRepeat above-mentioned elimination Fmoc protection and add corresponding aminoacid and carry out the step of coupling，Peptide sequence according to Sa Molu peptide main chain，It is sequentially completed Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、There is the compound (preparation method that embodiment 13 provides prepares) of structure shown in Formulas I-1、Fmoc-Ala-Ala-OH (derives from gill biochemistry)、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser (tBu)-Ser (tBu)-OH (derives from gill biochemistry)、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、The coupling of Fmoc-Aib-OH and Boc-His (Trt)-OH.Being washed 3 times by products therefrom DMF, DCM washes 5 times, and methanol shrinks, and dried in vacuum overnight obtains peptide resin 18.5g.
The preparation of the thick peptide of embodiment 19 Sa Molu peptide
The peptide resin 18.2g obtained in Example 15, joins in 250mL reactor, prepares lytic reagent 182mL by the volume ratio of TFA thioanisole methyl phenyl ethers anisole EDT=85 636, is poured into by lytic reagent in peptide resin, room temperature reaction 2.5 hours.Reaction terminates, and filters resin, collects filtrate.Using 10mLTFA washing resin, merging filtrate, filtrate joined in 1.82L absolute ether and precipitate, centrifugal, absolute ether washs, and vacuum drying, obtains solid 4.85g, yield 78.7%.
Solid precipitation is dissolved in 2L25% acetonitrile/water solution, adds 10mL1NLiOH solution, stirring reaction 1 hour under ice bath, obtain the thick peptide of Sa Molu peptide, adjust pH to 6.0 with acetic acid.
The preparation of the thick peptide of embodiment 20 Sa Molu peptide
The peptide resin obtained in Example 16, joins in 250mL reactor, prepares lytic reagent 185mL by the volume ratio of TFA thioanisole methyl phenyl ethers anisole EDT=92 413, is poured into by lytic reagent in peptide resin, room temperature reaction 2.5 hours.Reaction terminates, and filters resin, collects filtrate.Using 10mLTFA washing resin, merging filtrate, filtrate joined in 1.85L absolute ether and precipitate, centrifugal, absolute ether washs, and vacuum drying, obtains solid 4.80g, yield 77.9%.
Solid precipitation is dissolved in 2L25% acetonitrile/water solution, adds 10mL1NLiOH solution, stirring reaction 1 hour under ice bath, obtain the thick peptide of Sa Molu peptide, adjust pH to 6.0 with acetic acid.
The preparation of the thick peptide of embodiment 21 Sa Molu peptide
The peptide resin obtained in Example 17, joins in 250mL reactor, prepares lytic reagent 185mL by the volume ratio of TFA thioanisole methyl phenyl ethers anisole EDT=90 523, is poured into by lytic reagent in peptide resin, room temperature reaction 2.5 hours.Reaction terminates, and filters resin, collects filtrate.Using 10mLTFA washing resin, merging filtrate, filtrate joined in 1.85L absolute ether and precipitate, centrifugal, absolute ether washs, and vacuum drying, obtains solid 4.90g, yield 79.5%.
Solid precipitation is dissolved in 2L25% acetonitrile/water solution, adds 10mL1NLiOH solution, stirring reaction 1 hour under ice bath, obtain the thick peptide of Sa Molu peptide, adjust pH to 6.0 with acetic acid.
The preparation of the thick peptide of embodiment 22 Sa Molu peptide
The peptide resin obtained in Example 18, joins in 250mL reactor, prepares lytic reagent 185mL by the volume ratio of TFA thioanisole methyl phenyl ethers anisole EDT=90 532, is poured into by lytic reagent in peptide resin, room temperature reaction 2.5 hours.Reaction terminates, and filters resin, collects filtrate.Using 10mLTFA washing resin, merging filtrate, filtrate joined in 1.85L absolute ether and precipitate, centrifugal, absolute ether washs, and vacuum drying, obtains solid 4.93g, yield 80.1%.
Solid precipitation is dissolved in 2L25% acetonitrile/water solution, adds 10mL1NLiOH solution, stirring reaction 1 hour under ice bath, obtain the thick peptide of Sa Molu peptide, adjust pH to 6.0 with acetic acid.
The preparation of embodiment 23 Sa Molu peptide fine peptide
The thick peptide solution of Sa Molu peptide that Example 19 prepares, adopts Waters2545RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C8 post of 50 × 250mm, column temperature is 50 DEG C, conventional 0.1%TFA/ acetonitrile mobile phase purification, collects purpose peak fraction (30-40min).
Adopting Mass Spectrometer Method purpose peak fraction, result is shown in Fig. 6-A, Ms, m/z4113.4 (M+H)+；Its HPLC spectrogram is shown in Fig. 6-B.Through resolving, containing Sa Molu peptide in gained purpose peak fraction, its purity is 98.6%.After lyophilizing, obtaining Sa Molu peptide 1.47g, yield is 23.85%.
The preparation of embodiment 24 Sa Molu peptide fine peptide
The thick peptide solution of Sa Molu peptide that Example 20 prepares, adopts Waters2545RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C8 post of 50 × 250mm, column temperature is 50 DEG C, conventional 0.1%TFA/ acetonitrile mobile phase purification, collects purpose peak fraction (30-40min).
Adopting Mass Spectrometer Method purpose peak fraction, result is shown in Fig. 7-A, Ms, m/z4113.5 (M+H)+；Its HPLC spectrogram is shown in Fig. 7-B.Through resolving, containing Sa Molu peptide in gained purpose peak fraction, its purity is 98.7%.After lyophilizing, obtaining Sa Molu peptide 1.45g, yield is 23.53%.
The preparation of embodiment 25 Sa Molu peptide fine peptide
The thick peptide solution of Sa Molu peptide that Example 21 prepares, adopts Waters2545RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C8 post of 50 × 250mm, column temperature is 50 DEG C, conventional 0.1%TFA/ acetonitrile mobile phase purification, collects purpose peak fraction (30-40min).
Adopting Mass Spectrometer Method purpose peak fraction, result is shown in Fig. 8-A, Ms, m/z4113.4 (M+H)+；Its HPLC spectrogram is shown in Fig. 8-B.Through resolving, containing Sa Molu peptide in gained purpose peak fraction, its purity is 98.5%.After lyophilizing, obtaining Sa Molu peptide 1.50g, yield is 24.33%.
The preparation of embodiment 26 Sa Molu peptide fine peptide
The thick peptide solution of Sa Molu peptide that Example 22 prepares, adopts Waters2545RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C8 post of 50 × 250mm, column temperature is 50 DEG C, conventional 0.1%TFA/ acetonitrile mobile phase purification, collects purpose peak fraction (30-40min).
Adopting Mass Spectrometer Method purpose peak fraction, result is shown in Fig. 9-A, Ms, m/z4113.2 (M+H)+；Its HPLC spectrogram is shown in Fig. 9-B.Through resolving, containing Sa Molu peptide in gained purpose peak fraction, its purity is 98.7%.After lyophilizing, obtaining Sa Molu peptide 1.56g, yield is 25.31%.
Comparative example
Weigh Fmoc-Gly-king's resin 2g (0.3mmol) that substitution degree is 0.15mmol/g of embodiment 14 preparation; add in solid state reaction post; wash 2 times with DMF; after the swelling Fmoc-Gly-king's resin of DMF 30 minutes, remove Fmoc protection with DBLK, then wash 4 times with DMF; DCM washes 2 times; detecting color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 0.98gFmoc-Arg (Pbf)-OH (1.5mmol), 0.44gHOBt (1.8mmol), 0.65gPyBOP (1.5mmol) is dissolved in DCM and the DMF mixed solution that volume ratio is 11, adding under ice-water bath after 0.38mLDIPEA (1.8mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that would react completely；Resin develops the color, then it represents that reaction not exclusively, needs coupling reaction 1 hour again, and this criterion judges reaction end with ninhydrin method detection suitable in subsequent content.
nullRepeat above-mentioned elimination Fmoc protection and add corresponding aminoacid and carry out the step of coupling，Peptide sequence according to Sa Molu peptide main chain，It is sequentially completed Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、The coupling of Fmoc-Ala-OH and Boc-His (Trt)-OH.
Add 2mL dichloromethane, add 0.44mL phenyl silane, react 3 minutes, add 0.09gPd (PPh3)4, room temperature reaction 45 minutes, to take out reactant liquor, detect color of resin with ninhydrin method, resin has color, represents that Alloc removes.
Weigh Fmoc-AEEA-OH0.58g (1.5mmol), PyBOP0.65g (1.5mmol), HOBt0.44g (36mmol), dissolve with 2mLDMF, add 0.58mLDIPEA (0.3mmol) under ice-water bath and activate 3 minutes, add reaction column to react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Same method coupling Fmoc-AEEA-OH again, Fmoc-Glu-OtBu, octadecane diacid, reaction is shunk with methanol after terminating, and dried in vacuum overnight obtains peptide resin 3.5g.
The peptide resin obtained, joins in 50mL reactor, prepares lytic reagent 35mL by the volume ratio of TFA thioanisole methyl phenyl ethers anisole EDT=90 523, is poured into by lytic reagent in peptide resin, room temperature reaction 2.5 hours.Reaction terminates, and filters resin, collects filtrate.Using 3mLTFA washing resin, merging filtrate, filtrate joined in 250mL absolute ether and precipitate, centrifugal, absolute ether washs, and vacuum drying, obtains solid 0.92g, yield 74.6%.
The thick peptide solution of Sa Molu peptide prepared, adopts Waters2545RP-HPLC system, wavelength 214nm, and chromatographic column is the anti-phase C8 post of 50 × 250mm, and column temperature is 50 DEG C, conventional 0.1%TFA/ acetonitrile mobile phase purification, collects purpose peak fraction (30-40min).Through resolving, containing Sa Molu peptide in gained purpose peak fraction, its purity is 98.5%.After lyophilizing, obtaining Sa Molu peptide 0.19g, yield is 15.41%.
Below being only the preferred embodiment of the present invention, it is noted that above-mentioned preferred implementation is not construed as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. the preparation method of Yi Zhong Sa Molu peptide, it is characterised in that comprise the following steps:
Step 1: obtain the compound with structure shown in Formulas I；
Step 2: take resin, after activation, progressively coupling L-glycine, L-arginine, L-glycine, L-arginine, Valine, L-Leu, L-Trp, ALANINE, ILE, L-phenylalanine, Pidolidone, described in there is the compound of structure shown in Formulas I, obtain the first peptide resin；
Step 3: taking described first peptide resin, progressively coupling amino acid or amino acid derivativges obtain the second peptide resin；
Step 4: take described second peptide resin, through cracking, purification, to obtain final product；
Wherein, described R1Selected from Fmoc, Boc, ivDde；
Described R2Selected from tBu, Dmab, Bzl.
2. preparation method according to claim 1, it is characterised in that in coupling described in step 3, the Ala-Ala in the peptide sequence of Sa Molu peptide is with the form coupling of dipeptide fragment.
3. preparation method according to claim 1 and 2, it is characterised in that in coupling described in step 3, the Ser-Ser in the peptide sequence of Sa Molu peptide is with the form coupling of dipeptide fragment.
4. preparation method according to claim 1, it is characterised in that resin described in step 2 is king's resin or 2-CTC resin.
5. preparation method according to claim 1, it is characterised in that the lytic reagent used of cracking described in step 4 is the mixture of TFA, thioanisole, methyl phenyl ethers anisole and EDT.
6. preparation method according to any one of claim 1 to 5, it is characterised in that step 1 has the preparation method of the compound of structure shown in Formulas I particularly as follows:
Take resin, after activation, the 1B of coupling side chain protected, obtain amino-acid resin；
Take described amino-acid resin; de-1B Side chain protective group; progressively coupling 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid, 2-(2-(2-ammonia ethyoxyl) ethyoxyl) acetic acid, Pidolidone, octadecane diacid mono-methyl, obtain primary product；
Take described primary product, through cracking, purification, must have the compound of structure shown in Formulas I；
Wherein, R1Selected from Fmoc, Boc, ivDde；
R2Selected from tBu, Dmab, Bzl.
7. preparation method according to claim 6, it is characterised in that resin described in step 1 is 2-trityl chloride resin or HMPB-AM resin.
8. preparation method according to claim 6, it is characterised in that the Side chain protective group of the 1B of side chain protected described in step 1 is allyloxycarbonyl or 1-(4,4-dimethyl-2,6-dioxocyclohexyl methylene)-3-methyl butyl.
9. preparation method according to claim 6, it is characterised in that crack the mixture that lytic reagent used is TFE and DCM described in step 1.
10. the intermediate of Yi Zhong Sa Molu peptide, it is characterised in that it is the compound with structure shown in Formulas I；
Wherein, R1Selected from Fmoc, Boc, ivDde；
R2Selected from tBu, Dmab, Bzl.
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| WO2019120639A1 (en) | 2017-12-21 | 2019-06-27 | Bachem Holding Ag | Solid phase synthesis of acylated peptides |
| CN110498849A (en) * | 2019-09-16 | 2019-11-26 | 南京迪维奥医药科技有限公司 | A kind of main peptide chain of Suo Malu peptide and preparation method thereof |
| WO2020190757A1 (en) | 2019-03-15 | 2020-09-24 | Novetide Ltd. | Improved processes for the preparation of semaglutide |
| CN111748019A (en) * | 2019-03-29 | 2020-10-09 | 深圳翰宇药业股份有限公司 | Synthetic method of polypeptide derivative compound |
| CN112028986A (en) * | 2020-09-11 | 2020-12-04 | 哈尔滨吉象隆生物技术有限公司 | Synthesis method of semaglutide |
| US11744873B2 (en) | 2021-01-20 | 2023-09-05 | Viking Therapeutics, Inc. | Compositions and methods for the treatment of metabolic and liver disorders |
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| CN112028986A (en) * | 2020-09-11 | 2020-12-04 | 哈尔滨吉象隆生物技术有限公司 | Synthesis method of semaglutide |
| US11744873B2 (en) | 2021-01-20 | 2023-09-05 | Viking Therapeutics, Inc. | Compositions and methods for the treatment of metabolic and liver disorders |
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