CN114085269B - All-solid-phase synthesis method of Hoshinoamide A - Google Patents
All-solid-phase synthesis method of Hoshinoamide A Download PDFInfo
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- CN114085269B CN114085269B CN202111342045.3A CN202111342045A CN114085269B CN 114085269 B CN114085269 B CN 114085269B CN 202111342045 A CN202111342045 A CN 202111342045A CN 114085269 B CN114085269 B CN 114085269B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an all-solid-phase synthesis method of Hoshinoamide A, which comprises the steps of coupling Fmoc-Pro-OH to 2-CTC resin; sequentially coupling the Fmoc-Pro-2-CTC resin according to the Hoshinoamide A peptide sequence to obtain polypeptide resin 1; splitting the polypeptide resin 1 to obtain a polypeptide 2; methylation treatment is carried out on the polypeptide 2; removing Fmoc protecting groups of the obtained crude Hoshinoamide A product to obtain Hoshinoamide A. The all-solid-phase synthesis method of the Hoshinoamide A provided by the invention is used for artificially synthesizing the natural product of the Hoshinoamide A for the first time, preparing the compound simply and rapidly, reducing synthesis steps, being applicable to large-scale production, providing a sufficient amount of the compound for related pharmacodynamics and pharmacokinetics research and future application in antimalarial aspect.
Description
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and relates to an all-solid-phase synthesis method of Hoshinoamide A.
Background
The university of tokyo, japan, professor Kiyotake sunaga in 2018 reported that a linear peptide compound Hoshinoamide a was isolated from marine cyanobacteria Caldora penicillata and its structure was analyzed by spectroscopic analysis and degradation reactions (j.nat. Prod.2018,81, 2545-2552). Hoshinoamide A is a polypeptide with a molecular weight of 1146.48Da, and the peptide sequence of Hoshinoamide A is Hba-Aha-N-Me-Leu-Ile-N-Me-D-Val-Gln-Val-N-Me-D-Phe-Pro-OMe: contains two N-Me-D configuration amino acids, namely N-Me-D-Phe at the 2 nd position and N-Me-D-Val at the fifth position, and besides the two N-Me amino acids, the N-Me-Leu at the 7 th position is also connected with two non-amino acid fragments of Aha and Hba through N segments. Although Hoshinoamide a did not show any cytotoxicity to HeLa cells, it showed moderate antimalarial activity (ic50=0.52), and was a lead compound with a certain development prospect.
However, the content of the Hoshinoamide a in the natural product is very low, and it is difficult to meet the requirements of pharmacological research, mass production application and the like, so that a simple, efficient and low-cost synthetic method of the Hoshinoamide a is needed for subsequent application requirements, and no document or patent has been reported about the synthesis of the Hoshinoamide a at present.
Disclosure of Invention
In order to achieve the above purpose, the invention provides an all-solid-phase synthesis method of Hoshinoamide A, which is to synthesize the natural product of Hoshinoamide A for the first time, take simple and cheap amino acid as raw materials, simply and rapidly prepare Hoshinoamide A, and solve the problem that no artificial synthesis technology of Hoshinoamide A exists in the prior art.
The technical scheme adopted by the invention is that the all-solid-phase synthesis method of Hoshinoamide A comprises the following steps:
step one: coupling Fmoc-Pro-OH to the 2-CTC resin to obtain Fmoc-Pro-2-CTC resin;
step two: removing Fmoc protecting groups of Fmoc-Pro-2-CTC resin, and coupling the obtained H-Pro-2-CTC resin with Fmoc-N-Me-D-Phe-OH under condensation conditions to obtain Fmoc-N-Me-D-Phe-Pro-2-CTC resin;
step three: according to Hoshinoamide A peptide sequence, fmoc-N-Me-D-Phe-Pro-2-CTC resin is coupled in sequence according to the method of the step two, wherein the coupling sequence is as follows: fmoc-Val-OH, fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-Ile-OH, fmoc-N-Me-Leu-OH, fmoc-Aha-OH, and Fmoc-Hba (tBu) -OH to obtain polypeptide resin 1;
step four: splitting the polypeptide resin 1 to obtain a polypeptide 2;
step five: under the action of an acid binding agent, carrying out methylation treatment on the polypeptide 2 to obtain a Hoshinoamide A crude product;
step six: removing Fmoc protecting groups of the crude product of the Hoshinoamide A, and purifying and freeze-drying by HPLC to obtain the Hoshinoamide A.
Further, the first step specifically includes the following steps: placing the 2-CTC resin in a mixed solvent of DCM and DMF, adding Fmoc-Pro-OH and alkali liquor, and performing coupling reaction for 2-10 h; purifying the obtained coupling product by a mixture of DIPEA, meOH, DMF for 2-8 hours to obtain Fmoc-Pro-2-CTC resin; the volume ratio of DCM to DMF in the mixed solvent of DCM and DMF is 1 (1-5); the loading of the 2-CTC resin is 0.2 mmol/g-1.2 mmol/g; lye includes any one of DIPEA, NMM, TEA; the volume ratio of DIPEA, meOH, DMF in the mixture of DIPEA, meOH, DMF is 1 (1-5): 5-10.
Further, the molar ratio of phenylalanine, fmoc-Pro-OH and alkali liquor in the 2-CTC resin is 1 (2-5): 2-6; the mass volume ratio of the mixture of DIPEA, meOH, DMF and the 2-CTC resin is 3 mL-4 mL:1g.
Further, in the second step, fmoc protecting groups of Fmoc-Pro-2-CTC resin are removed specifically as follows: removing Fmoc protecting groups of the Fmoc-Pro-2-CTC resin by adopting a DMF solution containing piperidine; the volume fraction of piperidine in DMF solution containing piperidine is 10% -20%; the mass volume ratio of Fmoc-Pro-2-CTC to the piperidine-containing DMF solution is 1g:18 mL-30 mL.
Further, in the second step, the condensing agent used under the condensing condition includes: any one of PyBOP/HOAt/DIPEA, HATU/DIPEA, bop-Cl/NMM, bop-Cl/DIPEA, DIC/HOBt, HBTU/HOAt/DIPEA; the coupling time is 0.5 h-5 h.
Further, the molar ratio of the condensing agent to Fmoc-N-Me-D-Phe-OH under the condensing condition was (1-6): 1.
Further, in the third step, the Fmoc-Val-OH is coupled and condensed by using a Bop-Cl/DIPEA condensing agent; coupling Fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-N-Me-Leu-OH and Fmoc-Hba (tBu) -OH, and condensing by adopting a HATU/DIPEA condensing agent; when Fmoc-Ile-OH and Fmoc-Aha-OH are coupled, bop-Cl/NMM condensing agent is adopted for condensation.
In the fourth step, the cracking agent adopted in the cracking is a DCM solution of TFA with the mass percent of 0.5-1.0%; the mass volume ratio of the polypeptide resin 1 to the cracking agent is 1g (20 mL-50 mL); the cracking time is 1.5-3 h.
Further, in the fifth step, the acid binding agent includes: DIPEA, K 2 CO 3 、TEA、Na 2 CO 3 Any one or more of the following; the methylating agent used for methylation comprises MeI, DMS, CH 3 OTs、CH 3 Any one of OTf; the molar ratio of the methylating agent to the polypeptide 2 is (1-5): 1; acid bindingThe molar ratio of the agent to the methylating agent is 1:1; the methylation treatment time is 10-18 h.
Further, in the sixth step, the reagent for removing Fmoc protecting group of the crude Hoshinoamide A is TFA or Et 3 SiH、H 2 Mixing the mixed reagent obtained by mixing 95%, 2.5% and 2.5% of O by volume; HPLC purification is reversed-phase C4, C8 or C18 high performance liquid chromatography purification, and the column temperature is 20-50 ℃.
The beneficial effects of the invention are as follows: according to the embodiment of the invention, the Hoshinoamide A natural product is synthesized for the first time, and the compound can be prepared simply and rapidly by taking simple and cheap amino acid as a raw material, so that the synthesis steps are reduced; and the method can be used for scale-up production, providing sufficient amounts of the compounds for relevant pharmacodynamic, pharmacokinetic studies, and future use in antimalarial applications.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a structural formula of each material used in the synthesis process of Hoshinoamide A according to an embodiment of the present invention.
FIG. 2 is a flow chart of an all solid phase synthesis process of Hoshinoamide A according to embodiment 1 of the invention.
FIG. 3 is a hydrogen spectrum of Hoshinoamide A obtained in example 1 of the present invention.
FIG. 4 is a carbon spectrum of Hoshinoamide A obtained in example 1 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In order to verify the structure of Hoshinoamide A and complete the first total synthesis of Hoshinoamide A, a synthesis scheme is provided for the research of the structure-activity relationship of Hoshinoamide A, and the invention provides a total solid-phase synthesis method of Hoshinoamide A, which comprises the following steps:
step one: fmoc-Pro-OH was coupled to the 2-CTC resin to give Fmoc-Pro-2-CTC resin.
The first step specifically comprises the following steps: placing 2-CTC resin in a mixed solvent of DCM and DMF, and then adding Fmoc-Pro-OH and alkali liquor for coupling reaction; the resulting coupled product was purified by a mixture of DIPEA, meOH, DMF to give Fmoc-Pro-2-CTC resin.
The molar ratio of phenylalanine, fmoc-Pro-OH and alkali liquor in the 2-CTC resin is 1 (2-5) to 2-6.
The loading of the 2-CTC resin is 0.2 mmol/g-1.2 mmol/g; the volume ratio of DCM to DMF in the mixed solvent of DCM and DMF is 1 (1-5); lye includes any one of DIPEA, NMM, TEA; the volume ratio of DIPEA, meOH, DMF in the mixture of DIPEA, meOH, DMF is 1 (1-5) (5-10); the mass volume ratio of the mixture of DIPEA, meOH, DMF and the 2-CTC resin is 3 mL-4 mL:1g.
The time range of the coupling reaction is 2-10 h; the purification treatment time ranges from 2 hours to 8 hours; the reaction process of the whole step one is carried out at normal temperature.
Step two: removing Fmoc protecting groups of Fmoc-Pro-2-CTC resin, and coupling the obtained H-Pro-2-CTC resin with Fmoc-N-Me-D-Phe-OH under condensation conditions to obtain Fmoc-N-Me-D-Phe-Pro-2-CTC resin;
wherein, fmoc protecting group of Fmoc-Pro-2-CTC resin is removed by adopting DMF solution containing piperidine to obtain H-Pro-2-CTC resin. The volume fraction of piperidine in DMF solution containing piperidine is 10% -20%; the mass volume ratio of Fmoc-Pro-2-CTC to the piperidine-containing DMF solution is 1g:18 mL-30 mL.
Wherein the condensing agent used under the condensing condition comprises: any one of PyBOP/HOAt/DIPEA, HATU/DIPEA, bop-Cl/NMM, bop-Cl/DIPEA, DIC/HOBt, HBTU/HOAt/DIPEA.
The molar ratio of the condensing agent to the amino acid under the condensing condition is (1-6): 1; the condensation time is 0.5 to 5 hours.
Step three: according to Hoshinoamide A peptide sequence, fmoc-N-Me-D-Phe-Pro-2-CTC resin is coupled in sequence according to the method of the step two, wherein the coupling sequence is as follows: fmoc-Val-OH, fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-Ile-OH, fmoc-N-Me-Leu-OH, fmoc-Aha-OH, and Fmoc-Hba (tBu) -OH to give polypeptide resin 1.
Wherein, when Fmoc-Val-OH is coupled, bop-Cl/DIPEA condensing agent is adopted for condensation; coupling Fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-N-Me-Leu-OH and Fmoc-Hba (tBu) -OH, and condensing by adopting a HATU/DIPEA condensing agent; when Fmoc-Ile-OH and Fmoc-Aha-OH are coupled, bop-Cl/NMM condensing agent is adopted for condensation.
The polypeptide resin 1 is:
Hba-Aha-N-Me-Leu-Ile-N-Me-D-Val-Gln (Trt) -Val-N-Me-D-Phe-Pro-2-CTC resin.
Step four: cleaving the polypeptide resin 1 to obtain polypeptide 2.
Wherein, the cracking agent adopted for cracking is a DCM solution of TFA with the mass percent of 0.5-1.0%; the mass volume ratio of the polypeptide resin 1 to the cracking agent is 1g (20 mL-50 mL); the cracking time is 1.5-3 h.
Polypeptide 2: hba (tBu) -Aha-N-Me-Leu-Ile-N-Me-D-Val-Gln (Trt) -Val-N-Me-D-Phe-Pro-OH.
Step five: under the action of an acid binding agent, carrying out methylation treatment on the polypeptide 2 to obtain a Hoshinoamide A crude product;
wherein the acid binding agent comprises: DIPEA, K 2 CO 3 、TEA、Na 2 CO 3 Any one or more of the following.
The holhinoamide A crude product is specifically:
Hba(tBu)-Aha-N-Me-Leu-Ile-N-Me-D-Val-Gln(Trt)-Val-N-Me-D-Phe-Pro-OMe。
wherein the methylation reagent comprises MeI, DMS, CH 3 OTs、CH 3 Any one of OTf.
The molar ratio of the methylating agent to the polypeptide 2 is (1-5): 1.
The molar ratio of the acid binding agent to the methylation reagent is 1:1; the methylation treatment time is 10-18 h.
Step six: removing Fmoc protecting groups of the crude product of the Hoshinoamide A, and purifying and freeze-drying by HPLC to obtain the Hoshinoamide A.
Wherein the reagents for removing Fmoc protecting groups are TFA and Et 3 SiH、H 2 The mixed reagent obtained by mixing 95%, 2.5% and 2.5% of O by volume ratio.
HPLC purification is reversed-phase C4, C8 or C18 high performance liquid chromatography purification, and the column temperature is 20-50 ℃.
The method can take simple and cheap amino acid as raw material, simply and rapidly prepare the compound, solves the problem of low condensation efficiency of N-Me-amino acid by adopting a specific condensing agent, can be used for large-scale production, provides a sufficient amount of compound for related pharmacodynamics and pharmacokinetics research, and is applied to malaria resistance in the future.
The structural formula of each material adopted in the synthesis process of Hoshinoamide A is shown in figure 1. The Chinese name corresponding to English written in the application is:
Fmoc-X (Rx) -OH represents a protected amino acid; fmoc is fluorenylmethoxycarbonyl, representing the amino protecting group of an amino acid; x represents an amino acid; the amino acids referred to in this application are: pro proline, phe phenylalanine, val valine, gln glutamine, ile isoleucine, leu leucine; rx represents a side chain protecting group of an amino acid; rx referred to in this application is tBu t-butyl, trt trityl. Fmoc-Aha-OH represents amino-protected 6-aminocaproic acid; hba (tBu) -OH represents side chain protected 4- (4-hydroxyphenyl) butanoic acid.
CTC resin is also called 2-CTC resin, chinese name is 2-chlorotrityl chloride resin, english name is 2-Chlorotrityl ChlorideResin; DCM represents dichloromethane; DMF means N, N-dimethylformamide; DIPEA represents N, N-diisopropylethylamine; NMM represents N-methylmorpholine; TEA represents triethylamine; meOH represents methanol; HOAt represents N-hydroxy-7-azabenzeneAnd triazole; HATU represents 2- (7-azabenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate; bop-Cl represents bis (2-oxo-3-oxazolidinyl) hypophosphorous acid chloride; pyBOP represents benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate; DIC represents N, N' -diisopropylcarbodiimide; HOBt represents 1-hydroxybenzotriazole; HBTU represents O-benzotriazol-tetramethylurea hexafluorophosphate; me represents methyl; meI represents methyl iodide; DMS represents dimethyl sulfate; NMP represents N-methylpyrrolidone; DMSO represents dimethyl sulfoxide; CH (CH) 3 OTs represents methyl p-toluenesulfonate; piperidine represents Piperidine; CH (CH) 3 OTf represents methyl triflate; TFA represents trifluoroacetic acid; TIS represents triisopropylsilane; et (Et) 3 SiH represents triethylsilane.
Example 1
As shown in FIG. 2, 1.0mmol/g Fmoc-Pro-2-CTC resin was prepared: 10g of 2-CTC resin (1.0 mmol/g loading) was added to the flask, fmoc-Pro-OH (8.5 g,25 mmol) and DIPEA (8.6 mL,49 mmol) were added as solvents in DCM (30 mL) and DMF (30 mL) and stirred slowly at room temperature for 6h; the resin and reaction solution were filtered through a solid phase synthesis tube, and the resulting resin was alternately washed three times with MeOH (3X 50 mL) and DCM (3X 50 mL) and the resin was drained; pouring the obtained resin into a reaction bottle, adding 30mL of DIPEA, meOH, DMF mixed solution (volume ratio is 1:5:10), and slowly stirring for 4 hours at room temperature to seal the site which is not combined with amino acid in the 2-CTC resin; the reaction and resin were poured into a solid phase synthesis tube and washed three times alternately with MeOH (3X 50 mL) and DCM (3X 50 mL) and dried to give Fmoc-Pro-2-CTC resin.
Preparation of polypeptide resin 1: adding Fmoc-Pro-2-CTC resin into a solid phase synthesis tube, adding DMF (20 mL) with 20% of piperidine by volume fraction, blowing air by an air pump, finishing the reaction after 30min, alternately washing the resin three times with DCM (50 mL) and MeOH (50 mL), and pumping to obtain H-Pro-2-CTC resin; DMF (20 mL), fmoc-N-Me-D-Phe-OH (7 g,20 mmol), HATU (7.6 g,20 mmol) and DIPEA (3.5 mL,20 mmol) were added to the solid phase synthesis tube, and the reaction was completed after 1h of air pump blowing; the resin was washed with DCM (50 mL) and MeOH (50 mL) three times alternately, a small amount of the resin was added to a 1mL reaction flask after washing, kaiser reagent (one drop each of 80% phenol in ethanol, piperidine and 5% ninhydrin in ethanol) was added, and the mixture was heated to 110℃and reacted for 3min, and no discoloration of the resin indicated that the N-terminus had reacted completely, to give Fmoc-N-Me-D-Phe-Pro-2-CTC resin.
Adding DMF (50 mL) solution of piperidine with the volume fraction of 20% into a solid phase synthesis tube, and after 30min of air pump air blowing, ending the reaction; resin was washed with DCM (50 mL) and MeOH (50 mL) alternately three times, a small amount of resin was added to a 1mL reaction flask after washing, kaiser reagent was added, the reaction was carried out for 3min at 110℃until the resin turned blue or purple indicating that Fmoc had been removed, to give H-N-Me-D-Phe-Pro-2-CTC resin; to a solid phase synthesis tube containing H-N-Me-D-Phe-Pro-2-CTC resin, dimethylformamide DMF (20 mL), fmoc-Val-OH (6.7 g,20 mmol), bop-Cl (5.1 g,20 mmol) and DIPEA (3.5 mL,20 mmol) were added, the reaction was terminated after 2H, the resin was alternately washed three times with DCM (50 mL) and MeOH (5 mL), a small amount of the resin was added to a 1mL reaction flask after washing, kaiser reagent (80% phenol in ethanol, piperidine and 5% ninhydrin in ethanol) were added one drop each) and heated to 110℃for 3min, and no discoloration of the resin indicated that the N-terminus had reacted completely, yielding Fmoc-Val-N-Me-D-Phe-Pro-2-CTC resin.
Repeating the method for removing Fmoc protecting groups, adding the subsequent corresponding amino acid to continue the coupling step, and sequentially completing the connection according to the Hoshinoamide A main chain peptide sequence, wherein the coupling of Fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-N-Me-Leu-OH and Fmoc-Hba (tBu) -OH is performed by adopting a HATU/DIPEA condensing agent; and (3) condensing by using a Bop-Cl/NMM condensing agent when Fmoc-Ile-OH and Fmoc-Aha-OH are coupled to obtain the polypeptide resin 1.
Preparation of polypeptide 2: the polypeptide was cleaved from polypeptide resin 1 by stirring with 0.5% TFA in DCM (50 mL) for 2h, filtering, collecting the filtrate, washing the resin three times with DCM (10 mL) and MeOH (10 mL), combining the organic phases, and spin-drying to give polypeptide 2 as a foamy white solid (7.1 g,4.9mmol, 49% yield).
Preparation of a Hoshinoamide A crude product: polypeptide 2 (7.1 g,4.9 mmol) was dissolved in 30mL dry DMF and K was added 2 CO 3 (284 mg,6.0 mmol) and MeI (373. Mu.L, 6.0 mmol), were stirred at room temperature for 12h, 60mL of DCM was added to the reaction, washed five times with 30mL of water,the organic layer was dried by spin to give a white solid, crude Hoshinoamide A.
Preparation of Hoshinoamide A: the crude Hoshinoamide A was added to TFA: et (Et) 3 SiH：H 2 O (30 mL,95/2.5/2.5 v/v/v) for 3h at room temperature, and spin-drying to obtain oily compound; the oily compound was dissolved in MeOH (5 mL), and iced diethyl ether (95 mL) was added and centrifuged at 7000rpm with a centrifuge to obtain a Hoshinoamide A primary product; the initial Hoshinoamide A product was separated by preparative HPLC using a preparative HPLC column model Thermo Hypersil Gold (5 μm,150x 21.2 mm) of Scientific Ultimate, apparatus model used for preparative high performance liquid chromatography, mobile A phase: aqueous 0.5% tfa; mobile phase B: acetonitrile solution of 0.5% TFA at a flow rate of 8mL/min, detected using a wavelength of 220nm or 254 nm. Mobile phase gradient 10% -90% -90% -10% CH 3 CN/H 2 O,40min (10-90% MeCN 30min, 90-90% 5min, 90-10% 5 min) gave Hoshinoamide A (3.5 mg,3.1 mmol) in 31% overall yield. The Hoshinoamide A nuclear magnetic resonance hydrogen spectrum and the carbon spectrum of the main peak material are shown in fig. 3 and 4: 1 H NMR(400MHz,Methanol-d 4 )δ7.30-7.11(m,6H),7.02-6.94(m,2H),6.72-6.65(m,2H),5.73(dd,J＝9.2,6.4Hz,1H),4.82-4.73(m,1H),4.64–4.53(m,2H),4.43-4.25(m,2H),3.69(s,2H),3.50(dt,J＝11.2,6.0Hz,1H),3.41-3.31(m,1H),3.20-3.06(m,9H),2.99–2.92(m,3H),2.91-2.86(m,1H),2.52(t,J＝7.6Hz,2H),2.47-2.38(m,2H),2.29-2.22(m,3H),2.18(q,J＝7.7Hz,3H),2.05-1.99(m,1H),1.99-1.71(m,9H),1.68-1.46(m,6H),1.46-1.34(m,4H),1.11-0.97(m,3H),0.97-0.81(m,16H),0.68-0.55(m,6H). 13 C NMR(101MHz,MeOD)δ177.43,176.52,176.00,174.55,173.86,173.15,173.09,172.87,171.89,170.47,156.60,138.40,133.75,130.69,130.40,129.45,127.70,116.18,64.21,60.84,57.23,55.69,55.64,55.52,54.32,52.69,49.68,49.46,49.25,49.25,49.04,48.83,48.81,48.61,48.40,40.23,38.43,37.82,36.62,35.76,35.51,34.59,32.62,31.80,31.59,31.56,30.22,29.98,29.20,27.72,26.21,26.17,26.06,25.95,25.90,25.11,23.80,22.17,20.39,19.90,19.49,18.13,16.50,11.60.HRMS:(+ESI)Calc.for C 61 H 95 N 9 O 12 :1146.7173[M+H] + ,Found:1146.7173[M+H] + 。
example 2
10g of 2-CTC resin (loading 0.2 mmol/g) was added to the flask, fmoc-Pro-OH (10 mmol) and NMM (12 mmol) were added as solvents in DCM (10 mL) and DMF (50 mL) and stirred slowly at room temperature for 2h; filtering the resin and the reaction liquid by using a solid phase synthesis tube, alternately washing the obtained resin for three times, and pumping the resin; pouring the obtained resin into a reaction bottle, adding 40mL of DIPEA, meOH, DMF mixed solution (volume ratio is 1:1:5), and slowly stirring for 2 hours at room temperature to seal the sites which are not combined with amino acid in the 2-CTC resin; and pouring the reaction solution and the resin into a solid phase synthesis tube, alternately washing for three times, and pumping to obtain Fmoc-Pro-2-CTC resin.
Adding Fmoc-Pro-2-CTC resin into a solid phase synthesis tube, adding 18mL of 10% piperidine DMF solution, pumping air by an air pump, ending the reaction after 30min, washing the resin for three times alternately, and pumping to obtain H-Pro-2-CTC resin; DMF (20 mL), fmoc-N-Me-D-Phe-OH (7 g,20 mmol), bop-Cl (10 mmol) and NMM (10 mmol) are added into the solid phase synthesis tube, and the reaction is finished after the air pump is inflated for 1 h; and (3) washing the resin for three times alternately, taking a small amount of resin after washing, adding the resin into a 1mL reaction bottle, adding a Kaiser reagent, heating to 110 ℃, and reacting for 3min, wherein no color change of the resin indicates that the N end has reacted completely, so as to obtain Fmoc-N-Me-D-Phe-Pro-2-CTC resin.
Adding DMF (50 mL) solution with 10% piperidine by volume fraction into a solid phase synthesis tube, and after 30min of air pump air blowing, ending the reaction; alternately washing the resin for three times, adding a small amount of resin into a 1mL reaction bottle after washing, adding Kaiser reagent, heating to 110 ℃, reacting for 3min, and obtaining H-N-Me-D-Phe-Pro-2-CTC resin after the resin turns blue or becomes purple to indicate that Fmoc is removed; to a solid phase synthesis tube containing H-N-Me-D-Phe-Pro-2-CTC resin, dimethylformamide DMF (20 mL), fmoc-Val-OH (6.7 g,20 mmol), bop-Cl (60 mmol) and DIPEA (60 mmol) were added to the tube, the reaction was completed after 2 hours, the resin was alternately washed three times, a small amount of the resin was added to a 1mL reaction flask after washing, kaiser reagent was added, the reaction was heated to 110℃for 3 minutes, and no discoloration of the resin indicated that the N-terminal had reacted completely, to give Fmoc-Val-N-Me-D-Phe-Pro-2-CTC resin.
Repeating the method for removing Fmoc protecting groups, adding the subsequent corresponding amino acid to continue the coupling step, and sequentially completing the connection according to the Hoshinoamide A main chain peptide sequence, wherein the coupling of Fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-N-Me-Leu-OH and Fmoc-Hba (tBu) -OH is performed by adopting a HATU/DIPEA condensing agent; and (3) condensing by using a Bop-Cl/NMM condensing agent when Fmoc-Ile-OH and Fmoc-Aha-OH are coupled to obtain the polypeptide resin 1.
The polypeptide was cleaved from polypeptide resin 1 by stirring with 0.5% TFA in DCM (50 mL) for 2h, filtering, collecting the filtrate, washing the resin three times each, combining the organic phases, and spin-drying to give polypeptide 2 as a foamy white solid (4.3 mmol).
Polypeptide 2 (4.3 mmol) was dissolved in 30mL dry DMF and K was added 2 CO 3 (4.3 mmol) and MeI (4.3 mmol), stirred at room temperature for 10h, 60mL of DCM was added to the reaction, and the organic layer was dried by five times with 30mL of water to give a white solid as crude Hoshinoamide A.
The crude Hoshinoamide A was added to TFA: et (Et) 3 SiH：H 2 O (30 mL,95/2.5/2.5 v/v/v) for 3h at room temperature, and spin-drying to obtain oily compound; the oily compound was dissolved in MeOH (5 mL), and iced diethyl ether (95 mL) was added and centrifuged at 7000rpm with a centrifuge to obtain a Hoshinoamide A primary product; the initial product of Hoshinoamide A was isolated by preparative HPLC to give Hoshinoamide A (2.9 mmol) in 29% overall yield.
Example 3
10g of 2-CTC resin (1.2 mmol/g loading) was added to the flask, fmoc-Pro-OH (24 mmol) and NMM (24 mmol) were added as solvents in DCM (20 mL) and DMF (40 mL) and stirred slowly at room temperature for 10h; filtering the resin and the reaction liquid by using a solid phase synthesis tube, alternately washing the obtained resin for three times, and pumping the resin; pouring the obtained resin into a reaction bottle, adding 36mL of DIPEA, meOH, DMF mixed solution (volume ratio is 1:3:8), and slowly stirring at room temperature for 8 hours to seal the site which is not combined with amino acid in the 2-CTC resin; and pouring the reaction solution and the resin into a solid phase synthesis tube, alternately washing for three times, and pumping to obtain Fmoc-Pro-2-CTC resin.
Adding Fmoc-Pro-2-CTC resin into a solid phase synthesis tube, adding 30mL of DMF solution with 15% of piperidine volume fraction, blowing air by an air pump, finishing the reaction after 30min, washing the resin for three times alternately, and pumping to obtain H-Pro-2-CTC resin; DMF (20 mL), fmoc-N-Me-D-Phe-OH (7 g,20 mmol), pyBOP (40 mmol), HOAt (40 mmol) and DIPEA (40 mmol) are added into the solid phase synthesis tube, and the reaction is finished after the air pump is inflated for 1 h; and (3) washing the resin for three times alternately, taking a small amount of resin after washing, adding the resin into a 1mL reaction bottle, adding a Kaiser reagent, heating to 110 ℃, and reacting for 3min, wherein no color change of the resin indicates that the N end has reacted completely, so as to obtain Fmoc-N-Me-D-Phe-Pro-2-CTC resin.
Adding DMF (50 mL) solution with 10% piperidine by volume fraction into a solid phase synthesis tube, and after 30min of air pump air blowing, ending the reaction; alternately washing the resin for three times, adding a small amount of resin into a 1mL reaction bottle after washing, adding Kaiser reagent, heating to 110 ℃, reacting for 3min, and obtaining H-N-Me-D-Phe-Pro-2-CTC resin after the resin turns blue or becomes purple to indicate that Fmoc is removed; to a solid phase synthesis tube containing H-N-Me-D-Phe-Pro-2-CTC resin, dimethylformamide DMF (20 mL), fmoc-Val-OH (6.7 g,20 mmol), bop-Cl (10 mmol) and DIPEA (10 mmol) were added to the tube, the reaction was completed after 2 hours, the resin was alternately washed three times, a small amount of the resin was added to a 1mL reaction flask after washing, kaiser reagent was added, the reaction was heated to 110℃for 3 minutes, and no discoloration of the resin indicated that the N-terminal had reacted completely, to give Fmoc-Val-N-Me-D-Phe-Pro-2-CTC resin.
Repeating the method for removing Fmoc protecting groups, adding the subsequent corresponding amino acid to continue the coupling step, and sequentially completing the connection according to the Hoshinoamide A main chain peptide sequence, wherein the coupling of Fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-N-Me-Leu-OH and Fmoc-Hba (tBu) -OH is performed by adopting a HATU/DIPEA condensing agent; and (3) condensing by using a Bop-Cl/NMM condensing agent when Fmoc-Ile-OH and Fmoc-Aha-OH are coupled to obtain the polypeptide resin 1.
The polypeptide was cleaved from polypeptide resin 1 by stirring with 0.5% TFA in DCM (20 mL) for 3h, filtering, collecting the filtrate, washing the resin three times each, combining the organic phases, and spin-drying to give polypeptide 2 as a foamy white solid (4.5 mmol).
Polypeptide 2 was dissolved in 30mL of dry DMF and K was added 2 CO 3 (9 mmol) and MeI (9 mmol), stirred at room temperature for 18h, 60mL of DCM was added to the reaction, and the mixture was washed with 30mL of waterFive times, spin-dry the organic layer to give a white solid, crude Hoshinoamide A.
The crude Hoshinoamide A was added to TFA: et (Et) 3 SiH：H 2 O (30 mL,95/2.5/2.5 v/v/v) for 3h at room temperature, and spin-drying to obtain oily compound; the oily compound was dissolved in MeOH (5 mL), and iced diethyl ether (95 mL) was added and centrifuged at 7000rpm with a centrifuge to obtain a Hoshinoamide A primary product; the initial product of Hoshinoamide A was isolated by preparative HPLC to give Hoshinoamide A (3.3 mmol) in a total yield of 33%.
Comparative example
The procedure of example 1 was followed except that Fmoc-Val-OH was followed using HCTU condensing agent (40 mmol).
The yield of Hoshinoamide A obtained in this comparative example was 10%.
Experimental results illustrate: hoshinoamide A was successfully prepared. The all-solid-phase synthesis method of the Hoshinoamide A has the characteristics of simplicity in operation, few synthesis steps, short period, low cost and the like, and is favorable for the all-production research of the Hoshinoamide A and the synthesis of derivatives thereof.
It should be noted that in this application relational terms such as first, second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
In this specification, each embodiment is described in a related manner, and identical and similar parts of each embodiment are all referred to each other, and each embodiment mainly describes differences from other embodiments.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.
Claims (1)
1. The all-solid-phase synthesis method of the Hoshinoamide A is characterized by comprising the following steps of:
step one: coupling Fmoc-Pro-OH to the 2-CTC resin to obtain Fmoc-Pro-2-CTC resin;
step two: removing Fmoc protecting groups of Fmoc-Pro-2-CTC resin, and coupling the obtained H-Pro-2-CTC resin with Fmoc-N-Me-D-Phe-OH under condensation conditions to obtain Fmoc-N-Me-D-Phe-Pro-2-CTC resin;
step three: according to Hoshinoamide A peptide sequence, fmoc-N-Me-D-Phe-Pro-2-CTC resin is coupled in sequence according to the method of the step two, wherein the coupling sequence is as follows: fmoc-Val-OH, fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-Ile-OH, fmoc-N-Me-Leu-OH, fmoc-Aha-OH, and Fmoc-Hba (tBu) -OH to obtain polypeptide resin 1;
step four: splitting the polypeptide resin 1 to obtain a polypeptide 2;
step five: under the action of an acid binding agent, carrying out methylation treatment on the polypeptide 2 to obtain a Hoshinoamide A crude product;
step six: removing Fmoc protecting groups of the crude Hoshinoamide A product, purifying by HPLC, and freeze-drying to obtain Hoshinoamide A;
the first step specifically comprises the following steps: placing the 2-CTC resin in a mixed solvent of DCM and DMF, adding Fmoc-Pro-OH and alkali liquor, and performing coupling reaction for 2-10 h; purifying the obtained coupling product by a mixture of DIPEA, meOH, DMF for 2-8 hours to obtain Fmoc-Pro-2-CTC resin; the volume ratio of DCM to DMF in the mixed solvent of DCM and DMF is 1 (1-5); the loading capacity of the 2-CTC resin is 0.2 mmol/g-1.2 mmol/g; the lye includes any one of DIPEA, NMM, TEA; the volume ratio of DIPEA, meOH, DMF in the mixture of DIPEA, meOH, DMF is 1 (1-5) (5-10);
the molar ratio of phenylalanine, fmoc-Pro-OH and alkali liquor in the 2-CTC resin is 1 (2-5) (2-6); the mass volume ratio of the mixture of DIPEA, meOH, DMF and the 2-CTC resin is (3 mL-4 mL): 1g;
in the second step, the condensing agent used under the condensing condition comprises: any one of PyBOP/HOAt/DIPEA, HATU/DIPEA, bop-Cl/NMM, bop-Cl/DIPEA, DIC/HOBt, HBTU/HOAt/DIPEA; the coupling time is 0.5 h-5 h;
in the second step, the Fmoc protecting group for removing the Fmoc-Pro-2-CTC resin is specifically as follows: removing Fmoc protecting groups of the Fmoc-Pro-2-CTC resin by adopting a DMF solution containing piperidine; the volume fraction of piperidine in the DMF solution containing piperidine is 10% -20%; the mass volume ratio of the Fmoc-Pro-2-CTC to the DMF solution containing piperidine is 1g:18 mL-30 mL;
the molar ratio of the condensing agent to Fmoc-N-Me-D-Phe-OH under the condensing condition is (1-6): 1;
in the third step, when Fmoc-Val-OH is coupled, a Bop-Cl/DIPEA condensing agent is adopted for condensation; coupling the Fmoc-Gln (Trt) -OH, fmoc-N-Me-D-Val-OH, fmoc-N-Me-Leu-OH and Fmoc-Hba (tBu) -OH, and condensing by adopting a HATU/DIPEA condensing agent; when Fmoc-Ile-OH and Fmoc-Aha-OH are coupled, a Bop-Cl/NMM condensing agent is adopted for condensation;
in the fourth step, the cracking agent adopted in the cracking is a DCM solution of TFA with the mass percent of 0.5-1.0%; the mass volume ratio of the polypeptide resin 1 to the cracking agent is 1g (20 mL-50 mL); the cracking time is 1.5-3 h;
in the fifth step, the acid binding agent comprises: DIPEA, K 2 CO 3 、TEA、Na 2 CO 3 Any one or more of the following; the methylation reagent comprises MeI, DMS, CH 3 OTs、CH 3 Any one of OTf; the molar ratio of the methylation reagent to the polypeptide 2 is (1-5): 1; the molar ratio of the acid binding agent to the methylation reagent is 1:1; the methylation treatment time is 10-18 h;
in the sixth step, fmoc protection of the Hoshinoamide A crude product is removedThe reagent of the group is TFA, et 3 SiH、H 2 Mixing the mixed reagent obtained by mixing 95%, 2.5% and 2.5% of O by volume; the HPLC purification is reversed-phase C4, C8 or C18 high performance liquid chromatography purification, and the column temperature is 20-50 ℃.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0388982A1 (en) * | 1989-03-24 | 1990-09-26 | Bristol-Myers Squibb Company | Pradimicin amide derivatives |
| CN107827973A (en) * | 2017-09-15 | 2018-03-23 | 吴忠臣 | A kind of solid phase synthesis process of Liraglutide |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0388982A1 (en) * | 1989-03-24 | 1990-09-26 | Bristol-Myers Squibb Company | Pradimicin amide derivatives |
| CN107827973A (en) * | 2017-09-15 | 2018-03-23 | 吴忠臣 | A kind of solid phase synthesis process of Liraglutide |
Non-Patent Citations (2)
| Title |
|---|
| First total synthesis of Hoshinoamides A;Haipin Zhou et al;《BEILSTEIN ARCHIVES》;方案1-2，5-13 * |
| 克林沙星酰胺衍生物的简易合成及其体内外生物活性;韩海燕;陈力;徐兴然;范莉;杨艳;杨大成;;中国科学:化学(第03期);全文 * |
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