CN113929749A - Sixteen-peptide compound and preparation method thereof - Google Patents
Sixteen-peptide compound and preparation method thereof Download PDFInfo
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- CN113929749A CN113929749A CN202111371773.7A CN202111371773A CN113929749A CN 113929749 A CN113929749 A CN 113929749A CN 202111371773 A CN202111371773 A CN 202111371773A CN 113929749 A CN113929749 A CN 113929749A
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Abstract
The invention discloses a sixteen peptide compound and a preparation method thereof, wherein the sixteen peptide has the sequence as follows: Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Met-Gln-Arg-Ala, wherein Xaa (1) ═ Thr or Ser, Xaa (2) ═ Glu or Asp, Xaa (3) ═ Lys or Arg, Xaa (4) ═ Leu, Ile or Val. The sixteen-peptide is divided into 3 sections of full-protection fragments, a 7+4+5 strategy is adopted, solid-phase fragment assembly connection, cutting and cracking are sequentially carried out, and a crude product is prepared and purified to obtain the target sixteen-peptide. The invention adopts the modes of long peptide segmentation and short peptide assembly, and overcomes the problems of low yield, poor purity of crude products and difficult purification of the traditional solid-phase synthesis method by one-by-one extension synthesis. Greatly improves the synthesis yield, is easy to purify crude products, and can be used for preparing the sixteen-peptide in a large scale.
Description
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a hexadecapeptide compound and a preparation method thereof.
Background
The botulinum toxin is a neurotransmitter-inhibiting peptide, and can participate in competition for SNAP-25 at the site of a vacuolar complex to promote the vesicle not to effectively release the neurotransmitter, so that the effect of weakening muscle contraction is achieved. The prior art of the botulinum toxin polypeptide, such as acetyl hexapeptide-8 (commercial hexapeptide), is a famous wrinkle-removing active ingredient, and the mechanism thereof is to inhibit the release of neurotransmitter acetylcholine (acetylcholine), so that the contraction of muscles can be reduced, and the generation of dynamic lines and expression lines can be reduced. However, the problems of small effective binding area and poor affinity exist between the acetyl hexapeptide-8 and the target point Rabphilin-3A.
In the field of polypeptide synthesis, solid-phase synthesis is mostly adopted for synthesis, specifically, C-terminal amino acid is bonded on a solid-phase carrier, and the C-terminal amino acid is extended one by one and sequentially synthesized to form an amido bond. The disadvantage of the polypeptide one-by-one extension method is that: the solid phase reaction is difficult to realize that each step can be fully recrystallized or subjected to column chromatography operation, the by-products and the deletion peptides of each step of condensation can only be accumulated until the sequence synthesis is finished, the purity of the crude peptide after cracking is poor, and the preparation and purification are difficult; meanwhile, for a polypeptide sequence consisting of sixteen amino acids, the loading capacity of a solid phase carrier for multiple selection is SD (0.4-0.6 mmol/g), the synthesis yield is low, the purity of a crude product is not high, the preparation and purification loss is large, and the overall synthesis yield is low; the synthesis also uses liquid phase fragment synthesis and solid phase assembly to synthesize polypeptide, and the method is suitable for short peptide and medium short peptide sequences. This approach is not applicable to sixteen peptides with longer sequences and more difficult amino acid sequences.
Disclosure of Invention
In view of the above, the invention provides a hexadecapeptide compound and a preparation method thereof, wherein the hexadecapeptide compound has stronger affinity with Rabphilin-3A, has more excellent anti-wrinkle, compact and repairing effects, and simultaneously provides a preparation method of the hexadecapeptide which has high synthesis yield, is easy to purify and can be scaled.
In one embodiment, the polypeptide sequence of the sixteen peptide compound is: C-Xaa (1) -Xaa (2) -FM-Xaa (3) -NE-Xaa (4) -EEMQRRA, wherein Xaa (1) ═ Thr or Ser, Xaa (2) ═ Glu or Asp, Xaa (3) ═ Lys or Arg, Xaa (4) ═ Leu, Ile or Val.
Different from the prior art, the sixteen-peptide is an active polypeptide containing sixteen amino acids, and the sequence of the active polypeptide is as follows: Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Glu-Met-Gln-Arg-Ala, wherein Xaa (1) ═ Thr or Ser (Thr and Ser are both hydroxyl amino acids), Xaa (2) ═ Glu or Asp (Glu and Asp are both acidic amino acids), Xaa (3) ═ Lys or Arg (Lys and Arg are both basic amino acids), Xaa (4) ═ Leu, Ile or Val (Leu, Ile and Val are both alkyl branched chain amino acids), the polypeptide sequence is designed by computer-aided simulation and structure, and the key amino acids are intercepted according to the effective binding amino acid site between SNAP25 and Rabphilin-3A, and recombined and designed to obtain a new polypeptide sequence.
The hexadecapeptide is a botulinum toxin-like polypeptide, has an action mechanism similar to that of acetyl hexapeptide-8 (commercially available hexapeptide), has strong affinity with Rabphilin-3A, is improved by 5-6 orders of magnitude, has a special-SH structure capable of being combined with gold nanoparticles, and greatly enhances the transdermal property and the degradation resistance. Compared with the currently marketed hexapeptide, the hexadecapeptide has more excellent anti-wrinkle, tightening and repairing effects.
In one embodiment, the method of making the sixteen-peptide compound comprises the steps of:
fully protected pentapeptide resin was used: Fmoc-Met-Gln (Trt) -Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin; fully protected tetrapeptide fragments: Fmoc-Glu (OtBu) -Leu-Glu (OtBu) -COOH; with fully protected heptapeptide fragments: Fmoc-Cys (Trt) -Ser (tBu) -Glu (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -OH; solid phase assembling to obtain fully-protected hexadecapeptide resin, removing side chain protection, separating and purifying to obtain the target hexadecapeptide compound: Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Glu-Met-Gln-Arg-Arg-Ala.
The prior art is distinguished: the preparation method of the hexadecapeptide compound adopts a solid phase sectional assembly 5+4+7 strategy, and compared with a solid phase synthesis one-by-one extension method, the purity of the polypeptide crude product is increased, and the total preparation yield is obviously improved. The advantages of the segment assembly strategy of the present invention are: the sixteen-peptide is divided into three peptide segments, namely three amino acids are bonded on a carrier, so that the continuous accumulation of impurities is controlled, the three peptide segments have shorter sequences and higher synthetic purity, and fragment recrystallization or column chromatography is not required. The yield and the purity of the polypeptide crude product after solid phase assembly are obviously improved, and the prepared and purified by-product has larger structural difference with the main product and is easy to purify; the self-designed segmentation mode of the tripeptide segment is determined according to the reactive activity of an amino component and a carboxyl component in the synthesis process of the polypeptide, avoids difficult amino acid sequences such as Arg (Lys), Leu (Ile) and Gln, selects Met and Glu with the highest reactive activity as the amino component or the carboxyl component, and directly improves the reaction conversion rate of segment assembly.
In one embodiment, the fully protected pentapeptide resin is synthesized by:
the method comprises the following steps: adopting 2-Chlorotrityl Chloride Resin as a carrier, swelling the 2-Chlorotrityl Chloride Resin with dichloromethane, adding Fmoc-Ala-OH and N, N-diisopropylethylamine, reacting at normal temperature for 1-2 hours under the protection of nitrogen, performing suction filtration, and washing with isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Ala-2-Chlorotrityl Chloride Resin;
step two: removing Fmoc protecting groups once from the obtained Fmoc-Ala-2-Chlorotrityl Chloride Resin by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, adding N, N-dimethylformamide, Fmoc-Arg (Pbf) -OH, 1-hydroxyphenyltriazole and N, N' -diisopropylcarbodiimide, reacting at normal temperature for 2-3 hours under the protection of nitrogen, performing suction filtration, and washing a filter cake by using isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin;
step three: by adopting a second step method, Fmoc-Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin is connected with Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH and Fmoc-Met-OH in sequence to obtain Fmoc-Met-Gln (Trt) -Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin.
In one embodiment, the fully protected tetrapeptide fragment is synthesized by:
adopting 2-Chlorotrityl Chloride Resin as a carrier, swelling the 2-Chlorotrityl Chloride Resin with dichloromethane, adding Fmoc-Glu (OtBu) -OH and N, N-diisopropylethylamine, reacting at normal temperature for 1-2 hours under the protection of nitrogen, performing suction filtration, and washing with isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Glu (OtBu) -2-Chlorotrityl Chloride Resin;
removing Fmoc protecting groups of the obtained Fmoc-Glu (OtBu) -2-Chlorotrityl Chloride Resin once by using a mixed solution with the volume ratio of piperidine to N, N-dimethylformamide being 1:4, adding N, N-dimethylformamide, 1-hydroxy phenylpropyl triazole and N, N' -diisopropyl carbodiimide, sequentially connecting Fmoc-Glu (OtBu) -OH, Fmoc-Xaa (4) -OH and Fmoc-Glu (OtBu) -OH, reacting for 2-3 hours at normal temperature under the protection of nitrogen, performing suction filtration, and washing a filter cake by using isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -2-Chlorotrityl Chloride Resin;
adding lysis solution of trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 20:7.25:72.5 into Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -Met-2-Chlorotrityl Chloride Resin, stirring at normal temperature for 2-3 hours, filtering, adding NaHCO3Adjusting the filtrate to be neutral by using an aqueous solution, combining aqueous phases, and removing the organic solvent under reduced pressure to obtain Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -COOH.
In one embodiment, the fully protected heptapeptide fragment is synthesized by:
adopting 2-Chlorotrityl Chloride Resin as a carrier, swelling the 2-Chlorotrityl Chloride Resin with dichloromethane, adding Fmoc-Asn (Trt) -OH and N, N-diisopropylethylamine, reacting at normal temperature for 1-2 hours under the protection of nitrogen, performing suction filtration, and sequentially washing with isopropanol and N, N-dimethylformamide to obtain Fmoc-Asn (Trt) -2-Chlorotrityl Chloride Resin;
removing Fmoc protecting groups from the obtained Fmoc-Glu (OtBu) -2-Chlorotrityl Chloride Resin once by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, adding N, N-dimethylformamide, 1-hydroxy-phenylpropyl triazole and N, N' -diisopropyl carbodiimide, sequentially connecting Fmoc-Xaa (3) -OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Xaa (2) -OH, Fmoc-Xaa (1) -OH and Fmoc-Cys (Trt) -OH under the protection of nitrogen, reacting at normal temperature for 2-3 hours, filtering, washing the filter cake with isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Cys (Trt) -Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn (Trt) -2-Chlorotrityl Chloride Resin;
adding trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 20:7.25:72.5 into Fmoc-Cys (Trt) -Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn (Trt) -2-Chlorotrityl Chloride Resin, and carrying out post-reaction treatment to obtain Fmoc-Cys (Trt) -Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn (Trt) -OH.
In one embodiment, the fully protected hexadecapeptide resin is synthesized by the following steps:
removing Fmoc once from the fully protected pentapeptide Resin by using a mixed solution with a volume ratio of piperidine to N, N-dimethylformamide of 1:4, adding N, N-dimethylformamide, a fully protected tetrapeptide fragment, 1-hydroxyphenyltriazole, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate and N-methylmorpholine, stirring at normal temperature for 2-4 hours under the protection of nitrogen to obtain Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin, and connecting the Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Ala-2-Chlorotrityl Resin by the same method on the basis of Fmoc-Glu (OtBu) -Xaa (4) -Glu) (OtBu) -Glu) (OtBu-Met-Gln) (Trt) and Arg (Pbf) -Arg-Pbf) -Ala-2-Chlorotrityl Resin And (4) fully protecting the heptapeptide fragment to obtain the fully protected hexadecapeptide resin.
The preferable condensing agent is 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate, and the reaction activity is high; the preferable organic base is N-methylmorpholine, and has strong alkalinity and strong catalytic capability. The combination of the condensing agent and the organic base can carry out fragment assembly more quickly and efficiently, inhibit the epimeric racemization side reaction of the polypeptide synthetic amino acid, and have high reaction rate and high yield.
In one embodiment, the hexadecapeptide compound is synthesized by the following steps:
adding a cutting fluid with the volume ratio of trifluoroacetic acid to triisopropylsilane to phenylmethylsulfide to pure water of 88:5:4:3 into the fully protected hexadecyl peptide resin, reacting for 2-3 hours in an ice bath, carrying out suction filtration, carrying out vacuum concentration on the filtrate, adding glacial ethyl ether into the concentrated solution for crystallization to obtain a hexadecyl peptide crude product, purifying the crude product by reverse phase chromatography, carrying out salt transformation on an ammonium acetate system, and carrying out freeze drying to obtain the target hexadecyl peptide, namely Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Met-Gln-Arg-Arg-Ala.
In one embodiment, the molar ratio of the 2-Chlorotrityl Chloride Resin to Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Met-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Xaa (4) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Xaa (3) -OH, Fmoc-Phe-OH, Fmoc-Xaa (2) -OH, Fmoc-Xaa (1) -OH, Fmoc-Cys (Trt) -OH is 1 to 3; the molar ratio of the 2-Chlorotrityl Chloride Resin to the 1-hydroxy phenylpropyl triazole to the N, N' -diisopropyl carbodiimide is 1 (2-4): (2-4).
In one embodiment, the molar ratio of the fully-protected pentapeptide resin to the fully-protected tetrapeptide fragment, the fully-protected heptapeptide fragment, 1-hydroxy benzotriazole, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate and N-methylmorpholine is 1:2:2:3:4 (4-6).
In one embodiment, the 2-Chlorotrityl Chloride Resin has an SD of 1.0 to 1.6 mmol/g. It is understood that, when 2-chlorotitinyl Chloride Resin with SD of 1.0-1.6mmol/g is used, the substitution degree of the solid phase carrier is only 0.4-0.5 mmol/g compared with the one-by-one extension method, and the synthesis yield is increased by 1-1.5 times under the same feeding condition from the atom economy point of view.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is an HPLC chromatogram of the acetate salt of hexadecapeptide obtained in example 2;
FIG. 2 is an ESI-MS spectrum of the acetate salt of hexadecapeptide obtained in example 2;
FIG. 3 is an HPLC chromatogram of crude hexadecapeptide synthesized by the solid-phase one-by-one extension method of comparative example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The abbreviations in the specification are as follows, and 2-Chlorotrityl Chloride Resin has degrees of substitution of 1.1mmol/g, 1.6mmol/g and 0.47 mmol/g; OtBu: a tert-butoxy group; trt: a trityl group; pbf: 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl; tBu: a tertiary butyl group; the analytical high performance liquid chromatograph is Agilent 1260; the preparative high performance liquid chromatograph is Innovative Hengtong LC3000, the C18 analytical chromatographic column is Dalian physical chemistry research institute 4.6mm multiplied by 250mm, and the C18 preparative chromatographic column is Chengdu science popularization biology Limited company 10cm multiplied by 65 cm; the mass spectrometer was a Waters UPLC-Mass spectrometer.
Example 1
The polypeptide sequence of the hexadecapeptide compound is as follows: Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Met-Gln-Arg-Ala, wherein Xaa (1) ═ Thr or Ser, Xaa (2) ═ Glu or Asp, Xaa (3) ═ Lys or Arg, Xaa (4) ═ Leu, Ile or Val. .
Different from the prior art, the sixteen-peptide is an active polypeptide containing sixteen amino acids, and the sequence of the active polypeptide is as follows: Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Glu-Met-Gln-Arg-Ala, wherein Xaa (1) ═ Thr or Ser (Thr and Ser are both hydroxyl amino acids), Xaa (2) ═ Glu or Asp (Glu and Asp are both acidic amino acids), Xaa (3) ═ Lys or Arg (Lys and Arg are both basic amino acids), Xaa (4) ═ Leu, Ile or Val (Leu, Ile and Val are both alkyl branched chain amino acids), the polypeptide sequence is designed by computer-aided simulation and structure, and the key amino acids are intercepted according to the effective binding amino acid site between SNAP25 and Rabphilin-3A, and recombined and designed to obtain a new polypeptide sequence.
The hexadecapeptide is a botulinum toxin-like polypeptide, has an action mechanism similar to that of acetyl hexapeptide-8 (commercially available hexapeptide), has strong affinity with Rabphilin-3A, is improved by 5-6 orders of magnitude, has a special-SH structure capable of being combined with gold nanoparticles, and greatly enhances the transdermal property and the degradation resistance. Compared with the currently marketed hexapeptide, the hexadecapeptide has more excellent anti-wrinkle, tightening and repairing effects.
Example 2
The preparation method of the hexapeptide compound comprises the following steps:
1. synthesizing a fully protected pentapeptide resin:
(1.1): weighing 2-Chlorotrityl Chloride Resin (SD is 1.1mmol/g), adding into a synthetic column, adding 50mL of dichloromethane, swelling for 1 hour, carrying out suction filtration, adding 60mL of dichloromethane, 3.76g (12.1mmol) of Fmoc-Ala-OH and 7.3mL (44mmol) of N, N-diisopropylethylamine, reacting for 2 hours at normal temperature under the protection of nitrogen, carrying out suction filtration on the reaction solution, adding 5mL of anhydrous methanol, 45mLN, N-diisopropylethylamine, blocking unreacted active sites for 30min, carrying out suction filtration, washing the filter cake twice with isopropanol and N, N-dimethylformamide in sequence, and obtaining Fmoc-Ala-2-Chlorotrityl Chloride Resin after 50mL each time.
(1.2): adding 60mL of N, N-dimethylformamide solution of 20% piperidine into Fmoc-Ala-2-Chlorotrityl Chloride Resin, removing the Fmoc protecting group for 30min, performing suction filtration, washing a filter cake twice with isopropanol and N, N-dimethylformamide sequentially, wherein each time is 60mL, ninhydrin detects that the Resin is positive, and the Resin is black; 60mL of N-dimethylformamide, 14.3g (22mmol) of Fmoc-Arg (Pbf) -OH, 4.5g (33mmol) of 1-hydroxyphenyltriazole and 5.1mL (33mmol) of N, N' -diisopropylcarbodiimide are added into a synthetic column, and the mixture is reacted for 2 hours at normal temperature under the protection of nitrogen, ninhydrin detects that the Resin is negative, the Resin is colorless and transparent, the mixture is filtered by suction, and a filter cake is washed twice by isopropanol and N, N-dimethylformamide in sequence and 60mL of the filter cake each time, so that Fmoc-Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin is obtained.
(1.3): according to the method of step (1.2), Fmoc-Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin was sequentially linked with 14.3g (22mmol) of Fmoc-Arg (Pbf) -OH, 13.4g (22mmol) of Fmoc-Gln (Trt) -OH, and 8.2g of Fmoc-Met-OH to obtain a fully protected pentapeptide Resin Fmoc-Met-Gln (Trt) -Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin.
2. Synthesis of fully protected tetrapeptide fragments:
(2.1) 15g of 2-Chlorotrityl Chloride Resin (SD ═ 1.6mmol/g) was charged, and 11.2g (26.4mmol) of Fmoc-Glu (OtBu) -OH was ligated upward according to the method of step (1.1) to give Fmoc-Glu (OtBu) -2-Chlorotrityl Chloride Resin.
(2.2) 20.4g (48mmol) of Fmoc-Glu (OtBu) -OH, 16.9g (48mmol) of Fmoc-Leu-OH, 20.4g (48mmol) of Fmoc-Glu (OtBu) -OH were sequentially ligated according to the procedure of step (1.2) to obtain Fmoc-Glu (OtBu) -Leu-Glu (OtBu) -2-Chlorotrityl Chloride Resin.
(2.3) to Fmoc-Glu (OtBu) -Leu-Glu (OtBu) -Met-2-Chlorotrityl Chloride Resin, 50mL of trifluoroethanol, 18.8mL of acetic acid, and 181.2mL of dichloromethane were added, reacted at ordinary temperature for 2 hours, filtered, the cake was washed three times with 30mL of dichloromethane, the filtrates were combined, NaHCO was added3Adjusting the pH value of the filtrate to be 7 by using an aqueous solution, merging water phases, removing an organic solvent at 40 ℃ under reduced pressure, separating out a white solid in the water phase, filtering, washing filter cakes by using saturated saline and pure water respectively, and drying at 40 ℃ to obtain 20.7g of the fully-protected tetrapeptide fragment, namely Fmoc-Glu (OtBu) -Leu-Glu (OtBu) -COOH with the yield of 95%.
3. Synthesis of fully protected heptapeptide fragments:
(3.1) feeding 2-Chlorotrityl Chloride Resin (SD ═ 1.6mmol/g)15g, and connecting 15.7g (26.4mmol) Fmoc-Asn (Trt) -OH according to the method of the step (1.1) to obtain Fmoc-Asn (Trt) -2-Chlorotrityl Chloride Resin;
(3.2) connecting 31.1g (48mmol) of Fmoc-Arg (Pbf) -OH, 17.8g (48mmol) of Fmoc-Met-OH, 18.5g (48mmol) of Fmoc-Phe-OH, 20.4g (48mmol) of Fmoc-Glu (OtBu) -OH, 18.4g (48mmol) of Fmoc-Ser (tBu) -OH, 28.1g (48mmol) of Fmoc-Cys (Trt) -OH in this order according to the method of step (1.2) to obtain Fmoc-Cys (Trt) -Ser tBu-Glu (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -2-Chlorotrityl Chloride Resin;
(3.3) to Fmoc-Cys (Trt) -Ser (tBu) -Glu (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -2-Chlorotrityl Chloride Resin, 80mL of trifluoroethanol, 30mL of acetic acid, and 1290mL of dichloromethane were added, and after-treatment of the reaction, 38.2g of the all-protected heptapeptide fragment, i.e., Fmoc-Cys (Trt) -Ser (tBu) -Glu (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -OH, was obtained in a yield of 93%.
4. Assembly of sixteen peptides:
removing Fmoc protecting groups of fully-protected pentapeptide Resin Fmoc-Met-Gln (Trt) -Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin by using 80mL of N, N-dimethylformamide solution of 20% piperidine, performing suction filtration on reaction liquid, washing the reaction liquid for 2 times by using isopropanol and N, N-dimethylformamide sequentially, wherein 70mL of each time is used, ninhydrin detects that the Resin is positive, and the Resin is black; weighing 20g (22mmol) of Fmoc-Glu (OtBu) -Leu-Glu (OtBu) -COOH and 4.5g (33mmol) of 1-hydroxy phenylpropanetriazole and 18.2g (44mmol) of 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate into 100mLN, N-dimethylformamide, dissolving and clarifying, adding 4.6mL (44mmol) of N-methylmorpholine for activation for 5min, adding the activated reaction solution into a resin carrier, reacting for 3 hours at normal temperature under the protection of nitrogen, detecting that the resin is negative through ninhydrin, the resin is colorless and transparent, filtering the reaction solution, washing filter cakes with isopropanol and N, N-dimethylformamide sequentially, and obtaining 80mL of Fmoc-Glu (OtBu) -Leu-Glu (OtBu) -Arg (Met-Gln Trt) -Pbf) -Arg at each time to obtain Pbf-Ala -2-Chlorotrityl Chloride Resin;
the same method is used to connect 37.7g (22mmol) of fully protected heptapeptide fragment 37.7g (22mmol) of Fmoc-Cys (Trt) -Ser (tBu) -Glu (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -OH on the basis of Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -Met (Trt) -Ala-2-Chlorotrityl Chloride Resin, after the assembly is completed, the Fmoc protecting group is removed by 100mL of 20% piperidine solution in N, N-dimethylformamide for 30min, isopropanol and N, N-dimethylformamide are washed twice in turn, each time for 90mL, ninhydrin methanol Resin is positive, and the Resin is washed by methanol and dichloromethane respectively, each time for 80mL, and dried to obtain the hexadecapeptide Resin.
5. Preparation of the target sixteen-peptide:
adding 264mL of trifluoroacetic acid, 15mL of triisopropylsilane, 12mL of benzylsulfide and 9mL of pure water into the dried fully-protected hexadecyl peptide resin, reacting for 2 hours in an ice bath, carrying out suction filtration, washing a filter cake with a little acetonitrile, merging filtrate, concentrating the filtrate under reduced pressure until no fraction overflows basically, adding glacial ethyl ether into the concentrated solution, separating out a white solid, filtering, and drying the filter cake in vacuum to obtain crude hexadecyl peptide trifluoroacetate.
The crude product was dissolved in 20% acetonitrile in water, filtered and the volume was determined. Purification by reverse phase chromatography (Filler: C18), phase A0.1% TFA/H2O, B phase 0.1% TFA/MeOH, a phase equilibrium loading, elution procedure: 20-80% of the total amount of the eluent is collected for 60 min. Transsalification using 50mmol/L ammonium acetate system, analytical procedure: 5 to 65 percent of the solution for 30min with the wavelength of 215nm, and freeze-drying to obtain 15.1g of target hexadecapeptide acetate, namely Cys-Ser-Glu-Phe-Met-Arg-Asn-Glu-Leu-Glu-Met-Gln-Arg-Arg-Ala in 65.8% yield and 97% purity.
The structure of the synthesized product was characterized by mass spectrometry, and the molecular weight of the hexadecapeptide was M-2029.3. As can be seen from fig. 1 and 2, the molecular weight and molecular ion peaks of the synthesized product were consistent with those of hexadecapeptide, indicating that the synthesized product was the target hexadecapeptide.
Example 3
In this example, the same portions of the scheme of the method for preparing a hexadecapeptide compound in this example as those in example 2 are not repeated, and the differences in the method for preparing a hexadecapeptide compound in this example will be described with emphasis.
In this example, the 2-Chlorotrityl Chloride Resin (SD ═ 1.1mmol/g) was dosed at 10g for the synthesis of fully protected pentapeptide resins.
In this example, the amount of 2-Chlorotrityl Chloride Resin (SD ═ 1.6mmol/g) charged for the synthesis of the fully protected tetrapeptide fragments was 15g, to give 20.9g of fully protected tetrapeptide fragments Fmoc-Glu (OtBu) -Ile-Glu (OtBu) -COOH.
In this example, the total protected heptapeptide fragment was synthesized by 15g of 2-Chlorotrityl Chloride Resin (SD ═ 1.6mmol/g) in which the protected amino acids Fmoc-Xaa (1) -OH were Fmoc-Thr (tBu) -OH, Fmoc-Xaa (2) -OH were Fmoc-Asp (OtBu) -OH and Fmoc-Xaa (3) -OH were Fmoc-Arg (Fmf) -OH, to obtain 38.6g of total protected heptapeptide fragment oc-Cys (Trt) -Thr-tBu) -Asp (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -OH.
In this example, the assembly of the sixteen peptide solid phase fragment was performed to obtain the fully protected sixteen peptide Resin H-Cys (Trt) -Thr (tBu) -Asp (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -Glu (OtBu) -Ile-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin.
In this example, the desired sixteen peptide was prepared to yield 14.2g of hexadecapeptide acetate, Cys-Thr-Asp-Phe-Met-Arg-Asn-Glu-Ile-Glu-Glu-Met-Gln-Arg-Arg-Ala in 62% yield and 95% purity.
Comparative example 1
The comparative example adopts a polypeptide solid phase synthesis one-by-one extension method, and the condensing agent selects benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate to synthesize the hexapeptide, and the sequence is the same as that in the example 1.
Weighing 2-Chlorotrityl Chloride Resin (SD ═ 0.47mmol/g), and connecting 1.6g (5.17mmol) of Fmoc-Ala-OH according to the technical scheme of the first step of the example 2 to synthesize Fmoc-Ala-2-Chlorotrityl Chloride Resin;
adding 60mL of N, N-dimethylformamide solution of 20% piperidine into Fmoc-Ala-2-Chlorotrityl Chloride Resin, removing the Fmoc protecting group for 30min, performing suction filtration, washing a filter cake twice with isopropanol and N, N-dimethylformamide sequentially, wherein each time is 60mL, ninhydrin detects that the Resin is positive, and the Resin is black; adding 60mL of N, N-dimethylformamide, 6.1g (9.4mmol) of Fmoc-Arg (Pbf) -OH, 1.9g (14.1mmol) of 1-hydroxy phenylpropyl triazole, 5.4g (14.1mmol) of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 3.1mL (18.8mmol) of N, N-diisopropylethylamine into a synthesis column, reacting at normal temperature for 2 hours under the protection of nitrogen, detecting the negative of Resin by ninhydrin, performing suction filtration, washing a filter cake twice by using isopropanol and N, N-dimethylformamide in sequence, and obtaining Fmoc-Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin by 60mL each time;
successively, 6.1g (9.4mmol) of Fmoc-Arg (Pbf) -OH, 5.7g (9.4mmol) of Fmoc-Gln (Trt) -OH, 3.5g (9.4mmol) of Fmoc-Met-OH, 4g (9.4mmol) of Fmoc-Glu (OtBu) -OH, 3.3g (9.4mmol) of Fmoc-Leu-OH, 4g (9.4mmol) of Fmoc-Glu (OtBu) -OH, 5.6g (9.4mmol) of Fmoc-Asn (Trt) -OH, 6.1g (9.4mmol) of Fmoc-Arg (Pbf) -OH, 3.5g (9.4mmol) of Fmoc-Met-OH, 3.6g (9.4mmol) of Fmoc-Phe-OH, 4g (9.4mmol) of Trt-OH, 4mmol) of Ser-Glu (Ser-Glu) (9.4mmol) of Fmoc-Glu-OH, 3.6g (9.4mmol) of Ser-Glu) (9.4mmol) of Fmoc-Glu-OH, obtaining the hexadecapeptide Resin H-Cys (Trt) -Ser (tBu) -Glu (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -Glu (OtBu) -Leu-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Ala-2-Chlorotrityl Chloride Resin;
250mL of cleavage solution was added to the hexadecapeptide resin, and the crude product of hexadecapeptide was obtained after the reaction and treatment, with a purity of 60%, as shown in FIG. 3. The crude product of the hexadecapeptide is subjected to reverse phase chromatography, purification, salt conversion and freeze drying to obtain 2.74g of hexadecapeptide acetate, the yield is 28 percent, and the purity is 95 percent.
From comparative example 1, it can be seen that: the target hexapeptide is synthesized by adopting a solid-phase one-by-one extension method, the loading amount of a solid-phase carrier is small, and the synthesis yield is greatly reduced. The purity of the crude product is poor, and compared with the technical scheme of the invention, the synthesis yield is improved by more than 2 times.
The sixteen-peptide compound and the preparation method thereof disclosed by the embodiments of the present invention are described in detail above, and the principle and the embodiment of the present invention are explained in the present document by applying specific embodiments, and the description of the above embodiments is only used to help understanding the sixteen-peptide compound, the preparation method thereof and the core concept thereof of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (10)
1. A sixteen-peptide compound, wherein the polypeptide sequence of the sixteen-peptide compound is: Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Met-Gln-Arg-Ala, wherein Xaa (1) ═ Thr or Ser, Xaa (2) ═ Glu or Asp, Xaa (3) ═ Lys or Arg, Xaa (4) ═ Leu, Ile or Val.
2. The method for preparing a hexadecapeptide compound according to claim 1, characterized in that: the method comprises the following steps:
s1, synthesizing a fully protected pentapeptide resin: Fmoc-Met-Gln (Trt) -Arg (Pbf) -Ala-2-chlorotritylChlororiResin;
s2, fully protected tetrapeptide fragment: Fmoc-Glu (OtBu) -Leu-Glu (OtBu) -COOH;
s3, synthesis of fully protected heptapeptide fragments: Fmoc-Cys (Trt) -Ser (tBu) -Glu (OtBu) -Phe-Met-Arg (Pbf) -Asn (Trt) -OH;
s4, solid phase assembly to obtain the hexadecapeptide resin: H-Cys (Trt) -Xaa (1) -Xaa (2) -Phe-Met- -Xaa (3) -Asn (Trt) -Glu (OtBu) -Xaa (4) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Ala-2-chlorotritylChlororeResin. Wherein Xaa (1) side chain tBu protection, Xaa (2) side chain OtBu protection, Xaa (3) side chain Boc or Pbf protection;
s5, removing side chain protection, separating and purifying to obtain the target hexadecapeptide compound: Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Glu-Met-Gln-Arg-Arg-Ala.
3. The method for preparing a hexadecapeptide compound according to claim 2, characterized in that: the fully protected pentapeptide resin in step S1 is synthesized by the following steps:
the method comprises the following steps: adopting 2-chlorotrityle chloride resin as a carrier, swelling the 2-chlorotrityle chloride resin by using dichloromethane, adding Fmoc-Ala-OH and N, N-diisopropylethylamine, reacting for 1-2 hours at normal temperature under the protection of nitrogen, carrying out suction filtration, and washing by using isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Ala-2-chlorotrityle chloride resin;
step two: removing Fmoc protecting groups once from the obtained Fmoc-Ala-2-chlorotrityl Chloriresin by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, adding N, N-dimethylformamide, Fmoc-Arg (Pbf) -OH, 1-hydroxyphenyltriazole and N, N' -diisopropylcarbodiimide, reacting at normal temperature for 2-3 hours under the protection of nitrogen, performing suction filtration, and washing a filter cake by using isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Arg (Pbf) -Ala-2-chlorotrityl Chloriresin;
step three: the Fmoc-Arg (Pbf) -Ala-2-chlorotritylChroriresin is connected with Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH and Fmoc-Met-OH in sequence by adopting a second step method to obtain Fmoc-Met-Gln (Trt) -Arg (Pbf) -Ala-2-chlorotritylChroriresin.
4. The method for preparing a hexadecapeptide compound according to claim 3, characterized in that: the fully protected tetrapeptide fragment in step S2 was synthesized by the following steps:
connecting Fmoc-Glu (OtBu) -OH to the 2-chlorotrityChlorariResin by adopting the method of the step one to obtain Fmoc-Glu (OtBu) -2-chlorotrityChlorariResin;
sequentially connecting Fmoc-Glu (OtBu) -OH, Fmoc-Xaa (4) -OH and Fmoc-Glu (OtBu) -OH to the obtained Fmoc-Glu (OtBu) -2-chlorotritylChloriresin by using the method of the second step to obtain Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -2-chlorotritylChloriresin;
adding lysis solution of trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 20:7.25:72.5 into Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -Met-2-chlorotritylChloriresin, stirring for 2-3 hours at normal temperature, filtering, adding NaHCO3Adjusting the filtrate to be neutral by using an aqueous solution, combining aqueous phases, and removing the organic solvent under reduced pressure to obtain Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -COOH.
5. The method for preparing a hexadecapeptide compound according to claim 3, characterized in that: the fully protected heptapeptide fragment in step S3 was synthesized by:
connecting Fmoc-Asn (Trt) -OH to the 2-chlorotrityChlorhredriResin by adopting a method of the step one to obtain Fmoc-Asn (Trt) -2-chlorotrityChlordriResin;
sequentially connecting Fmoc-Xaa (3) -OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Xaa (2) -OH, Fmoc-Xaa (1) -OH and Fmoc-Cys (Trt) -OH to obtain Fmoc-Cys (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn (Trt) -2-chlorotritylChroriderResin by adopting the method of the step two on the basis of obtaining Fmoc-Asn (Trt) -2-chlorotritylChroriderResin;
adding trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 20:7.25:72.5 into Fmoc-Cys (Trt) -Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn (Trt) -2-chlorotritylChroriresin, and carrying out post-reaction treatment to obtain Fmoc-Cys (Trt) -Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn (Trt) -OH.
6. The method for preparing a hexadecapeptide compound according to claim 2, characterized in that: the fully protected hexadecapeptide resin in step S4 was synthesized by the following steps:
removing Fmoc once from the fully protected pentapeptide resin with a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, adding N, N-dimethylformamide, a fully protected tetrapeptide fragment, 1-hydroxyphenyltriazole, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate and N-methylmorpholine, stirring at room temperature for 2-4 hours under nitrogen protection to obtain Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Ala-2-chlorotritylChroridin, and re-connecting the Fmoc-Glu (OtBu) -Xaa (4) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Ala-2-chlorotypidin base to obtain a fully protected tetrapeptide fragment by the same method To obtain the fully protected hexadecapeptide resin.
7. The method for preparing a hexadecapeptide compound according to claim 2, characterized in that: the hexadecapeptide compound in step S5 is synthesized by the following steps:
adding a cutting fluid with the volume ratio of trifluoroacetic acid to triisopropylsilane to phenylmethylsulfide to pure water of 88:5:4:3 into the fully protected hexadecyl peptide resin, reacting for 2-3 hours in an ice bath, carrying out suction filtration, carrying out vacuum concentration on the filtrate, adding glacial ethyl ether into the concentrated solution for crystallization to obtain a hexadecyl peptide crude product, purifying the crude product by reverse phase chromatography, carrying out salt transformation on an ammonium acetate system, and carrying out freeze drying to obtain the target hexadecyl peptide, namely Cys-Xaa (1) -Xaa (2) -Phe-Met-Xaa (3) -Asn-Glu-Xaa (4) -Glu-Met-Gln-Arg-Arg-Ala.
8. The method for preparing a hexadecapeptide compound according to claim 3 or 4 or 5, characterized in that: the molar ratio of the 2-chlorotrityle ChlororiResin to Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Met-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Xaa (4) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Xaa (3) -OH, Fmoc-Phe-OH, Fmoc-Xaa (2) -OH, Fmoc-Xaa (1) -OH and Fmoc-Cys (Trt) -OH is 1 (2-3); the molar ratio of the 2-chlorotrityleresin, the 1-hydroxy phenylpropyl triazole and the N, N' -diisopropylcarbodiimide is 1 (2-4) to (2-4).
9. The method for preparing a hexadecapeptide compound according to claim 6, characterized in that: the molar ratio of the fully-protected pentapeptide resin to the fully-protected tetrapeptide fragment, the fully-protected heptapeptide fragment, the 1-hydroxy phenylpropyl triazole, the 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate and the N-methylmorpholine is 1:2:2:3:4 (4-6).
10. The method for preparing a hexadecapeptide compound according to claim 2, characterized in that: the 2-chlorotritylchloriderresin has an SD of 1.0 to 1.6 mmol/g.
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