Liquid phase carrier for polypeptide synthesis as well as preparation method and application of liquid phase carrier
By preparing a novel liquid-phase carrier to condense and cleave amino acids, the problems of high pollution, complex reactions, and high costs in liquid-phase peptide synthesis were solved, achieving efficient and low-cost peptide synthesis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-28
- Publication Date
- 2026-03-10
AI Technical Summary
Existing liquid-phase peptide synthesis methods suffer from problems such as high pollution, complex reactions, high costs, and low efficiency. In particular, solid-phase synthesis methods are difficult to scale up and have high costs.
A novel liquid-phase carrier was prepared by reacting 4-hydroxymethyl-1,1-biphenyl-4-ol with hexadecyl alcohol in the presence of triphenylphosphine and diethyl azodicarbonate. The carrier was then condensed with amino acids, followed by cleavage with a cleavage fluid to obtain peptides. The condensation reaction was a homogeneous reaction with few byproducts and easy purification.
This method enables low-cost, high-efficiency peptide synthesis with fewer byproducts, simple operation, high condensation efficiency, reduced reaction costs, and high yield.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug synthesis technology, and particularly relates to a liquid carrier for polypeptide synthesis, its preparation method and application. Background Technology
[0002] The chemical synthesis of peptides is mainly achieved through the dehydration condensation reaction of amino acids. During the reaction, the protecting groups of amino acids in the raw materials that do not need to participate in the reaction are temporarily protected, thereby enabling the targeted synthesis. Chemical synthesis is further divided into solid-phase and liquid-phase synthesis, the main difference being whether or not a solid-phase support resin is used.
[0003] For liquid-phase synthesis, there are two main strategies: stepwise synthesis and fragment synthesis. Stepwise synthesis is simple and rapid, suitable for most peptide synthesis, while fragment synthesis is advantageous for larger peptides (containing more than 10 amino acids). Overall, liquid-phase methods have been largely replaced by solid-phase synthesis methods due to their higher pollution levels and more complex reaction development. Currently, solid-phase synthesis is still widely used for peptide synthesis of peptides with fewer than 10 amino acids due to its large scale and lower unit cost.
[0004] Solid-phase synthesis, specifically solid-phase peptide synthesis, involves immobilizing the C-terminus of amino acids onto an insoluble resin, followed by a condensation reaction to elongate the peptide chain. Solid-phase synthesis can be further divided into the Fmoc method and the Boc method. The Fmoc method offers advantages such as milder reaction conditions, simpler operation, and easier automation, making it the preferred method for peptide synthesis. However, solid-phase synthesis still suffers from relatively low reaction efficiency, difficulty in scaling up, and higher costs. Summary of the Invention
[0005] The purpose of this invention is to provide a liquid carrier for polypeptide synthesis, its preparation method and application. The method of this invention has low cost, high synthesis efficiency and few by-products.
[0006] This invention provides a liquid carrier for polypeptide synthesis, having the chemical formula shown in Formula I:
[0007]
[0008] This invention provides a method for preparing a liquid support for peptide synthesis as described above, comprising the following steps:
[0009] 4-Hydroxymethyl-1,1-biphenyl-4-ol was reacted with hexadecyl alcohol in the presence of triphenylphosphine and diethyl azodicarbonate to obtain the liquid support shown in Formula I.
[0010] Preferably, at -5 to 5°C, 4-hydroxymethyl-1,1-biphenyl-4-ol and hexadecyl alcohol are mixed in a solvent, triphenylphosphine and diethyl azodicarbonate are added, and the mixture is heated to 20 to 30°C and stirred to carry out the reaction.
[0011] This invention provides a method for synthesizing polypeptides, comprising the following steps:
[0012] A) The liquid carrier is reacted with the nth amino acid in a solvent to obtain the first intermediate product;
[0013] The liquid carrier has the structure shown in Formula I:
[0014]
[0015] B) Under alkaline conditions and in the presence of a condensing reagent, the first intermediate product is sequentially condensed with the (n-1)th amino acid, the (n-2)th amino acid, ..., the first amino acid in a solvent to obtain the second intermediate product; 2≤n≤10, where n is an integer;
[0016] C) The second intermediate product is cleaved using a cleavage fluid to obtain a polypeptide.
[0017] Preferably, in step B), alkaline conditions are achieved by adding an alkaline reagent, which includes one or more of potassium phosphate, potassium carbonate, sodium hydroxide, sodium carbonate, sodium bicarbonate, N,N-diisopropylethylamine, triethylamine, 1,8-diazabicycloundec-7-ene, N-methylimidazolium, and N-methylmorpholine.
[0018] Preferably, the molar ratio of the liquid carrier to the first to nth amino acids is 1:(0.5 to 3).
[0019] Preferably, the condensing agent is one or more of N-hydroxysuccinimide, N-hydroxy5-norbornene-2,3-dihydroxyimide, 1-hydroxybenzotriazole, benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate, N,N-diisopropylcarbodiimide, N′,N′-dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and propylphosphoric anhydride.
[0020] The molar ratio of the condensation reagent to the first to nth amino acids is (1 to 3):1.
[0021] Preferably, the temperature of the condensation reaction in step B) is 15–30°C.
[0022] Preferably, the cutting fluid comprises trifluoroacetic acid, triisopropylsilane, and water, wherein the mass ratio of trifluoroacetic acid, triisopropylsilane, and water is (93-98):(0.5-1.5):(0.5-6.5).
[0023] Preferably, after cleaving the second intermediate product with a cleaving fluid, the cleaved product is recrystallized to obtain a polypeptide.
[0024] This invention provides a liquid-phase carrier for peptide synthesis, having the chemical formula shown in Formula I. The liquid-phase carrier of this invention can be used for liquid-phase peptide synthesis. Taking the synthesis of a dipeptide as an example, this invention reacts the liquid-phase carrier with the second amino acid, protecting the C-terminus to obtain a first intermediate. Then, under alkaline conditions, the first amino acid is condensed with the first intermediate to obtain a second intermediate. The second intermediate is then cleaved to obtain the dipeptide. The liquid-phase carrier of this invention has a structure of two parallel benzene rings, and the synthesized peptide intermediate is easily purified into a solid. Simultaneously, the alkane chain has low polarity, and the liquid-phase carrier is soluble in solvents. Therefore, the condensation reaction is a homogeneous reaction, characterized by high condensation efficiency. Attached Figure Description
[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0026] Figure 1 The above is the 1H NMR spectrum of the liquid support prepared in Example 1 of this invention;
[0027] Figure 2 The 1H NMR spectrum of the smedipeptide prepared in Example 4 of this invention;
[0028] Figure 3 The 1H NMR spectrum of the collagen dipeptide prepared in Example 7 of this invention;
[0029] Figure 4 The hydrogen nuclear magnetic resonance spectrum of Ala-Pro prepared in Example 11 of this invention;
[0030] Figure 5 The image shows the hydrogen nuclear magnetic resonance spectrum of His-Gly-Ala-Pro prepared in Example 12 of this invention. Detailed Implementation
[0031] This invention provides a liquid carrier for peptide synthesis, having the chemical formula shown in Formula I:
[0032]
[0033] The present invention also provides a method for preparing the liquid support for peptide synthesis described above, as shown in the following formula, comprising the following steps:
[0034] 4-Hydroxymethyl-1,1-biphenyl-4-ol was reacted with hexadecyl alcohol in the presence of triphenylphosphine and diethyl azodicarbonate to obtain the liquid support shown in Formula I.
[0035]
[0036] In this invention, 4-hydroxymethyl-1,1-biphenyl-4-ol and hexadecyl alcohol are mixed in dichloromethane at -5 to 5°C, and triphenylphosphine (PPh3) and diethyl azodicarbonate (DEAD) are added. The mixture is then heated to 20 to 30°C and stirred to carry out the reaction.
[0037] In this invention, the molar ratio of hexadecyl alcohol to 4-hydroxymethyl-1,1-biphenyl-4-ol is preferably 1:(1-2.0), more preferably 1:(1.3-1.8), such as 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, preferably a range of values with any of the above as the upper or lower limit; the molar ratio of the amount of triphenylphosphine to 4-hydroxymethyl-1,1-biphenyl-4-ol is preferably 1:(1-2.5), more preferably 1:(1-2.0). The molar ratio of diethyl azodicarbonate to 4-hydroxymethyl-1,1-biphenyl-4-ol is preferably 1:(1 to 3.0), more preferably 1:(1.5 to 2.5), even more preferably 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, and preferably within the range of values mentioned above.
[0038] In this invention, the reaction temperature is preferably 20-30°C, more preferably 25-30°C, and the reaction time is preferably 5-10 hours, more preferably 8-9 hours.
[0039] After the reaction is complete, the reaction solution is washed with water, dried with anhydrous sodium sulfate, and then petroleum ether (PE) is added to precipitate a solid, which is a liquid carrier with the structure of Formula I.
[0040] This invention also provides an application of the liquid carrier described above in liquid-phase synthesis of peptides, namely, a method for synthesizing peptides, comprising the following steps:
[0041] A) The liquid carrier is reacted with the nth amino acid in a solvent to obtain the first intermediate product;
[0042] The liquid carrier has the structure shown in Formula I:
[0043]
[0044] B) Under alkaline conditions and in the presence of a condensing reagent, the first intermediate product is sequentially condensed with the (n-1)th amino acid, the (n-2)th amino acid, ..., the first amino acid in a solvent to obtain the second intermediate product; n is an integer ≥2.
[0045] C) The second intermediate product is cleaved using a cleavage fluid to obtain a polypeptide.
[0046] Preferably, the present invention first reacts the halogenating reagent of the liquid carrier to obtain the halogenated product of the liquid carrier, and then reacts the halogenated product with the nth amino acid in a solvent to obtain the first intermediate product.
[0047] In this invention, the halogenating agent is preferably sulfoxide dichloride, and the molar ratio of the liquid support to sulfoxide dichloride is preferably 1:(1-1.2), more preferably 1:(1.05-1.1). The halogenation reaction is preferably carried out under ice bath conditions.
[0048] In this invention, the solvent is preferably one or more of dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile (ACN), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), H2O, ethyl acetate (EA), and methanol (MeOH).
[0049] In this invention, the molar ratio of the liquid carrier to the nth amino acid is preferably 1:(0.5-3), more preferably 1:(1-2.5), such as 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, and preferably a range of values with any of the above values as the upper or lower limit.
[0050] In this invention, the reaction temperature of the halogenated product with the nth amino acid is preferably 20-30°C, more preferably 25°C, and the reaction time is determined by monitoring the completion of the reaction.
[0051] After completing the reaction between the halogenated product and the nth amino acid, the present invention preferably purifies the reaction product, as follows:
[0052] Water was added to the reaction solution, and the mixture was extracted multiple times with dichloromethane. The oil phase was collected, washed multiple times with water, dried with anhydrous sodium sulfate, and concentrated to obtain an oily substance, which is the first intermediate product.
[0053] After obtaining the first intermediate product, the present invention, under alkaline conditions and in the presence of a condensing reagent, performs a condensation reaction between the first intermediate product and the (n-1)th amino acid in a solvent to obtain product 1.
[0054] Then, under alkaline conditions and in the presence of a condensing agent, product 1 and the (n-2)th amino acid were condensed in a solvent to obtain product 2.
[0055] ...
[0056] This process continues until product n-1 and the first amino acid undergo a condensation reaction in a solvent under alkaline conditions and in the presence of a condensing agent, yielding the second intermediate product.
[0057] In this invention, n is preferably an integer ≥2, more preferably an integer between 2 and 10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, and preferably a range of values with any of the above values as the upper or lower limit.
[0058] In this invention, the alkaline conditions are achieved by adding an alkaline reagent. The alkaline reagent is preferably one or more of potassium phosphate, potassium carbonate, sodium hydroxide, sodium carbonate, sodium bicarbonate, N,N-diisopropylethylamine (DIEA), triethylamine (Et3N), 1,8-diazabicycloundec-7-ene (DBU), N-methylimidazolium (NMI), and N-methylmorpholine (NMM). This invention does not impose any special restrictions on the amount of the alkaline reagent used, and the optimal pH of the reaction system is maintained at 8-11.
[0059] In this invention, the condensing agent is preferably one or more of N-hydroxysuccinimide (HOSu), N-hydroxy5-norbornene-2,3-dihydroxyimide (HONB), 1-hydroxybenzotriazole (HOBt), benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU), 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU), N,N-diisopropylcarbodiimide (DIC), N′,N′-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), and propylphosphoric anhydride (T3P); the amount of the condensing agent is preferably selected from the following:
[0060] In this invention, the solvent used in the condensation reaction is preferably one or more of dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile (ACN), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), H2O, ethyl acetate (EA), and methanol (MeOH).
[0061] In this invention, the molar ratio of the liquid carrier to the 1st to nth amino acids is 1:(0.5-3), that is, the molar ratio of the liquid carrier to the (n-1)th amino acid is preferably 1:(0.5-3), more preferably 1:(1-2.5), such as 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, preferably within the range of any of the above values as the upper or lower limit. The molar ratio of the liquid carrier to the (n-2)th amino acid is also within the above range, which is 1:(0.5-3), and the molar ratio of the liquid carrier to the amino acids used, such as the (n-3)th amino acid, ..., the 1st amino acid, is also within the above range, which is 1:(0.5-3), more preferably 1:(1-2.5), such as 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, preferably within the range of any of the above values as the upper or lower limit.
[0062] In this invention, since the liquid carrier of this invention has a wide range of applications, there are no special restrictions on the types of amino acids. Specifically, in the embodiments of this invention, the amino acids can be two or more of Aib, His (Trt), Pro, Gly, Ala and His.
[0063] In this invention, the temperature of the condensation reaction is preferably 15-30°C, more preferably 20-25°C. This invention does not impose any special restrictions on the time of the condensation reaction, but rather on monitoring the completion of the reaction.
[0064] After obtaining the second intermediate product, the present invention uses a cleavage fluid to cleave the second intermediate product to obtain a polypeptide.
[0065] In this invention, the cutting fluid preferably comprises trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water, wherein the mass ratio of the trifluoroacetic acid, triisopropylsilane, and water is (93-98):(0.5-1.5):(0.5-6.5), more preferably 94:1:4; the molar ratio of the cutting fluid to the liquid carrier is preferably 1:(0.5-1), more preferably 1:(0.6-0.8), such as 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, and preferably a range of values with any of the above values as the upper or lower limit.
[0066] In this invention, the cutting temperature is preferably 0 to 25°C, more preferably 10 to 20°C, such as 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, preferably a range of values with the above values as the upper or lower limit; the cutting time is preferably 0.5 to 3 hours, more preferably 1 to 2.5 hours, and most preferably 1.5 to 2 hours.
[0067] After the cutting fluid performs the cutting, the present invention preferably performs crystallization purification on the reaction solution after cutting, and the specific steps are as follows:
[0068] The pH of the reaction solution after cutting was adjusted to 3-4, and the solution was washed several times with water. The organic phase was dried with anhydrous sodium sulfate and then concentrated to obtain a crude oily product. The crude product was mixed with purification reagent and recrystallized to obtain the polypeptide.
[0069] In this invention, the water washing, anhydrous sodium sulfate drying and concentration are all conventional post-processing methods used in the synthesis of polypeptides in this field, and will not be described in detail here.
[0070] In this invention, the purification reagent is preferably one or more of dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile (ACN), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), H2O, ethyl acetate (EA), methanol (MeOH), ethanol (EtOH), isopropanol (iPrO2), and petroleum ether (PE). The recrystallization temperature is preferably 0–40°C, more preferably 10–30°C, such as 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C, and preferably a range of values with any of the above values as the upper or lower limit.
[0071] Compared with the prior art, the present invention has the following advantages:
[0072] The technical effects of this invention are as follows:
[0073] This method uses a liquid-phase carrier to protect the C-terminus of the nth amino acid, which is then condensed with other amino acids and cleaved to obtain the target polypeptide. It produces few byproducts, is novel and simple, reduces reaction costs, and has a high yield.
[0074] Since the liquid carrier is soluble in the solvent, the condensation is a homogeneous reaction with high condensation efficiency.
[0075] Protecting the C-terminus of the nth amino acid using a liquid carrier allows for a wider selection of condensation reagents, fewer byproducts, and easier purification during condensation with subsequent amino acids.
[0076] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes a liquid carrier for polypeptide synthesis provided by the present invention, its preparation method, and its application, but this should not be construed as limiting the scope of protection of the present invention.
[0077] Example 1
[0078] At 0°C, 10 g of 4-hydroxymethyl-1,1-biphenyl-4-ol and 9.2 g of cetyl alcohol were added. Using DCM as a solvent, 13.0 g of PPh3 and 14.3 g of DEAD were added. The mixture was heated to 25°C and stirred for 8 hours until the reaction was complete. The reaction solution was washed with water, dried over anhydrous sodium sulfate, and then PE was added to precipitate a solid.
[0079]
[0080] Example 2 Synthesis of Smedipeptide by Liquid Phase Carrier Method
[0081]
[0082] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution.
[0083] H-Aib-OH (2.67 g, 1.1 eq) was dissolved in DMF (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0084] The oily substance was dissolved in DCM (50 mL), and Boc-His(Trt)-OH (11.50 g, 0.98 eq), DIC (3.57 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and DIEA (4.56 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean sema dipeptide product with a yield of 70% and a purity of 99.2%.
[0085] Example 3: Synthesis of Smedipeptide using a liquid-phase carrier method
[0086]
[0087] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution (the reaction formula above is a simplified representation and this step is not shown).
[0088] H-Aib-OH (3.64 g, 1.5 eq) was dissolved in DMSO (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0089] The oily substance was dissolved in DCM (50 mL), and Boc-His(Trt)-OH (11.50 g, 0.98 eq), DCC (5.83 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and DIEA (4.56 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean sema dipeptide product with a yield of 75% and a purity of 99.0%.
[0090] Example 4: Synthesis of Smedipeptide using a liquid-phase carrier method
[0091]
[0092] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution.
[0093] H-Aib-OH (3.64 g, 1.5 eq) was dissolved in DMSO (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0094] The oily substance was dissolved in DCM (50 mL), and Boc-His(Trt)-OH (11.50 g, 0.98 eq), EDCI (5.07 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and DIEA (4.56 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean sema dipeptide product with a yield of 80% and a purity of 99.4%.
[0095] Example 5: Synthesis of Collagen Dipeptides by Liquid Phase Carrier Method
[0096]
[0097] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution.
[0098] H-Pro-OH (2.98 g, 1.1 eq) was dissolved in DMF (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0099] The oily substance was dissolved in DCM (50 mL), and H-Gly-OH (1.73 g, 0.98 eq), EDCI (5.07 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and DIEA (4.56 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean collagen dipeptide product with a yield of 72% and a purity of 98.6%.
[0100] Example 6 Synthesis of Collagen Dipeptides by Liquid Phase Carrier Method
[0101]
[0102] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution.
[0103] H-Pro-OH (4.07 g, 1.5 eq) was dissolved in THF (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0104] The oily substance was dissolved in DCM (50 mL), and H-Gly-OH (1.73 g, 0.98 eq), EDCI (5.07 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and DIEA (4.56 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean collagen dipeptide product with a yield of 78% and a purity of 98.5%.
[0105] Example 7 Synthesis of Collagen Dipeptides by Liquid Phase Carrier Method
[0106]
[0107] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution.
[0108] H-Pro-OH (5.42 g, 2.0 eq) was dissolved in DMSO (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0109] The oily substance was dissolved in DCM (50 mL), and H-Gly-OH (1.73 g, 0.98 eq), DCC (5.83 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and DIEA (4.56 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance. The oily substance was recrystallized from DCM / MTBE (5V) to obtain a clean collagen dipeptide product with a yield of 84% and a purity of 98.9%.
[0110] Example 8: Synthesis of Ala-Pro by Liquid Phase Support Method
[0111]
[0112] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution.
[0113] H-Pro-OH (2.93 g, 1.1 eq) was dissolved in DMF (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0114] The oily substance was dissolved in DCM (50 mL), and H-Ala-OH (2.06 g, 0.98 eq), DCC (5.83 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and DIEA (4.56 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean H-Ala-Pro-OH product with a yield of 78% and a purity of 99.1%.
[0115] Example 9: Synthesis of Ala-Pro by Liquid Phase Support Method
[0116]
[0117] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution.
[0118] H-Pro-OH (2.93 g, 1.1 eq) was dissolved in ACN (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0119] The oily substance was dissolved in DCM (50 mL), and H-Ala-OH (2.06 g, 0.98 eq), DCC (5.83 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and Et3N (3.57 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean H-Ala-Pro-OH product with a yield of 82% and a purity of 99.4%.
[0120] Example 10 Synthesis of Ala-Pro by Liquid Phase Support Method
[0121]
[0122] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the SI reaction solution.
[0123] H-Pro-OH (2.93 g, 1.1 eq) was dissolved in DMSO (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0124] The oily substance was dissolved in DCM (50 mL), and H-Ala-OH (2.06 g, 0.98 eq), DCC (5.83 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and NMI (2.90 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean H-Ala-Pro-OH product with a yield of 75% and a purity of 98.6%.
[0125] Example 11 Synthesis of Ala-Pro by Liquid Phase Support Method
[0126]
[0127] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the SI reaction solution.
[0128] H-Pro-OH (2.93 g, 1.1 eq) was dissolved in DMSO (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0129] The oily substance was dissolved in DCM (50 mL), and H-Ala-OH (2.06 g, 0.98 eq), DCC (5.83 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and NMM (3.57 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the reaction solution, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance. The oily substance was recrystallized from DCM / PE (5V) to obtain a clean H-Ala-Pro-OH product with a yield of 77% and a purity of 98.3%.
[0130] Example 12 Synthesis of His-Gly-Ala-Pro by Liquid Phase Support Method
[0131]
[0132] The liquid carrier (10 g, 1.0 eq) prepared in Example 1 was added to a 250 mL bottle and dissolved in dichloromethane (100 mL). SOCl2 (3.09 g, 1.1 eq) was added dropwise under an ice bath and stirred for 30 min to obtain the S1 reaction solution.
[0133] H-Pro-OH (2.98 g, 1.1 eq) was dissolved in DMSO (10 mL), and S1 reaction solution was added dropwise. The reaction was spotted by TLC until the support was completely reacted. Water was added to the reaction solution, and the mixture was extracted three times with DCM (10 mL). The DCM phase was collected, washed three times with water (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance.
[0134] The oily substance was dissolved in DCM (50 mL), and H-Ala-OH (2.06 g, 0.98 eq), DCC (5.83 g, 1.2 eq), HOBt (3.82 g, 1.2 eq), and NMM (3.57 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. The reaction solution was filtered, and the filtrate was collected. The filtrate was washed three times with water (50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance.
[0135] The oily substance was dissolved in DCM (50 mL), and H-Gly-OH (1.73 g, 0.98 eq), DCC (5.83 g, 1.2 eq), HOAT (3.84 g, 1.2 eq), and Py (2.79 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. The reaction solution was filtered, and the filtrate was collected. The filtrate was washed three times with water (50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain the oily substance.
[0136] The oily substance was dissolved in THF (50 mL), and H-His-OH (3.58 g, 0.98 eq), DIC (3.56 g, 1.2 eq), Oxyma (5.09 g, 1.2 eq), and Et3N (3.5 g, 1.5 eq) were added. The mixture was spotted by TLC until the starting material completely disappeared. The reaction solution was filtered, and the filtrate was collected. TFA / TIS / H2O = 90 / 5 / 5 (5 mL) was added to the filtrate, and the mixture was spotted by TLC until the starting material completely disappeared. Water (50 mL) was added to the reaction solution to adjust the pH to 3. The mixture was extracted and separated, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance. The oily substance was recrystallized from MeOH / H2O (5V) to obtain a clean H-His-Gly-Ala-Pro-OH product with a yield of 89% and a purity of 99.3%.
[0137] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A liquid phase carrier for polypeptide synthesis, having a chemical formula shown in formula I. 2.A method for preparing the liquid phase carrier for polypeptide synthesis according to claim 1, comprising the following steps: reacting 4-hydroxymethyl-1, 1-biphenyl-4-ol with hexadecanol in the presence of triphenylphosphine and diethyl azodicarboxylate to obtain the liquid phase carrier shown in formula I.
3. The production method according to claim 2, characterized by, mixing 4-hydroxymethyl-1, 1-biphenyl-4-ol with hexadecanol in a solvent at -5-5 ℃, adding triphenylphosphine and diethyl azodicarboxylate, and then warming to 20-30 ℃ to react. 4.A method for synthesizing polypeptides, comprising the following steps: A) reacting the liquid phase carrier with the nth amino acid in a solvent to obtain a first intermediate product; the liquid phase carrier has a structure shown in formula I: B) sequentially condensing the first intermediate product with the (n-1)th amino acid, the (n-2)th amino acid, …, the 1st amino acid in a solvent under the presence of a basic condition and a condensing reagent to obtain a second intermediate product; 2≤n≤10, n is an integer; C) using a cleavage solution to cleave the second intermediate product to obtain a polypeptide.
5. The method of claim 4, wherein, the basic condition in step B) is achieved by adding a basic reagent, which includes one or more of potassium phosphate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, N, N-diisopropylethylamine, triethylamine, 1, 8-diazabicycloundec-7-ene, N-methylimidazole, and N-methylmorpholine.
6. The method of claim 4, wherein, the molar ratio of the liquid phase carrier to the 1st to nth amino acid is all 1: (0.5-3).
7. The method of claim 4, wherein, the condensing reagent is one or more of N-hydroxysuccinimide, N-hydroxy 5-norbornene-2, 3-dihydroxyl imine, 1-hydroxybenzotriazole, benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate, N, N-diisopropylcarbodiimide, N', N'-dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and propyl phosphonic anhydride; the molar ratio of the amount of the condensing reagent to the 1st to nth amino acid is all (1-3) :
1.
8. The method of claim 4, wherein, the temperature of the condensation reaction in step B) is 15-30 ℃.
9. The method of claim 4, wherein, the cleavage solution includes trifluoroacetic acid, triisopropylsilane, and water, and the mass ratio of trifluoroacetic acid, triisopropylsilane, and water is (93-98) : (0.5-1.5) : (0.5-6.5).
10. The method according to any one of claims 4 to 9, characterized in that, after the second intermediate product is cleaved using the cleavage solution, the cleavage product is recrystallized to obtain a polypeptide.