An iterative exponential synthesis method for long-chain collagen-like polypeptide

Abstract

The present invention provides an iterative exponential synthesis method for long-chain collagen-like polypeptides. The long-chain collagen-like polypeptides prepared by this method have precise chemical structures and uniform chain lengths. The present invention adopts a liquid-phase iterative exponential synthesis strategy, which enables the molecular weight of the polypeptides to increase exponentially during the synthesis process, reduces the reaction steps, improves the synthesis efficiency of long-chain collagen-like polypeptides, has advantages such as large synthesis scale and low cost, and can precisely prepare various functionalized long-chain collagen-like polypeptides.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis, and particularly relates to an iterative exponential synthesis method for long-chain collagen-like polypeptides. Background Art

[0002] Collagen is the most important protein in the natural extracellular matrix (ECM). It is also the most abundant and widely distributed functional protein in mammals, accounting for 25% - 30% of the total protein content. So far, a total of twenty-nine natural collagens have been discovered. Through research, it has been found that these collagens all have similar structural characteristics, a tight right-handed triple helix structure composed of three α-polypeptide chains. Each α-polypeptide contains one or more repeating sequences of three amino acids, which are Gly-Xaa-Yaa (where Xaa and Yaa can be any amino acids, but are usually proline and hydroxyproline). These repeating sequences form the central triple helix structure of collagen, which is the basis for the stability of collagen. Collagen is widely used in the biomedical field due to its excellent mechanical properties, hemostatic properties, cell growth promotion, weak antigenicity, biodegradability, collagen self-association, and other important biological characteristics. The large-scale use of collagen has led to an increasing demand. Currently, the mainstream methods for extracting and preparing collagen include animal source extraction and gene recombination. Animal-derived collagen is mainly extracted from animal tissues such as bovine tendon and pigskin. This long-chain collagen has extremely poor water solubility and risks such as immunogenic reactions, batch-to-batch instability, and potential pathogen transmission. For recombinant collagen, according to different technical routes, it can be divided into three categories: recombinant human collagen, recombinant humanized collagen, and recombinant long-chain collagen. However, recombinant collagen also faces problems such as poor sample stability, potential endotoxin release risk, and complex and difficult purification processes.

[0003] Collagen-like peptide (CLP) is a synthetic short peptide obtained by chemical synthesis, and its characteristic is that it contains (POG) nRepeating sequences. CLP has the same triple-helix conformation as native collagen and is thus widely used as a model peptide for studying the stability of native collagen triple-helices, guiding the self-assembly of higher-order structures. Previous studies have demonstrated the potential of CLP in improving cell adhesion, proliferation, and ECM production and its use as a thermoresponsive bioconjugate for tissue engineering and targeted drug delivery ([1] LUO J, TONG YW. Self-Assembly of Collagen-Mimetic Peptide Amphiphiles into Biofunctional Nanofiber[J]. ACS Nano, 2011, 5(10): 7739-47. [2] MO X, ZHAO S, ZHAO J, et al. Targeting collagen damage for sustained in situ antimicrobial activities[J]. Journal of controlled release: official journal of the Controlled Release Society, 2023, 360: 122-32). The current study established the relationship between hydrogel properties and polypeptide molecular structures by investigating the physicochemical properties of CLP hydrogels, such as mechanical strength, refractive index, transparency, water content, viscosity, swelling coefficient, etc., continuously optimized the molecular design of CLP, and screened in high throughput for biomimetic CLP similar to the composition, structure, and properties of native ECM to be used as a new biomimetic material.

[0004] However, it is currently difficult to prepare long-chain collagen-like proteins with accurate structures. Summary of the Invention

[0005] The object of the present invention is to provide an iterative exponential synthesis method for preparing long-chain collagen-like polypeptides with accurate structures.

[0006] The present invention provides a method for preparing long-chain collagen-like polypeptides, the method comprising the following steps:

[0007] (I) Preparing a protected tripeptide;

[0008] (II) Using the protected tripeptide as a raw material, preparing a long-chain collagen-like polypeptide by an iterative exponential synthesis method;

[0009] The structure of the protected tripeptide is The structure of the long-chain collagen-like polypeptide is R1 is a hydroxyl protecting group, R2 is a carboxyl protecting group, R3 is an amino protecting group, n is 2 to the power of x, and x is an integer greater than or equal to 1.

[0010] Furthermore, the method for preparing the protected tripeptide in step (I) includes the following steps:

[0011] (1) Dissolve the protected hydroxyproline, coupling agent, and basic catalyst in an organic solvent, then add the protected glycine for condensation reaction, and then add a protecting agent, an imidazole compound, and a catalyst for the protection reaction. After purification, a protected dipeptide product is obtained: R4 is an amino protecting group;

[0012] (2) Dissolve the protected dipeptide product and a deprotecting agent in an organic solvent for deprotection reaction. After purification, a deprotected dipeptide product is obtained:

[0013] (3) Dissolve the protected proline and a condensing agent in an organic solvent, add the deprotected dipeptide product, and react under a basic catalyst. After purification, a protected collagen-like tripeptide is obtained.

[0014] Furthermore, the hydroxyl protecting group is acetyl, tert-butoxycarbonyl, tert-butyldiphenylsilyl, or methoxymethyl; the carboxyl protecting group is benzyl, acetyl, tert-butoxycarbonyl, tert-butyldiphenylsilyl, or methoxymethyl; the amino protecting group is tert-butoxycarbonyl, acetyl, tert-butyldiphenylsilyl, or methoxymethyl.

[0015] Furthermore, in step (1), the protected hydroxyproline is Boc-L-hydroxyproline, the coupling agent is benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate; the basic catalyst is N,N-diisopropylethylamine, the protected glycine is glycine benzyl ester hydrochloride, the organic solvent is N,N-dimethylformamide, and the equivalent ratio of the protected hydroxyproline:benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate:N,N-diisopropylethylamine:protected glycine is 1:0.5 - 1:2 - 5:1 - 1.2, and the condensation reaction time is 1 - 5 hours; the protecting agent is acetic anhydride, the imidazole compound is imidazole, the catalyst is 4-dimethylaminopyridine, and the equivalent ratio of acetic anhydride:imidazole:4-dimethylaminopyridine is 1:1 - 3:1 - 3, and the protection reaction time is 1 - 3 hours;

[0016] In step (2), the deprotecting agent is 1,4 - epoxyhexane or its salt, the organic solvent is dichloromethane, the equivalent ratio of the protected dipeptide product to the deprotecting agent is 1:9 - 11; the mass - volume ratio of the protected dipeptide product to the organic solvent is 1:0.5 - 2; the time of the deprotection reaction is 0.5 - 2 hours;

[0017] In step (3), the protected proline is Boc - L - proline; the condensing agent is one or a mixture of two or more of ethyl 2 - oximecyanoacetate, O - benzotriazol - N,N,N',N' - tetramethyluronium hexafluorophosphate, 2-(7 - azabenzotriazol)-N,N,N',N' - tetramethyluronium hexafluorophosphate, N,N' - diisopropylcarbodiimide, the organic solvent is dichloromethane, and the basic catalyst is triethylamine; the equivalent ratio of the protected proline, the deprotected dipeptide product, the condensing agent, and the basic catalyst is 1:1 - 3:2 - 4:1 - 2; the mass - volume ratio of the protected proline to the organic solvent is 1:9 - 11; the reaction time is 2 - 6 hours.

[0018] Further, in step (1), the equivalent ratio of the protected hydroxyproline:benzotriazol - N,N,N',N' - tetramethyluronium hexafluorophosphate:N,N - diisopropylethylamine:protected glycine is 1:0.95:3:1.05, the condensation reaction time is 4 hours; the equivalent ratio of acetic anhydride:imidazole:4 - dimethylaminopyridine is 1:2:2; the time of the protection reaction is 2 hours;

[0019] In step (2), the deprotecting agent is 1,4 - epoxyhexane hydrochloride, the equivalent ratio of the protected dipeptide product to the deprotecting agent is 1:10; the mass - volume ratio of the protected dipeptide product to the organic solvent is 1:1; the deprotection reaction time is 1 hour;

[0020] In step (3), the condensing agent is ethyl 2 - oximecyanoacetate and N,N' - diisopropylcarbodiimide, the equivalent ratio of the protected proline, the deprotected dipeptide product, ethyl 2 - oximecyanoacetate, N,N' - diisopropylcarbodiimide, and the basic catalyst is 1:2:2:1.1:1.1; the mass - volume ratio of the protected proline to the organic solvent is 1:10, and the reaction time is 4 hours.

[0021] Further, the purification method in step (1) is: after the reaction is completed, water is added to the reaction solution, the product is extracted forward with ethyl acetate, and then back - extracted with water and saturated brine, and then anhydrous sodium sulfate is used for water absorption, and concentrated under reduced pressure to obtain the product;

[0022] The purification method described in step (2) is as follows: after the reaction is completed, the solvent is removed, and precipitation is carried out using ice-cold diethyl ether to obtain the product;

[0023] The purification method described in step (3) is as follows: after the reaction is completed, water is added to the reaction solution. First, the product is extracted forward using ethyl acetate, and then back-extracted using water and saturated brine. Then, anhydrous sodium sulfate is used to absorb water, and concentration under reduced pressure is carried out to obtain the product.

[0024] Furthermore, step (II) includes the following operations:

[0025] (a) Using a protected tripeptide as a raw material, a deprotection reaction is carried out with a metal catalyst in an organic solvent, and after purification, intermediate product 1 is obtained;

[0026] (b) Dissolving a protected tripeptide and a deprotecting agent in an organic solvent to carry out a deprotection reaction, and after purification, intermediate product 2 is obtained;

[0027] (c) Dissolving intermediate product 1 and a condensing agent in an organic solvent, adding intermediate product 2, and reacting under an alkaline catalyst, and after purification, the product is obtained;

[0028] (d) Using as a raw material, repeating steps (a)-(c) to obtain a long-chain collagen-like polypeptide; the number of repetitions is x - 1;

[0029] Preferably, the n is 2, 4, 8, 16, 32, 64.

[0030] Furthermore, in step (a), the metal catalyst is palladium on carbon; the equivalent ratio of the protected tripeptide to the metal catalyst is 1:0.005 - 0.02; the organic solvent is methanol, and the reaction time is 3 - 8 hours;

[0031] Preferably, in step (a), the equivalent ratio of the protected tripeptide to the metal catalyst is 1:0.01; the reaction time is 6 hours.

[0032] The present invention also provides a method for preparing a deprotected long-chain collagen-like polypeptide, and the method includes the following steps:

[0033] (i) Preparing a long-chain collagen-like polypeptide according to the above method:

[0034]

[0035] (ii) Dissolve the long-chain collagen polypeptide and inorganic carbonate in an organic solvent and react to remove R1; then react with a metal catalyst under the action of hydrogen to remove R2; finally react with a deprotecting agent to remove R3, and after purification, obtain the deprotected long-chain collagen polypeptide:

[0036] The present invention also provides a method for preparing an amino-terminal protected long-chain collagen polypeptide, and the method comprises the following steps:

[0037] I. Prepare the deprotected long-chain collagen polypeptide according to the above method;

[0038] II. React the deprotected long-chain collagen polypeptide with a protecting agent in an organic solvent to obtain an amino-terminal protected long-chain collagen polypeptide: R5 is an amino protecting group, preferably an acetyl group.

[0039] In summary, the present invention provides an iterative exponential synthesis method for preparing long-chain collagen polypeptides. By means of the liquid-phase polypeptide iterative exponential synthesis method, proline, hydroxyproline and glycine are connected according to a specific sequence to precisely prepare tripeptides; then, by using the liquid-phase iterative exponential synthesis strategy, the tripeptides are connected to form collagen polypeptide precursors with different lengths; finally, the protecting groups at the ends and side chains of the collagen polypeptide precursors are removed to prepare long-chain collagen polypeptides. The long-chain collagen polypeptides prepared by this method have precise chemical structures and uniform chain lengths. The present invention adopts the liquid-phase iterative exponential synthesis strategy, which enables the molecular weight of the polypeptide to increase exponentially during the synthesis process, reduces the reaction steps, improves the synthesis efficiency of long-chain collagen polypeptides, has the advantages of large synthesis scale and low cost, and can precisely prepare various functionalized long-chain collagen polypeptides.

[0040] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0041] The following further detailed description of the above content of the present invention is given in the form of specific embodiments by way of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a reaction flow chart for preparing a synthesis unit.

[0043] Figure 2 It is a schematic diagram for preparing a long-chain collagen polypeptide.

[0044] Figure 3 For the preparation of the HPLC and LC-MS spectra of the synthetic unit dipeptide (Boc-POG-OBn).

[0045] Figure 4 For the preparation of the HPLC and LC-MS spectra of the synthetic unit tripeptide (Boc-(POG)2-OBn).

[0046] Figure 5 For the HPLC and LC-MS spectra of the long-chain collagen-like polypeptide (Boc-(POG)8-OBn). Detailed implementation mode

[0047] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0048] Unless otherwise specified, this reaction is carried out at room temperature (25 ± 5 °C).

[0049] Example 1. Preparation of long-chain collagen-like polypeptide by iterative exponential synthesis method

[0050] 1. Preparation of Boc-O OAc G-OBn (abbreviated as O OAc G)

[0051] Dissolve 10 g of Boc-L-hydroxyproline (Boc-O OH -OH) with benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA) in 100 mL of N,N-dimethylformamide (DMF), and add glycine benzyl ester hydrochloride (HCl·NH2-G-OBn), where the equivalent ratio is: Boc-O OH -OH:HBTU:DIEA:HCl·NH2-G-OBn = 1:0.95:3:1.05, and react for 4 hours. After the reaction is complete, slowly dropwise add acetic anhydride liquid and imidazole, and add 4-dimethylaminopyridine solid (DMAP), where the equivalent ratio is: imidazole:DMAP:acetic anhydride = 2:2:1, and continue to react for 2 hours. The reaction flow chart is as Figure 1 shown.

[0052] After the reaction is complete, add an equal volume of water to the reaction solution. First, use ethyl acetate (EA) to extract the product forward, and then use pure water and saturated brine to back-extract 3 times to remove the remaining DMF in EA. Then, use anhydrous sodium sulfate to absorb water and concentrate under reduced pressure into an oily liquid to obtain O OAc G, with a yield of 96%. Confirm the purity and molecular weight by HPLC and LC-MS, and the HPLC purity is 98%.

[0053] 2. Preparation of HCl·NH2-O OAc G

[0054] Dissolve the O obtained in Step 1 OAc G in dichloromethane (DCM), add 1,4-epoxyhexane hydrochloride, where the equivalent ratio is O OAc G: 1,4-epoxyhexane hydrochloride = 1:10, O OAc The mass-volume ratio of G to DCM is 1:1 g / mL. Stir at room temperature for 1 hour. After the reaction is completed, remove the solvent, precipitate the above product with ice ether, and dry the filter cake to obtain HCl·NH2-O OAc G, with a yield of 99% and an HPLC purity of 98%. The reaction flow chart is as Figure 1 shown

[0055] 3. Preparation of Boc-PO OAc G-OBn (abbreviation: PO OAc G)

[0056] Mix Boc-L-proline (Boc-P-OH), ethyl 2-oximinoacetate (Oxyma), and N,N'-diisopropylcarbodiimide (EDCI) and dissolve them in DCM, add the HCl·NH2-O obtained in Step 2 OAc G, and react at room temperature for 4 hours under the alkaline condition (pH = 8) provided by triethylamine. The equivalent ratio is Boc-P-OH: Oxyma: EDCI: HCl·NH2-O OAc G: triethylamine = 1:2:2:1.1:1.1, and the mass-volume ratio of Boc-P-OH to DCM is 1:10 g / mL. The reaction flow chart is as Figure 1 shown

[0057] After the reaction is complete, add an equal volume of water to the reaction solution. First, extract the product with ethyl acetate (EA) in the forward direction, and then use pure water and saturated brine to back-extract 3 times to remove the remaining DMF in EA. Then, absorb water with anhydrous sodium sulfate and concentrate under reduced pressure to obtain an oily liquid to get PO OAc G, with a yield of 97%. Confirm the purity and molecular weight by HPLC and LC-MS. The HPLC purity is 95%. The HPLC spectrum and LC-MS spectrum are as Figure 3 shown

[0058] 4. Preparation of PO OAc G-OH

[0059] Dissolve the PO obtained in Step 3 OAc G in methanol (MeOH), add the catalyst palladium on carbon (Pd / C), where the equivalent ratio is PO OAc G: Pd / C = 1:0.01, PO OAcThe mass-volume ratio of G to MeOH is 1:10 g / mL. Hydrogen is introduced and the reaction proceeds for 5 hours. After the reaction, vacuum filtration is carried out, and the solvent is rotary evaporated to obtain PO OAC G-OH, with a yield of 100% and an HPLC purity of 98%. The reaction flow chart is as Figure 2 shown.

[0060] 5. Preparation of HCl·HN-PO OAc G

[0061] Dissolve the PO OAc G obtained in step 3 in DCM, and add 1,4-epoxyhexane hydrochloride, where the equivalent ratio is PO OAc G: 1,4-epoxyhexane hydrochloride = 1:10, and the mass-volume ratio of PO OAc G to DCM is 1:1 g / mL. Stir at room temperature for 1 hour. After the reaction, the solvent is removed, and the above product is precipitated with ice ether. The filter cake is dried to obtain HCl·HN-PO OAc G, with a yield of 99% and an HPLC purity of 95%. The reaction flow chart is as Figure 2 shown.

[0062] 6. Preparation of (PO OAc G)2

[0063] Dissolve the PO OAc G-OH obtained in step 4, Oxyma, and EDCI in DMF, and add the HCl·HN-PO OAc G obtained in step 5. React under the alkaline condition (pH = 8) provided by triethylamine for 4 hours. The equivalent ratio is PO OAc G-OH: Oxyma: EDCI: HCl·NH2-PO OAc G: triethylamine = 1:2:2:1.1:1.1, and the mass-volume ratio of PO OAc G-OH to DMF is 1:10 g / mL. The reaction flow chart is as Figure 2 shown.

[0064] After the reaction, the reaction solution is concentrated under reduced pressure, extracted 3 times with pure water and DCM (the ratio of pure water to DCM is 1:1), the DCM layer is collected, extracted 2 times with saturated Na2CO3 solution, washed 1 time with saturated sodium chloride aqueous solution, the organic phase is collected, dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain a pale yellow oily liquid. The obtained crude product is purified by silica gel column chromatography (petroleum ether / ethyl acetate = 60 / 40) to obtain (PO OAc G)2, with a yield of 82%. The purity and molecular weight of the product are confirmed by HPLC and LC-MS. The HPLC purity is 95%. The HPLC chromatogram and LC-MS chromatogram are as Figure 4 shown.

[0065] 7. Preparation of (PO OAc G)2-OH

[0066] Dissolve the product obtained in step 6, (PO OAc G)2, in MeOH, add Pd / C, with an equivalent ratio of (PO OAc G)2:Pd / C = 1:0.01, and the mass-volume ratio of (PO OAc G)2 to MeOH is 1:10 g / mL. Then pass hydrogen gas and react for 5 hours; after the reaction, perform vacuum filtration and rotary evaporation to obtain (PO OAc G)2-OH with a yield of 100% and an HPLC purity of 98%. The reaction flow chart is as shown in Figure 2 Figure

[0067] 8. Preparation of HCl·HN-(PO OAc G)2

[0068] Dissolve the product obtained in step 6, (PO OAc G), in DCM, add 1,4-epoxyhexane hydrochloride, with an equivalent ratio of (PO OAc G):1,4-epoxyhexane hydrochloride = 1:10, and the mass-volume ratio of (PO OAc G) to DCM is 1:1 g / mL. React at room temperature for 1 hour; after the reaction, perform vacuum rotary evaporation, and precipitate the above product with ice ether. Dry the filter cake to obtain HCl·HN-(PO OAc G)2 with a yield of 96% and an HPLC purity of 95%. The reaction flow chart is as shown in Figure 2 Figure

[0069] 9. Preparation of (PO OAc G)4

[0070] Dissolve the (PO OAc G)2-OH obtained in step 7, Oxyma, and EDCI in DMF, add the HCl·HN-(PO OAc G)2 obtained in step 8 to the reaction solution, and continue to react for 4 hours under the alkaline condition (pH = 8) provided by triethylamine. The equivalent ratio is (PO OAc G)2-OH:Oxyma:EDCI:HCl·HN-(PO OAc G)2:triethylamine = 1:2:2:1.1:1.1, and the mass-volume ratio of (PO OAc G)2-OH to DMF is 1:10 g / m. The reaction flow chart is as shown in Figure 2 Figure

[0071] After the reaction was completed, the reaction solution was concentrated under reduced pressure, and extracted three times with pure water and DCM. The ratio of pure water to DCM was 1:1. The DCM layer was collected, extracted twice with saturated Na2CO3 solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a pale yellow oily liquid. The obtained crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 60:40) to obtain (PO OAc G)4 with a yield of 82%. The purity and molecular weight of the product were confirmed by HPLC and LC-MS, and the HPLC purity was 98%.

[0072] 10. Repeat the above steps to obtain HCl·HN-(PO OAc G) n , Boc-(PO OAc G) n -OH, and prepare the corresponding (PO OAc G) 2n (n = 4, 8, 16, 32, etc.). The HPLC and LC-MS spectra of (PO OAc G)8 are as shown Figure 5 in the figure.

[0073] 11. Remove the protecting groups at the terminal and side chains of the (PO OR G) n precursor. Taking (PO OR G)4 as an example:

[0074] (a) First, dissolve (PO OR G)4 and potassium carbonate in methanol solution, stir for 6 hours to remove the acetyl group on the side chain, and obtain intermediate 1. Among them, the equivalent ratio of (PO OR G)4 to potassium carbonate is 1:3, and the mass-volume ratio of (PO OR G)4 to methanol is 1:10 g / mL.

[0075] (b) Then, under the action of hydrogen, react with the catalyst palladium on carbon (Pd / C) for 5 hours to remove the benzyl group on intermediate 1, and obtain intermediate 2. Among them, the equivalent ratio of intermediate 1 to Pd / C is 1:0.01.

[0076] (c) Finally, react with 1,4-epoxyhexane hydrochloride for 1 hour to remove the tert-butoxycarbonyl group on intermediate 2. Among them, the equivalent ratio of intermediate 2 to 1,4-epoxyhexane hydrochloride is 1:10. After the reaction is completed, concentrate the solvent, and precipitate the above product with ice ether. The filter cake is dried to obtain the corresponding product.

[0077] The total yield of steps (a)-(c) is 95%, and the product purity is 90%.

[0078] 12. Protection of the amino terminus of the CLP precursor. The product obtained in step 11 was dissolved in DMF, and acetic anhydride liquid was slowly added dropwise to the solution, and the reaction was carried out at room temperature for 1 hour. Among them, the equivalent ratio of the product obtained in step 11 to acetic anhydride was 1:4. After the reaction was completed, the solvent was concentrated to obtain CLP with the amino terminus of the precursor protected by an acetyl group. The yield of this step was 95%, and the product purity was 95%.

[0079] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0080] Experimental Example 1. Preparation of O OAc Screening experiment of G

[0081] Method 1:

[0082] 10 g of Boc-L-hydroxyproline (Boc-O OH -OH) and benzyl bromide were mixed and dissolved in N,N-dimethylformamide (DMF), potassium carbonate powder was added, and after reacting for 2 hours, 4-dimethylaminopyridine (DMAP) solid was added to the reaction solution, and then acetic anhydride was added as a protecting reagent and the reaction was continued for 4 hours. Among them, the equivalent ratio of Boc-O OH -OH, benzyl bromide, potassium carbonate, DMAP, and acetic anhydride was 1:2:2:1.1:1.1, and the mass-volume ratio of Boc-O OH -OH to DMF was 1:10 g / mL. After the reaction was completed, extraction and concentration under reduced pressure were carried out to obtain Boc-O OR -OBn. Boc-O OR -OBn was dissolved in methanol (MeOH), and palladium on carbon (Pd / C) was added as a catalyst and hydrogen was passed through. Among them, the Pd / C of Boc-O OR -OB to Pd / C, and the mass-volume ratio of Boc-O OR -OB to MeOH was 1:10 g / mL. After reacting for 5 hours, filtration under reduced pressure was carried out and the solvent was evaporated to dryness to obtain Boc-O OR -OH. The yield of these two steps was 97%.

[0083] Boc-O OR -OH, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (HBTU), and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in DMF, and glycine benzyl ester hydrochloride (HCl·NH2-G-OBn) was added, and the reaction was carried out for 3 hours; among them, after the reaction was complete, extraction and concentration under reduced pressure were carried out to obtain Boc-O OR G-OBn. The yield of this step was 96%, and the purity of Boc-O OR G-OBn was 95%.

[0084] Method 2:

[0085] Same as step 1 of Example 1.

[0086] The route in Method 1 is too cumbersome and time-consuming, so Method 2 is selected for the preparation of dipeptide Boc-O OR G-OBn.

[0087] Experimental Example 2, Preparation of HCl·NH2-O OR Screening experiment of G

[0088] Method 1:

[0089] Dissolve the O OR G obtained in step 1 of Example 1 in dichloromethane (DCM), add 1,4-epoxyhexane hydrochloride and stir at room temperature for 1 hour. The equivalent ratio of O OR G to 1,4-epoxyhexane hydrochloride is the same as that in step 2 of Example 1, and the mass-volume ratio of O OR G to DCM is the same as that in step 2 of Example 1. After the reaction is completed, remove the solvent to obtain HCl·NH2-O OR G. The yield of this step is 100%, and the HPLC purity is 98%.

[0090] Method 2:

[0091] Same as step 2 of Example 1.

[0092] The purity of the product obtained in Method 1 is not ideal, so Method 2 is selected for the preparation of HCl·NH2-O OR G.

[0093] Experimental Example 3, Preparation of PO OR Screening experiment of G

[0094] Method 1:

[0095] Mix Boc-L-proline (Boc-P-OH) with HOBT and DIEA and dissolve them in DMF. After activation for 0.5 hours, add HCl NH-O OR G-OBn and react at room temperature for 4 hours; after the reaction is completed, concentrate the reaction solution under reduced pressure, add pure water and DCM for extraction 3 times, the ratio of pure water to DCM is 1:1, collect the DCM layer, extract it 2 times with saturated Na2CO3 solution, and wash it 1 time with saturated sodium chloride aqueous solution. Collect the organic phase, add anhydrous Na2SO4 for drying, and concentrate under reduced pressure to obtain a light yellow oily liquid. The obtained crude product is purified by silica gel column chromatography (petroleum ether / ethyl acetate = 55:45) to obtain Boc-PO OR G-OBn. The yield of the product in this step is 82%, and the HPLC characterization purity is 98%.

[0096] Method 2:

[0097] Same as step 3 of Example 1.

[0098] The HOBT activator used in Method 1 produces more by-products, and the purification operation is complex and difficult. Column chromatography purification is required, and the product yield is low. Therefore, Method 2 is selected for the preparation of PO OR of G.

[0099] In summary, the present invention provides an iterative exponential synthesis method for preparing long-chain collagen-like polypeptides. The long-chain collagen-like polypeptides prepared by this method have precise chemical structures and uniform chain lengths. The present invention adopts a liquid-phase iterative exponential synthesis strategy, which enables the molecular weight of the polypeptide to increase exponentially during the synthesis process, reduces the reaction steps, improves the synthesis efficiency of long-chain collagen-like polypeptides, and has advantages such as large synthesis scale and low cost, and can precisely prepare various functionalized long-chain collagen-like polypeptides.

Claims

1. A method for preparing long-chain collagen-like polypeptides, characterized in that: The method includes the following steps: (I) Prepare a tripeptide protected by a protecting group; (II) Using the tripeptide protected by a protecting group as a raw material, prepare a long-chain collagen-like polypeptide by an iterative exponential synthesis method; The structure of the tripeptide protected by the protecting group is The structure of the long-chain collagen-like polypeptide is R1 is a hydroxyl protecting group, R2 is a carboxyl protecting group, R3 is an amino protecting group, n is 2 to the power of x, and x is an integer greater than or equal to 1; The method for preparing the tripeptide protected by a protecting group in step (I) includes the following steps: (1) Dissolve the protected hydroxyproline, coupling agent and basic catalyst in an organic solvent, then add the protected glycine for a condensation reaction, and then add a protecting agent, an imidazole compound and a catalyst for a protection reaction. After purification, a protected dipeptide product is obtained: R4 is an amino protecting group; R3 is the same as R4; the coupling agent is benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; (2) Dissolve the dipeptide product protected by the protecting group and the deprotecting agent in an organic solvent to carry out a deprotection reaction, and obtain the deprotected dipeptide product after purification: The deprotecting agent is 1,4-epoxyhexane hydrochloride; (3) Dissolve the protected proline and a condensing agent in an organic solvent, add the deprotected dipeptide product, react under an alkaline catalyst, and after purification, obtain a collagen-like tripeptide protected by a protecting group; Step (II) includes the following operations: (a) Using the protected tripeptide as a raw material, a deprotection reaction is carried out with a metal catalyst in an organic solvent, and after purification, intermediate 1 is obtained: (b) Dissolve the protecting group-protected tripeptide and the deprotecting agent in an organic solvent for deprotection reaction, and obtain intermediate 2 after purification: (c) Dissolve intermediate 1 and the condensing agent in an organic solvent, add intermediate 2, and react under an alkaline catalyst. After purification, the product is obtained: (d) Using as a raw material, repeat steps (a)-(c) to obtain long-chain collagen-like polypeptides; the number of repetitions is x-1; The hydroxyl protecting group is an acetyl group, the carboxyl protecting group is a benzyl group, and the amino protecting group is a tert-butoxycarbonyl group.

2. The method according to claim 1, wherein: The n is 2, 4, 8, 16, 32, 64.

3. The method according to claim 1, wherein: In step (1), the protected hydroxyproline is Boc-L-hydroxyproline; the alkaline catalyst is N,N-diisopropylethylamine, the protected glycine is glycine benzyl ester hydrochloride, the organic solvent is N,N-dimethylformamide, and the equivalent ratio of the protected hydroxyproline:benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate:N,N-diisopropylethylamine:protected glycine is 1:0.5-1:2-5:1-1.2, and the condensation reaction time is 1-5 hours; the protecting agent is acetic anhydride, the imidazole compound is imidazole, the catalyst is 4-dimethylaminopyridine, and the equivalent ratio of acetic anhydride:imidazole:4-dimethylaminopyridine is 1:1-3:1-3, and the time for the protection reaction is 1-3 hours; In step (2), the organic solvent is dichloromethane, and the equivalent ratio of the protected dipeptide product to the deprotecting agent is 1:9-11; the mass-volume ratio of the protected dipeptide product to the organic solvent is 1:0.5-2; the deprotection reaction time is 0.5-2 hours; In step (3), the protected proline is Boc-L-proline; the organic solvent is dichloromethane, and the alkaline catalyst is triethylamine; the equivalent ratio of the protected proline, deprotected dipeptide product, condensing agent, and alkaline catalyst is 1:1-3:2-4:1-2; the mass-volume ratio of the protected proline to the organic solvent is 1:9-11; the reaction time is 2-6 hours.

4. The method according to claim 3, wherein: In step (1), the equivalent ratio of the protected hydroxyproline:benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate:N,N-diisopropylethylamine:protected glycine is 1:0.95:3:1.05, and the condensation reaction time is 4 hours; the equivalent ratio of acetic anhydride:imidazole:4-dimethylaminopyridine is 1:2:2; the time for the protection reaction is 2 hours; In step (2), the equivalent ratio of the protected dipeptide product to the deprotecting agent is 1:10; the mass-volume ratio of the protected dipeptide product to the organic solvent is 1:1; the deprotection reaction time is 1 hour; In step (3), the condensing agent is ethyl 2-oximinoacetate and N,N'-diisopropylcarbodiimide, and the equivalent ratio of the protected proline, the deprotected dipeptide product, ethyl 2-oximinoacetate, N,N'-diisopropylcarbodiimide, and the basic catalyst is 1:2:2:1.1:1.1; the mass-volume ratio of the protected proline to the organic solvent is 1:10, and the reaction time is 4 hours.

5. The method according to claim 1, characterized in that: The purification method in step (1) is as follows: after the reaction is completed, water is added to the reaction solution, and the product is extracted forward with ethyl acetate, and then back-extracted with water and saturated brine, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the product. The purification method in step (2) is as follows: after the reaction is completed, the solvent is removed, and the product is precipitated with ice ether to obtain the product. The purification method in step (3) is as follows: after the reaction is completed, water is added to the reaction solution, and the product is first extracted forward with ethyl acetate, and then back-extracted with water and saturated brine, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the product.

6. The method according to claim 1, wherein: In step (a), the metal catalyst is palladium on carbon; the equivalent ratio of the protected tripeptide to the metal catalyst is 1:0.005 - 0.02; the organic solvent is methanol, and the reaction time is 3 - 8 hours.

7. The method according to claim 6, wherein: In step (a), the equivalent ratio of the protected tripeptide to the metal catalyst is 1:0.01; the reaction time is 6 hours.

8. A method for preparing a deprotected long-chain collagen-like polypeptide, characterized in that: The method comprises the following steps: (i) The long-chain collagen-like polypeptide is prepared by the method according to any one of claims 1-7: (ii) React the long-chain collagen polypeptide with inorganic carbonate in an organic solvent to remove R1; then react with a metal catalyst under the action of hydrogen to remove R2; finally react with a deprotecting agent to remove R3, and after purification, obtain the deprotected long-chain collagen polypeptide:

9. A method for preparing an amino-terminal protected long-chain collagen-like polypeptide, characterized in that: The method comprises the following steps:

1. Prepare the deprotected long-chain collagen-like polypeptide according to the method described in claim 8; Second, react the deprotected long-chain collagen polypeptide with a protecting agent in an organic solvent to obtain a long-chain collagen polypeptide with an amino-terminal protection: R5 is an amino protecting group.

10. The method according to claim 9, wherein: R5 is an acetyl group.

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