Synthesis method of polypeptide new coronavirus membrane fusion inhibitor
Through the solid-phase coupling method of full-protected fragment and coupling the 46 peptide sequence of HY3000, the problem of peptide chain folding and coupling is solved, and the purity and yield of the membrane fusion inhibitor of the peptide novel coronavirus is improved, which is suitable for large-scale Large-scale production.
Patent Information
- Application Number
- CN202310791487.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-30
- Publication Date
- 2024-12-31
AI Technical Summary
In the prior art, the chemical synthesis method of the polypeptide novel coronavirus membrane fusion inhibitor HY3000 has difficulty in folding and coupling of peptide chains, resulting in low purity and yield, making it difficult to produce on a large scale.
Using the solid phase coupling method of fully protected fragments, the 46 peptide sequence of HY3000 is divided into multiple short fragments and coupled one by one on the solid phase support. Through Fmoc solid phase condensation and cleavage technology, peptide chain folding and resin shrinkage are avoided, and the peptide chain folding and resin shrinkage are improved. Purity and yield.
It significantly improves the purity and yield of the 46-peptide crude peptide of HY3000, simplifies the production process, reduces the difficulty and cost of purification, and is suitable for large-scale production.
Smart Images
Figure CN119219746A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to a method for synthesizing a polypeptide coronavirus membrane fusion inhibitor. Background Art
[0002] HY3000 is a peptide-based novel coronavirus membrane fusion inhibitor. It binds to the HR1 region of the novel coronavirus spike protein, prevents the formation of the virus's six-helix bundle structure, and blocks the virus from infecting cells to achieve an antiviral effect. HY3000 has shown significant anti-novel coronavirus effects both in vivo and in vitro, and has significant inhibitory activity against the original strain of the novel coronavirus and its various popular variants, and its antiviral activity is not affected by viral mutations. According to the results of the Phase I clinical study, HY3000 has no serious adverse reactions to humans and has good safety.
[0003] HY3000 is composed of 46 amino acids and a cholesterol derivative, molecular formula: C243H410N60O81S molecular weight: 5500.35, peptide sequence Figure 1 shown.
[0004] There is no report on the chemical synthesis method of HY3000. Generally, the solid phase stepwise coupling method (Fmoc-SPPS) is selected to synthesize the 46-peptide part of HY3000, which is modified with cholesterol derivatives to obtain the crude HY3000 peptide, and then purified by reverse phase preparative chromatography to obtain the refined HY3000 peptide. The process is simple to operate, but the resin shrinks severely during the coupling process, making it difficult to couple smoothly and easily leading to missing peptide impurities. Summary of the invention
[0005] In order to solve the above-mentioned technical problems, the present invention provides a method for synthesizing a polypeptide coronavirus membrane fusion inhibitor.
[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0007] The present invention provides a method for synthesizing a polypeptide novel coronavirus membrane fusion inhibitor, wherein the polypeptide novel coronavirus membrane fusion inhibitor is HY3000. The method comprises dividing a 46-peptide portion of HY3000 into FP (38-46) fragments which are coupled one by one on a blank amide resin, and FP (28-37) fragments, FP (21-27) fragments, FP (9-20) fragments, and FP (1-8) fragments which are coupled one by one on 2-CTCResin and cleaved in a fully protective cleavage manner; and then coupling the fully protected fragments one by one by an Fmoc solid phase coupling manner to obtain the HY3000.
[0008] Furthermore, the blank amide resin is RinkAmide-AMResin or RinkAmideResin or RinkAmide-MBHAResin; the blank amide resin is preferably RinkAmide-AMResin; wherein the substitution degree of Rink Amide-AMResin is 0.2mmol / g to 0.5mmol / g, preferably 0.3mmol / g to 0.4mmol / g.
[0009] Furthermore, the FP (38-46) fragment includes a FP (38-46)-Resin fragment; the FP (38-46)-Resin fragment is synthesized using a blank amide resin as a solid phase carrier, and the resulting peptide resin peptide sequence is H-Gln (Trt) 38 -Glu(OtBu) 39 -Leu 40 -Gly 41 -Ser(tBu) 42 -Gly 43 -Ser(tBu) 44 -Gly 45 -Cys(Trt) 46 -Resin; the FP (28-37) fragment is treated with 2-CTCResin as a solid phase carrier with 20% to 30% TFE / DCM solution to separate the fully protected peptide from the resin carrier. The peptide sequence of the obtained fully protected fragment peptide is Fmoc-Lys (Boc) 28 -Asn(Trt) 29 -Leu 30 -Asn(Trt) 31 -Glu(OtBu) 32 -Ser(tBu) 33 -Leu 34 -Ile 35 -Asp(OtBu) 36 -Leu 37 -OH; the FP (21-27) fragment is treated with 2-CTCResin as a solid phase carrier, and the fully protected peptide and the resin carrier are separated by 20% to 30% TFE / DCM solution. The peptide sequence of the obtained fully protected fragment peptide is Fmoc-Asp (OtBu) 21 -Arg(Pbf) 22 -Leu 23 -Asn(Trt) 24 -Glu(OtBu) 25 -Val 26 -Ala 27-OH; the FP (9-20) fragment is treated with 2-CTCResin as a solid phase carrier, and the fully protected peptide and the resin carrier are separated by 20% to 30% TFE / DCM solution. The obtained fully protected fragment peptide sequence is Fmoc-Ile 9 -Asn(Trt) 10 -Ala 11 -Ser(Psi(Me,Me)pro) 12 -Val 13 -Val 14 -Asn(Trt) 15 -Ile 16 -Gln(Trt) 17 -Lys(Boc) 18 -Glu(OtBu) 19 -Ile 20 -OH; the FP (1-8) fragment is treated with 2-CTCResin as a solid phase carrier, and the fully protected peptide and the resin carrier are separated by 20% to 30% TFE / DCM solution. The peptide sequence of the obtained fully protected fragment peptide is Boc-Val 1 -Asp(OtBu) 2 -Leu 3 -Gly 4 -Asp(OtBu) 5 -Ile 6 -Ser(tBu) 7 -Gly 8 -OH.
[0010] Furthermore, the degree of substitution of 2-CTCResin is 1.0 mmol / g to 1.5 mmol / g, and preferably the degree of substitution is 1.1 mmol / g.
[0011] Furthermore, the method specifically comprises: subjecting the C-terminus of the FP(28-37) fragment to a coupling reaction with the N-terminus of the FP(38-46)-Resin fragment, and then removing the Fmoc protecting group to obtain the FP(28-46)-Resin fragment; subjecting the C-terminus of the FP(21-27) fragment to a coupling reaction with the N-terminus of the FP(28-46)-Resin fragment, and then removing the Fmoc protecting group to obtain the FP(21-46)-Resin fragment. sin fragment; after coupling reaction between the C-terminus of the FP(9-20) fragment and the N-terminus of the FP(21-46)-Resin fragment, the Fmoc protecting group is removed to obtain the FP(9-46)-Resin fragment; after coupling reaction between the C-terminus of the FP(1-8) fragment and the N-terminus of the FP(9-46)-Resin fragment, the FP(1-46)-Resin fragment is obtained, and the HY3000 46-peptide peptide resin is obtained.
[0012] Furthermore, the condensation system used for solid phase condensation of each protected amino acid or fully protected fragment by Fmoc method is one or more of DIC / HOBt or DIC / Oxyma or PyBOP / HOBt / DIEA, and the reaction solvent used is one or more of DCM, DMF, DMSO, and NMP; the Fmoc removal reagent is 20% Pip / DMF solution; the condensation system and its ratio are peptide resin / fragment peptide or / DIC / HOBt=1 / 2 / 2.4 / 2.4; the reaction solvent used is preferably DMF.
[0013] Furthermore, the method also includes: cleaving the HY3000 46-peptide peptide resin with a cleavage solution at room temperature for 2 hours to 4 hours to obtain the HY3000 46-peptide crude peptide, and then purifying the HY3000 46-peptide refined peptide by preparative chromatography.
[0014] Furthermore, the lysate is a mixture of TFA with a volume ratio of more than 80% and one or more of phenol, thioanisole, DTT, EDT, DODT, TES, Tis, and H2O in different proportions; the lysate is preferably TFA / EDT / Tis / H2O=91%:3%:3%:3%.
[0015] Furthermore, the method also includes: connecting the 46-peptide refined peptide of HY3000 with the cholesterol derivative BAPC, and then purifying it by preparative chromatography to obtain HY3000 API.
[0016] Furthermore, in the step of connecting the 46-peptide refined peptide of HY3000 with the cholesterol derivative BAPC, the step includes: dissolving the 46-peptide refined peptide of HY3000 with DMF, DMSO or THF, adding BAPC, and then adding DIEA, stirring and reacting at room temperature for 1 hour to 2 hours to obtain a HY3000 solution; wherein the 46-peptide refined peptide of HY3000 / BAPC / DIEA=1 / 1.05~1.3 / 2~20, preferably the 46-peptide refined peptide of HY3000 / BAPC / DIEA=1 / 1.05 / 15.
[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:
[0018] The present invention provides a method for synthesizing a polypeptide novel coronavirus membrane fusion inhibitor, wherein the 46-peptide portion of HY3000 is synthesized by a fully protected fragment peptide solid phase coupling method, specifically: firstly, several fully protected fragment polypeptides are obtained by solid phase synthesis, and then they are sequentially condensed on a solid phase carrier to obtain a fully protected long peptide chain, and finally the side chain protecting group is cleaved to obtain the 46-peptide portion of HY3000. The method can avoid the problems of peptide chain folding and coupling difficulties caused by solid phase coupling of amino acids one by one, and can greatly improve the purity and yield of the obtained 46-peptide crude peptide of HY3000, and finally, after modification with a cholesterol derivative (BAPC), the API refined peptide of HY3000 is obtained by purification through preparative chromatography.
[0019] 1) The present invention selects amino acid sites that are not prone to racemization reactions, such as Ala, Leu, Ile, and Gly. Due to the small steric hindrance, it is conducive to the rapid progress of the reaction and can reduce the occurrence of racemization side reactions.
[0020] 2) The present invention selects the cleavage site according to the shrinkage phenomenon of the peptide resin during the step-by-step coupling process, avoids the folding of multiple peptide chains in the HY3000 peptide sequence, overcomes the problem of slow coupling rate of protected amino acids one by one, and can obtain a 46-peptide crude peptide with purity and yield far higher than that obtained by coupling one by one.
[0021] 3) The fully protected fragment peptide sequence selected by the present invention is easy to couple the protected amino acids during the preparation process of the fully protected peptide, and the fully protected fragment peptide solid with high purity is easy to precipitate after the peptide resin is cracked, without the need for preparative chromatography purification, thus saving time and cost.
[0022] 4) The fully protected fragment peptide sequences selected by the present invention can show a high coupling rate when sequentially coupled to a solid phase carrier without special conditions, and can obtain a 46-peptide crude peptide with fewer impurities, higher purity and yield. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] One or more embodiments are exemplarily described by pictures in the corresponding drawings, and these exemplifications do not constitute limitations on the embodiments. Unless otherwise stated, the pictures in the drawings do not constitute proportional limitations.
[0024] Figure 1 This is the peptide sequence of HY3000;
[0025] Figure 2 This is the synthesis route of the 46-peptide partial fragment and API synthesis of HY3000 in the embodiment of the present invention Figure 1 ;
[0026] Figure 3 This is the HPLC spectrum of FP (28-37) of Example 3 of the present invention;
[0027] Figure 4This is the HPLC spectrum of FP (21-27) of Example 4 of the present invention;
[0028] Figure 5 This is the HPLC spectrum of Example 5FP (9-20) of the present invention;
[0029] Figure 6 This is the HPLC spectrum of Example 6FP (9-20a) of the present invention;
[0030] Figure 7 This is the HPLC spectrum of FP (1-8) of Example 7 of the present invention;
[0031] Figure 8 This is a solid phase synthesis method-HPLC spectrum of the 46-peptide portion of HY3000 in Example 8 of the present invention;
[0032] Fig. 9 This is the HPLC spectrum of the solid phase synthesis method II of the 46-peptide portion of HY3000 of Example 9 of the present invention;
[0033] Fig.10 This is the solid phase synthesis method three HPLC spectrum of the 46-peptide portion of Example 10HY3000 of the present invention;
[0034] Fig.11 This is a solid phase synthesis method four HPLC spectra of the 46-peptide portion of Example 11HY3000 of the present invention. DETAILED DESCRIPTION
[0035] In 1963, Merrifield first proposed the solid phase peptide synthesis method (SPPS). This synthesis method, which is a milestone in peptide chemistry, became the preferred method for peptide synthesis as soon as it appeared because of its convenient and rapid synthesis, and it also brought about a revolution in the organic synthesis of peptides. The step-by-step coupling synthesis of peptides is a process of repeated addition of amino acids. The solid phase synthesis sequence is generally from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus). However, when the step-by-step coupling method is used, amino acid coupling is often more difficult due to the long peptide sequence or the aggregation of peptide chains, and there are more impurities. The purity of the crude peptide is often low, resulting in a low yield of the finished product. In large-scale production, this risk brought about by continuous repeated operations will continue to increase.
[0036] The inventors found that the use of the fragment method for synthesis can effectively avoid the problem of coupling difficulties caused by long peptide sequences and peptide chain aggregation, and can also shorten the production cycle and improve production capacity. The fragment method here refers to first obtaining one or several shorter and higher purity fully protected fragments through solid phase step-by-step coupling, and then splicing the obtained fragments as a whole similar to protected amino acids through solid phase reaction.
[0037] HY3000 is a 46-mer peptide, which belongs to a long peptide. Its C-terminal cysteine amide is cholesterol-modified through a side chain thiol. Due to the long peptide chain, the peptide-based coronavirus membrane fusion inhibitor (HY3000) has a larger steric hindrance as the synthetic peptide sequence is extended on the resin carrier. Coupling difficulties, resin shrinkage, and reaction solution washing residues are all key factors that cause impurities, reduce the quality of crude peptides, and increase the difficulty of subsequent purification and removal. In addition, the use of a step-by-step coupling process restricts the efficiency and capacity of large-scale production.
[0038] In view of the above shortcomings, the linear 46-peptide part of HY3000 was synthesized from the C-terminus by solid phase synthesis of fragment FP(38-46)-Resin, and then the fragments (FP(28-37), fragment FP(21-27), fragment FP(9-20), and fragment FP(1-8) were coupled in sequence. The fragment sequences are shown in Table 1. The solid phase coupling process of the fragments is shown in Figure 2 As shown, the resin shrinkage and coupling problems of difficult sites during the peptide resin coupling process can be avoided, the number of impurities can be reduced, the coupling efficiency can be improved, the yield, purity and content of linear crude peptides can be improved, the purification difficulty can be reduced, and the synthesis cycle can be shortened; after modification with cholesterol derivatives, a final product with higher purity and yield can be obtained.
[0039] Table 1 Sequence list of 46 peptide fragments of HY3000
[0040]
[0041] The present invention is described in detail below in conjunction with specific implementation modes.
[0042] Example 1 Synthesis of FP(38-46)-Resin
[0043] Weigh 30g (scale 10.2mmol) of RinkAmide-AMResin with a substitution degree of 0.34mmol / g in a solid phase reaction column, swell the resin with 150mL DMF for 30 minutes, wash twice with 150mL DMF, deprotect with 150mL of 20% piperidine / DMF solution for 5+7min, wash 6 times with 150mL DMF, and detect the color of the resin with ninhydrin. Weigh 20.4mmol Fmoc-Cys(Trt)-OH and 24.48mmol HOBt, dissolve with 120mL DMF, add 24.48mmol DIC to activate for 5min in an ice-water bath, add the mixed solution to the reaction column, react with nitrogen gas at room temperature for 2h, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction, if the resin is colored, extend the reaction for 30min). After the reaction was completed, the resin was washed three times with 150 mL of DMF, deprotected for 5+7 min by adding 150 mL of 20% piperidine / DMF solution, and washed six times with 150 mL of DMF. The resin was detected by ninhydrin to see if it had color.
[0044] Repeat the above coupling operation and continue to couple Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Gln(trt)-OH in sequence according to the FP(38-46)-Resin peptide sequence. 20.4 mmol of each protected amino acid was added, 24.48 mmol of DIC and 24.48 mmol of HOBt were used. In the last step, 150 mL of 20% piperidine / DMF solution was added for deprotection for 5+7 min. After washing 6 times with 150 mL of DMF (the resin was colored when detected by ninhydrin), the peptide resin was washed 3 times with DCM, 3 times with MeOH and dried to obtain 44.115 g (10.2 mmol) of FP(38-46)-Resin peptide resin with a weight yield of 102.78%.
[0045] Example 2 Synthesis of FP(38-46a)-Resin
[0046] The difference from Example 1 is that Fmoc-(Hmb)Gly-OH is used to replace Fmoc-Gly-OH for coupling at the 41st Gly to obtain 45.714 g (10.2 mmol) of FP(38-46a)-Resin peptide resin with a weight yield of 103.17%.
[0047] Example 3 Synthesis of FP (28-37)
[0048] Weigh 40.0 g (scale 44.0 mmol) of 2-CTCResin with a substitution degree of 1.1 mmol / g into a solid phase reaction column, weigh 88.0 mmol Fmoc-Leu-OH, dissolve it with 160 mL DMF and add it to the solid phase reaction column, then add 176.0 mmol DIEA, and react with nitrogen gas at room temperature for 2 hours. After the reaction, add 40 mL MeOH for resin blocking reaction, and after 1 hour of reaction, remove the reaction liquid, wash the resin 3 times with 200 mL DMF, and then wash it 3 times with MeOH, and vacuum dry. The substitution degree of Fmoc-Leu-CTC Resin is 0.858 mmol / g, and the weight of the amino acid resin is 50.147 g.
[0049] Weigh 50.0g Fmoc-Leu-CTCResin (Sub=0.858mmol / g, scale 42.9mmol) into the solid phase reaction column, swell the resin with 250mL DMF for 30 minutes, wash twice with 250mL DMF, then deprotect with 250mL 20% piperidine / DMF solution for 5+7min, wash 6 times with 250mL DMF, and detect the color of the resin with ninhydrin. Weigh 85.8mmol Fmoc-Asp(OtBu)-OH, 103.0mmol HOBt, dissolve with 200mL DMF and add to the reaction column, then add 103.0mmol DIC to start the reaction, react with nitrogen gas at room temperature for 2h, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction, if the resin is colored, extend the reaction for 30min). After the reaction was completed, the resin was washed 3 times with 250 mL of DMF, deprotected for 5+7 min by adding 250 mL of 20% piperidine / DMF solution, and washed 6 times with 250 mL of DMF. The resin was detected by ninhydrin to see if it had color.
[0050] Repeat the above coupling operation, and continue to couple Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH in sequence according to the peptide sequence of FP(28-37). Each protected amino acid was fed with 85.8 mmol, DIC was used in an amount of 103.0 mmol, and HOBt was used in an amount of 103.0 mmol. Finally, without removing Fmoc, the peptide resin was washed with MeOH three times and dried by vacuum to obtain 110.472 g of peptide resin of FP(28-37), with a weight yield of 87.41%.
[0051] The obtained peptide resin of 110.0 g FP (28-37) was fully protected and cleaved with 1.1 L 20% TFE / DCM solution at room temperature, and the reaction was stirred for 2 h. After the reaction was completed, the filter cake was rinsed with DCM 3 times, 200 mL each time, and the filtrate was combined. The filtrate was subjected to reduced pressure rotary evaporation on a rotary evaporator to remove DCM, leaving about 250 mL of solution. 250 mL DMF was added to precipitate a white solid, and 750 mL H2O was added. After stirring and beating at room temperature for 1 h, the mixture was filtered. The filter cake was rinsed with H2O 3 times, 250 mL each time, and vacuum-dried to obtain 85.714 g (40.17 mmol) of FP (28-37) with a purity of 95.55%, as shown in FIG. Figure 3 As shown, the weight yield is 93.64%.
[0052] Example 4 Synthesis of FP (21-27)
[0053] Weigh 50.0 g (scale 55.0 mmol) of 2-CTCResin with a substitution degree of 1.1 mmol / g into a solid phase reaction column, weigh 110.0 mmol Fmoc-Ala-OH, dissolve it with 200 mL DMF and add it to the solid phase reaction column, then add 220.0 mmol DIEA, and react with nitrogen gas at room temperature for 2 hours. After the reaction is completed, add 50 mL MeOH for resin blocking reaction, and after 1 hour of reaction, remove the reaction liquid, wash the resin 3 times with 250 mL DMF, and then wash it 3 times with MeOH, and vacuum dry. The substitution degree of Fmoc-Ala-CTC Resin is 0.806 mmol / g, and the weight of the amino acid resin is 65.539 g.
[0054] Weigh 65.0g Fmoc-Ala-CTCResin (Sub=0.806mmol / g, scale 52.4mmol) into the solid phase reaction column, swell the resin with 325mL DMF for 30 minutes, wash twice with 325mL DMF, then deprotect with 325mL 20% piperidine / DMF solution for 5+7min, wash 6 times with 325mL DMF, and detect the color of the resin with ninhydrin. Weigh 104.8mmol Fmoc-Val-OH and 125.8mmol HOBt, dissolve with 260mL DMF and add to the reaction column, then add 125.8mmol DIC to start the reaction, react with nitrogen at room temperature for 2h, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction, if the resin is colored, extend the reaction for 30min). After the reaction was completed, the resin was washed three times with 325 mL of DMF, deprotected for 5+7 min with 325 mL of 20% piperidine / DMF solution, and washed six times with 325 mL of DMF. The resin was detected by ninhydrin to show color.
[0055] Repeat the above coupling operation, and continue to couple Fmoc-Glu(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Aap(OtBu)-OH in sequence according to the peptide sequence of FP(21-27). Each protected amino acid was fed with 104.8 mmol, DIC was used in an amount of 125.8 mmol, and HOBt was used in an amount of 125.8 mmol. Finally, without removing Fmoc, the peptide resin was washed with MeOH three times and dried by vacuum to obtain 121.322 g of the peptide resin of FP(21-27), with a weight yield of 80.57%.
[0056] The obtained peptide resin of 120.0g FP (21-27) was fully protected and cleaved with 1.2L 20% TFE / DCM solution at room temperature, and the reaction was stirred for 2h. After the reaction was completed, the filter cake was rinsed with DCM 3 times, 240mL each time, and the filtrate was combined. The filtrate was subjected to reduced pressure rotary evaporation on a rotary evaporator to remove DCM, leaving about 250mL of solution. 250mL DMF was added to precipitate a white solid, and 750mL H2O was added. After stirring and beating at room temperature for 1h, it was filtered, and the filter cake was rinsed with H2O 3 times, 250mL each time, and vacuum-dried to obtain FP (21-27) 72.475g (44.06mmol) with a purity of 96.67%. Figure 4 As shown, the weight yield is 84.08%.
[0057] Example 5 Synthesis of FP (9-20)
[0058] Weigh 50.0 g (scale 55.0 mmol) of 2-CTCResin with a substitution degree of 1.1 mmol / g into a solid phase reaction column, weigh 110.0 mmol Fmoc-Ile-OH, dissolve it with 200 mL DMF and add it to the solid phase reaction column, then add 220.0 mmol DIEA, and react with nitrogen gas at room temperature for 2 hours. After the reaction is completed, add 50 mL MeOH for resin blocking reaction, and after 1 hour of reaction, remove the reaction liquid, wash the resin 3 times with 250 mL DMF, and then wash it 3 times with MeOH, and vacuum dry. The substitution degree of Fmoc-Ile-CTC Resin is 0.814 mmol / g, and the weight of the amino acid resin is 62.482 g.
[0059] Weigh 30.0g Fmoc-Ile-CTCResin (Sub=0.814mmol / g, scale 24.4mmol) into a solid phase reaction column, swell the resin with 150mL DMF for 30 minutes, wash twice with 150mL DMF, deprotect with 150mL 20% piperidine / DMF solution for 5+7min, wash 6 times with 150mL DMF, and detect the color of the resin with ninhydrin. Weigh 48.8mmol Fmoc-Glu(OtBu)-OH, 58.6mmol HOBt, dissolve with 120mL DMF and add to the reaction column, then add 58.6mmol DIC to start the reaction, react with nitrogen at room temperature for 2h, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction, if the resin is colored, extend the reaction for 30min). After the reaction was completed, the resin was washed three times with 150 mL of DMF, deprotected for 5+7 min by adding 150 mL of 20% piperidine / DMF solution, and washed six times with 150 mL of DMF. The resin was detected by ninhydrin to see if it had color.
[0060] Repeat the above coupling operation, and continue to couple Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Ala-Ser(Psi(Me,Me)pro)OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH in sequence according to the peptide sequence of FP(9-20). Each protected amino acid was fed with 48.8 mmol, DIC was used in an amount of 58.6 mmol, and HOBt was used in an amount of 58.6 mmol. Finally, without removing Fmoc, the peptide resin was washed with MeOH three times and dried by vacuum to obtain 76.642 g of the peptide resin of FP(9-20), with a weight yield of 90.17%.
[0061] The obtained peptide resin of 75.0g FP (9-20) was fully protected and cleaved with 750mL 20% TFE / DCM solution at room temperature, and the reaction was stirred for 2h. After the reaction was completed, the filter cake was rinsed with DCM 3 times, 150mL each time, and the filtrate was combined. The filtrate was subjected to reduced pressure rotary evaporation on a rotary evaporator to remove DCM, and about 150mL of the remaining solution was added. 450mL H2O was added to precipitate a white solid, and the mixture was stirred and slurried at room temperature for 1h and then filtered. The filter cake was rinsed with H2O 3 times, 150mL each time, and vacuum-dried to obtain 52.388g (21.18mmol) of FP (9-20) with a purity of 94.01%. Figure 5 As shown, the weight yield is 86.82%.
[0062] Example 6 Synthesis of FP (9-20a)
[0063] 30.0 g of Fmoc-Ile-CTCResin (Sub = 0.814 mmol / g, scale 24.4 mmol) in Example 5 was weighed into a solid phase reaction column. The difference from Example 5 was that Fmoc-Ser(tBu)-OH and Fmoc-Ala-OH were used to replace Fmoc-Ala-Ser(Psi(Me,Me)pro)OH at the 12th and 11th positions of Ser and Ala, respectively, for coupling. Finally, 54.351 g (21.84 mmol) of FP(9-20a) was obtained with a purity of 79.07%. Figure 6 As shown, the weight yield is 89.49%.
[0064] Example 7 Synthesis of FP (1-8)
[0065] Weigh 60.0g (scale 66.0mmol) of 2-CTCResin with a substitution degree of 1.1mmol / g into a solid phase reaction column, weigh 132.0mmol Fmoc-Gly-OH, dissolve it with 240mL DMF and add it to the solid phase reaction column, then add 264.0mmol DIEA, and react with nitrogen gas at room temperature for 2h. After the reaction, add 60mL MeOH for resin blocking reaction, and remove the reaction liquid after 1h of reaction. Wash the resin 3 times with 300mL DMF, then wash it 3 times with MeOH, and vacuum dry it. The substitution degree of Fmoc-Gly-CTC Resin is 0.876mmol / g, and the weight of the amino acid resin is 72.144g.
[0066] Weigh 72.0g Fmoc-Gly-CTCResin (Sub=0.876mmol / g, scale 63.0mmol) into a solid phase reaction column, swell the resin with 315mL DMF for 30 minutes, wash twice with 315mL DMF, deprotect with 315mL 20% piperidine / DMF solution for 5+7min, wash 6 times with 315mL DMF, and detect the color of the resin with ninhydrin. Weigh 126.0mmol Fmoc-Ser(tBu)-OH, 151.2mmol HOBt, dissolve with 250mL DMF and add to the reaction column, then add 151.2mmol DIC to start the reaction, react with nitrogen at room temperature for 2h, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction, if the resin is colored, extend the reaction for 30min). After the reaction was completed, the resin was washed three times with 315 mL of DMF, deprotected for 5+7 min with 315 mL of 20% piperidine / DMF solution, and washed six times with 315 mL of DMF. The resin was detected by ninhydrin to see if it had color.
[0067] Repeat the above coupling operation, and continue to couple Fmoc-Ile-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Aap(OtBu)-OH, Boc-Val-OH in sequence according to the peptide sequence of FP(1-8). Each protected amino acid was charged with 126.0 mmol, DIC was used in an amount of 151.2 mmol, and HOBt was used in an amount of 151.2 mmol. Finally, the peptide resin was washed with MeOH three times and dried by vacuum to obtain 104.868 g of the peptide resin of FP(1-8), with a weight yield of 69.91%.
[0068] The obtained peptide resin of 100.0 g FP (1-8) was fully protected and cleaved with 1.0 L 20% TFE / DCM solution at room temperature, and the reaction was stirred for 2 h. After the reaction was completed, the filter cake was rinsed with DCM 3 times, 200 mL each time, and the filtrate was combined. The filtrate was subjected to reduced pressure rotary evaporation on a rotary evaporator to remove DCM, leaving about 200 mL of solution. 600 mL of H2O was added to precipitate a white solid, which was stirred and slurried at room temperature for 1 h and then filtered. The filter cake was rinsed with H2O 3 times, 200 mL each time, and vacuum-dried to obtain 44.605 g (42.76 mmol) of FP (1-8) with a purity of 98.53%. Figure 7 As shown, the weight yield is 71.17%.
[0069] Example 8 A solid phase synthesis method for the 46-peptide portion of HY3000
[0070] 8.1 Weigh 1.625 g (5.0 mmol) of FP(38-46)-Resin2 in Example 1 into a solid phase reaction column, swell the resin with 110 mL DMF for 30 minutes, and then wash it with 110 mL DMF twice. Weigh 10.0 mmol of FP(28-37), 10.0 mmol of PyBOP, and 12.0 mmol of HOBt in Example 3, dissolve them with 85 mL DMF, and add them to the reaction column. Then add 15.0 mmol of DIEA to start the reaction. React under nitrogen gas at room temperature for 2 hours, and use ninhydrin to detect the end point of the reaction (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 30 minutes). After the reaction, wash the resin with 110 mL DMF 3 times, add 110 mL 20% piperidine / DMF solution for deprotection for 5+7 minutes, and wash it with 110 mL DMF 6 times. Use ninhydrin to detect whether the resin is colored.
[0071] 8.2 Weigh 10.0mmol of FP (21-27), 9.5mmol of HBTU, and 12.0mmol of HOBt in Example 4, dissolve them in 85mL DMF, and add them to the reaction column. Then add 15.0mmol of DIEA to start the reaction. React under nitrogen gas at room temperature for 1h. Use ninhydrin to detect the end point of the reaction (if the resin is colorless and transparent, terminate the reaction. If the resin is colored, extend the reaction for 30min). After the reaction, wash the resin 3 times with 110mL DMF, add 110mL of 20% piperidine / DMF solution for deprotection for 5+7min, wash 6 times with 110mL DMF, and use ninhydrin to detect the color of the resin.
[0072] 8.3 Weigh 10.0mmol of FP (9-20a), 9.5mmol of HBTU, and 12.0mmol of HOBt in Example 6, dissolve them in 85mL DMF and add them to the reaction column, then add 15.0mmol of DIEA to start the reaction, and react under nitrogen at room temperature for 1h. Use ninhydrin to detect the end point of the reaction (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 30min). After the reaction, wash the resin 3 times with 110mL DMF, add 110mL of 20% piperidine / DMF solution for deprotection for 5+7min, wash 6 times with 110mL DMF, and use ninhydrin to detect the color of the resin.
[0073] 8.4 Weigh 10.0mmol of FP(1-8) and 12.0mmol of Oxyma in Example 7, dissolve them in 85mL DMF and add them to the reaction column, then add 12.0mmol of DIC to start the reaction, and react under nitrogen for 1h at room temperature. Use ninhydrin to detect the end point of the reaction (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 30min). After the reaction, wash the resin 3 times with 110mL DMF, then wash it 3 times with MeOH, and vacuum dry to obtain 51.663g of FP(1-46)-Resin peptide resin, i.e., HY3000 46 peptide partial peptide resin.
[0074] 8.5 According to the preparation method of 8 mL lysis buffer for 1 g of peptide resin, the volume percentage of TFA, Tis, EDT and H2O in the lysis buffer is 91:3:3:3. The lysis buffer is precooled to 0°C, and the 46-peptide peptide resin is added. The reaction is carried out at room temperature for 2 h. The resin is filtered, and the filtrate is added to MTBE below 0°C (90 mL pre-frozen is required for 1 g of peptide resin). A white solid is precipitated. After standing, it is centrifuged and dried to obtain 26.468 g of 46-peptide crude peptide of HY3000 with a purity of 42.68% and a weight yield of 109.37%. The purity detection chart of the crude peptide is as follows: Figure 8 shown.
[0075] 8.6 The obtained 46-peptide crude peptide of HY3000 was purified by preparative chromatography to obtain 46-peptide refined peptide. 1.0 g of 46-peptide refined peptide of HY3000 was dissolved in DMSO at 20 mg / mL, and 168 mg of BAPC (homemade) was added, followed by 0.5 mL of DIEA. The reaction was stirred at room temperature for 1 to 2 h to obtain HY3000 solution, which was then freeze-dried by preparative chromatography to obtain HY3000 API.
[0076] Example 9 A solid phase synthesis method for the 46-peptide portion of HY3000
[0077] The difference from Example 8 is that the starting peptide fragment used in Part 8.1 is 5mmol FP(38-46a)-Resin prepared in Example 2, and the subsequent steps of peptide fragment condensation, peptide resin cleavage, and refined peptide modification are all the same. 25.865g of HY3000 46-peptide crude peptide was obtained with a purity of 54.60% and a weight yield of 106.88%. The crude peptide purity test chart is as follows: Fig. 9 shown.
[0078] Example 10 A solid phase synthesis method for the 46-peptide portion of HY3000
[0079] The difference from Example 9 is that the fragment peptide used in operation 8.3 is 5mmolFP (9-20) prepared in Example 5, and the coupling reagents for each step of fragment condensation are changed to 12mmolHOBt and 12mmolDIC (the operation steps are similar to those described in 8.4), and the remaining peptide resin cleavage and refined peptide modification steps are the same. 25.133g of 46-peptide crude peptide of HY3000 was obtained, with a purity of 73.10% and a weight yield of 103.86%. The crude peptide purity detection chart is as follows: Fig.10 shown.
[0080] Example 11 A solid phase synthesis method for the 46-peptide portion of HY3000
[0081] The difference from Example 10 is that the starting peptide fragment used in operation 8.1 is 5 mmol FP (38-46) -Resin prepared in Example 1, and the subsequent peptide fragment condensation, peptide resin cleavage, and refined peptide modification steps are all the same. 25.316 g of HY3000 46-peptide crude peptide was obtained with a purity of 74.31% and a weight yield of 104.61%. The crude peptide purity test chart is as follows Fig.11 shown.
[0082] In summary, the present invention firstly adopts the solid phase synthesis of 5 fully protected fragments to obtain the 46-peptide peptide chain of HY3000. There are still some difficulties in the initial synthesis. Later, combined with the experience of solid phase step-by-step coupling, the Fmoc-Gly 41 -OH replaced by Fmoc-(Hmb)Gly 41 -OH, a crude peptide of 46 peptides with high purity was not obtained. Considering the difficulty in the preparation of FP (9-20) and the sequence in the solid phase stepwise coupling process, the pseudo-proline dipeptide Fmoc-Ala 11 -Ser(Psi(Me,Me)pro) 12 -OH was used to prepare fragment FP (9-20), and then a 46-peptide crude peptide of HY3000 with high purity was synthesized.
[0083] The present invention relates to the field of peptide synthesis, and more particularly to a method for preparing a peptide coronavirus membrane fusion inhibitor HY3000, and more particularly to fragmentation of a 46-peptide sequence portion of HY3000 and coupling thereof on a solid phase carrier, namely,
[0084] 1) The 46-peptide sequence of HY3000 was divided into five fully protected polypeptide fragments, namely FP (38-46) fragment, FP (28-37) fragment, FP (21-27) fragment, FP (9-20) fragment, and FP (1-8) fragment coupled on amino resin. The peptide sequences are shown in Table 1 above.
[0085] 2) The Ala10 and Ser11 sites of the FP (9-20) fragment were replaced by the dipeptide fragment Fmoc-Ala-Ser (Psi (Me, Me) pro) -OH during the solid phase synthesis process for the preparation of the fragment FP (9-20).
[0086] 3) Five peptide resins or fully protected fragment peptides of the fully protected polypeptide fragments were synthesized sequentially through the Fmoc protection strategy.
[0087] 4) The blank amide resin used in 3) is RinkAmide-AMResin or RinkAmideResin or RinkAmide-MBHAResin.
[0088] 5) The preferred RinkAmide-AMResin in 4) has a substitution degree of 0.2 to 0.5 mmol / g.
[0089] 6) The fully protected fragment peptides were coupled to the peptide resin one by one in sequence to obtain the fully protected HY3000 46-peptide peptide resin.
[0090] 7) The coupling method of 6) is a solid phase synthesis method of Fmoc / tBu strategy, the solvent for removing the Fmoc protecting group is a mixed solvent of 20% Pip / DMF, and the condensation system for amidation between the fragment and the peptide resin is DIC / HOBt or DIC / Oxyma or PyBOP / HOBt / DIEA or HBTU / HOBt / DIEA.
[0091] 8) The specific ratios between the condensation system of 7) and the peptide resin and the fragment peptide are: peptide resin / fragment peptide / DIC / HOBt=1 / 1.5~5 / 1.8~6 / 1.8~6 or peptide resin / fragment peptide / DIC / Oxyma=1 / 1.5~5 / 1.8~6 / 1.8~6 or peptide resin / fragment peptide / PyBOP / HOBt / DIEA=1 / 1.5~5 / 1.5~5 / 1.8~6 / 2.25~7.5 or peptide resin / fragment peptide / HBTU / HOBt / DIEA=1 / 1.5~5 / 1.4~4.7 / 1.8~6 / 2.25~7.5.
[0092] 9) The fully protected HY3000 46-peptide peptide resin was added to the cleavage solution and cleaved at room temperature for 2 to 4 hours to obtain the HY3000 46-peptide crude peptide.
[0093] 10) The lysis solution of 9) is a mixture of TFA with a volume ratio of more than 80% and one or more of phenol, thioanisole, DTT, EDT, DODT, TES, Tis, and H2O in different proportions.
[0094] 11) The 46-peptide crude peptide of HY3000 was purified by preparative chromatography and freeze-dried to obtain the corresponding refined peptide, which was then connected with the modified substance BAPC, purified and freeze-dried to obtain the HY3000 raw material.
[0095] 12) The connection method of 11) is to dissolve the 46-peptide refined peptide of HY3000 in DMF, DMSO or THF at any concentration, add BAPC (homemade), and then add DIEA, and stir the reaction at room temperature for 1 to 2 hours to obtain HY3000 solution.
[0096] 13) The ratio of 12) is HY3000 46-peptide refined peptide / BAPC / DIEA=1 / 1.05~1.3 / 2~20.
[0097] The abbreviations and their English meanings in this article are shown in Table 2.
[0098] Table 2 Abbreviations and English meanings
[0099]
[0100]
[0101] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.
Claims
1. A method for synthesizing a polypeptide novel coronavirus membrane fusion inhibitor, characterized in that: The polypeptide coronavirus membrane fusion inhibitor is HY3000. The method includes dividing the 46-peptide portion of HY3000 into FP (38-46) fragments that are coupled one by one on a blank amide resin, and FP (28-37) fragments, FP (21-27) fragments, FP (9-20) fragments, and FP (1-8) fragments that are coupled one by one on 2-CTC Resin and cleaved in a fully protected cleavage manner; and then coupling the fully protected fragments one by one by Fmoc solid phase coupling to obtain the HY3000.
2. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 1, characterized in that: The blank amide resin is RinkAmide-AM Resin or RinkAmide Resin or RinkAmide-MBHAResin; The blank amide resin is preferably RinkAmide-AM Resin; wherein the substitution degree of the RinkAmide-AM Resin is 0.2mmol / g to 0.5mmol / g, and preferably the substitution degree is 0.3mmol / g to 0.4mmol / g.
3. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 1 or 2, characterized in that: The FP(38-46) fragment includes a FP(38-46)-Resin fragment; The FP(38-46)-Resin fragment is synthesized using blank amide resin as a solid phase carrier, and the peptide resin peptide sequence obtained is H-Gln(Trt) 38 -Glu(OtBu) 39 -Leu 40 -Gly 41 -Ser(tBu) 42 -Gly 43 -Ser(tBu) 44 -Gly 45 -Cys(Trt) 46 -Resin; The FP (28-37) fragment is treated with 2-CTC Resin as a solid phase carrier, and the fully protected peptide and the resin carrier are separated by 20% to 30% TFE / DCM solution. The peptide sequence of the obtained fully protected fragment peptide is Fmoc-Lys (Boc) 28 -Asn(Trt) 29 -Leu 30 -Asn(Trt) 31 -Glu(OtBu) 32 -Ser(tBu) 33 -Leu 34 -Ile 35 -Asp(OtBu) 36 -Leu 37 -OH; The FP (21-27) fragment is treated with 2-CTC Resin as a solid phase carrier, and the fully protected peptide and the resin carrier are separated by 20% to 30% TFE / DCM solution. The peptide sequence of the obtained fully protected fragment peptide is Fmoc-Asp(OtBu) 21 -Arg(Pbf) 22 -Leu 23 -Asn(Trt) 24 -Glu(OtBu) 25 -Val 26 -Ala 27 -OH; The FP (9-20) fragment is treated with 2-CTC Resin as a solid phase carrier, and the fully protected peptide and the resin carrier are separated by 20% to 30% TFE / DCM solution. The peptide sequence of the obtained fully protected fragment peptide is Fmoc-Ile 9 -Asn(Trt) 10 -Ala 11 -Ser(Psi(Me,Me)pro) 12 -Val 13 -Val 14 -Asn(Trt) 15 -Ile 16 -Gln(Trt) 17 -Lys(Boc) 18 -Glu(OtBu) 19 -Ile 20 -OH; The FP (1-8) fragment was treated with 2-CTC Resin as a solid phase carrier, and the fully protected peptide and the resin carrier were separated by 20% to 30% TFE / DCM solution. The peptide sequence of the obtained fully protected fragment peptide was Boc-Val 1 -Asp(OtBu) 2 -Leu 3 -Gly 4 -Asp(OtBu) 5 -Ile 6 -Ser(tBu) 7 -Gly 8 -OH.
4. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 3, characterized in that: The substitution degree of the 2-CTC Resin is 1.0 mmol / g to 1.5 mmol / g, and preferably the substitution degree is 1.1 mmol / g.
5. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 3, characterized in that: The method specifically comprises: performing a coupling reaction on the C-terminus of the FP(28-37) fragment and the N-terminus of the FP(38-46)-Resin fragment, and then removing the Fmoc protecting group to obtain the FP(28-46)-Resin fragment; After coupling reaction between the C-terminus of the FP(21-27) fragment and the N-terminus of the FP(28-46)-Resin fragment, the Fmoc protecting group is removed to obtain the FP(21-46)-Resin fragment; After coupling reaction between the C-terminus of the FP(9-20) fragment and the N-terminus of the FP(21-46)-Resin fragment, the Fmoc protecting group is removed to obtain the FP(9-46)-Resin fragment; The C-terminus of the FP(1-8) fragment is coupled with the N-terminus of the FP(9-46)-Resin fragment to obtain the FP(1-46)-Resin fragment, and the 46-peptide peptide resin of HY3000 is obtained.
6. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 5, characterized in that: The condensation system used for solid phase condensation of each protected amino acid or fully protected fragment by Fmoc method is one or more of DIC / HOBt or DIC / Oxyma or PyBOP / HOBt / DIEA, and the reaction solvent used is one or more of DCM, DMF, DMSO, and NMP; the Fmoc removal reagent is 20% Pip / DMF solution; the condensation system and its ratio are peptide resin / fragment peptide or / DIC / HOBt=1 / 2 / 2.4 / 2.4; the reaction solvent used is preferably DMF.
7. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 5, characterized in that: The method further comprises: cleaving the HY3000 46-peptide peptide resin with a cleavage solution at room temperature for 2h to 4h to obtain the HY3000 46-peptide crude peptide, and then purifying the HY3000 46-peptide refined peptide by preparative chromatography.
8. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 7, characterized in that: The lysate is a mixture of 80% or more of TFA by volume and one or more of phenol, thioanisole, DTT, EDT, DODT, TES, Tis, and H2O in different proportions; The lysis solution is preferably TFA / EDT / Tis / H2O=91%:3%:3%:3%.
9. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 8, characterized in that: The method further comprises: connecting the 46-peptide refined peptide of HY3000 with the cholesterol derivative BAPC, and then purifying by preparative chromatography to obtain HY3000 API.
10. The method for synthesizing a polypeptide coronavirus membrane fusion inhibitor according to claim 9, characterized in that: The step of connecting the 46-peptide refined peptide of HY3000 with the cholesterol derivative BAPC comprises: dissolving the 46-peptide refined peptide of HY3000 with DMF, DMSO or THF, adding BAPC, and then adding DIEA, stirring and reacting at room temperature for 1 hour to 2 hours to obtain a HY3000 solution; wherein the 46-peptide refined peptide of HY3000 / BAPC / DIEA=1 / 1.05~1.3 / 2~20, preferably the 46-peptide refined peptide of HY3000 / BAPC / DIEA=1 / 1.05 / 15.