Bone Glue,Bone Glue Gelatin,Animal Bone Glue,Industrial Grade Bone Glue Hebei Haodong Biological Technology Co.,Ltd. , https://www.hdgelatin.com
Fmoc-Pro-Merrifield Resin (replacement value 0.291mmol/g, cross-linking degree 1%, 100-200 mesh), BocValWang Resin (replacement value 0.523mmol/g, cross-linking degree 1%, 100～) 200 mesh), Fmoc-Pro-CTC Resin (replacement value 0.42mmol/g, cross-linking degree 1%, 100-200 mesh), BocTyr(tBu)OH, BocProOH, BocIleOH , BocLeuOH, FmocTyr(tBu)OH, FmocProOH, FmocIleOH, FmocLeuOH, Bocstatine(3s,4s)OH, HATU[O( 7-azobenzotriazole 1oxy)N,N,N′,N′tetramethylurea hexafluorophosphate], HBTU (benzotriazole1tetramethylhexafluorophosphate Ester), HOBt (N-hydroxybenzotriazole), BOP (phthalyl octyl octyl ester), DIEA (N, N-diisopropylethylamine; diisopropylethylamine), TFA (three Fluoroacetic acid), DMF (dimethylformamide), DMSO (dimethyl sulfoxide), DCM (dichloromethane), ACN (acetonitrile), EDC [1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide hydrochloride], NMM (N-methylmorpholine) and phenyl sulfide are provided by Hangzhou Zhongpept Biochemical Co., Ltd., piperidine is used after re-distillation, and water is removed twice.
1.2 Chemical synthesis of HS1 and its analogues The five cyclic peptide sequences synthesized in this paper are:
HS1:cyclo(Ile1Ile2Pro3Pro4Tyr5Val6Pro7
Leu8);
A1HS1:cyclo(Ile1Ile2statine3Pro4Tyr5Val6
Pro7Leu8);
A2HS1:cyclo(Ile1Ile2Pro3statine4Tyr5Val6
Pro7Leu8);
A3HS1:cyclo(Ile1Ile2Pro3Pro4Tyr5Val6
Statine7Leu8);
A4HS1:cyclo(Ile1Ile2statine3statine4Tyr5
Val6Pro7Leu8).
Although the sequences of the five antigenic peptide cyclic peptides or cyclic peptide analogs are very similar as indicated above, and each amino acid residue is numbered in order, considering the hydrophobicity and steric hindrance of each compound It is not exactly the same, and the synthetic linear peptide chain is finally cyclized into an antigenic peptide cyclic peptide. Therefore, in order to conveniently and efficiently synthesize the target product, the first amino acid we select for the peptide reaction is also different.
(1) The synthetic route of HS1 and the swelling of the purified resin are called Self-programming Method1. The specific method of resin swelling is to soak FmocProCTC Resin 4.76g (synthesis scale 2.0mmol) with 200ml DMF for 30min to make it fully Swell and then drain.
Removal of the amino protecting group The self-programming Method 2 was invoked by adding 20 ml of a 20% piperidine-containing DMF solution, stirring the reaction for 30 min, and then draining. It was then washed 5 times with DMF to remove residual deprotecting reagent. Manual sampling, ninhydrin detection, if the resin is dark blue, it indicates that the Fmoc protecting group has been removed.
The peptide reaction was called Self-programming Method3 by adding 200 ml DMF solution dissolved in 3.33 g of Fmoc-Val-OH to the reactor, mechanically stirring for 2 min, then adding HBTU 3.57 g, and finally adding NMM 2.02 g [AA: HBTU : NMM (mol/mol) = 3: 2.85: 6], and reacted for 30 min. It was drained, washed 3 times with DMF, and then the resin was washed with methanol to remove the amino acid solution and DMF, and the ninhydrin was detected by hand. If the resin was colorless, the reaction was complete. The program Method2 and Method3 are sequentially called, and the peptide reaction is repeated by Fmoc-Tyr(2BrZ)OH, FmocProOH, FmocProOH, etc. according to the amino acid sequence of the peptide chain until the final synthesis. The desired peptide chain. The order of synthesis is: Pro, Val, Tyr, Pro, Pro, Ile, Ile, L order: Pro, Val, Tyr, Pro, Pro, Ile, Ileeu.
In the synthesis of subsequent analogs, the parameters listed in Table 1 were essentially unchanged. The feed ratio of BocstatineOH was 2, the reaction time of peptide was 40 min, and the deprotection time was 30 min.
Cleavage of the peptide chain The synthetic peptide chain was cleaved with a cutting solution [TFA:TIS (triisopropylsilane):H2O=95.0:2.5:2.5] at a temperature below 20 ° C for a reaction time of 2.5 h, then filtered under reduced pressure to collect the filtrate. The polypeptide dissolved in the cutting solution was precipitated with cold diethyl ether, and the ratio of each amino acid in the synthesis of HS-1 in Table 1 was centrifuged for 3 min at r/min, and dried under vacuum for 12 h to obtain a crude linear peptide chain of about 1.5 g.
Cyclization of the antigenic peptide peptide chain 1.4 g of the crude linear polypeptide was dissolved in 200 ml of DMF, followed by EDC (2 eq, 0.6 g), BOP (1 eq, 0.7 g), HOBt (2 eq, 0.42 g) and DIEA (5 eq, 1.1 g). ), cyclization overnight. The addition of EDC accelerates the condensation reaction. The DMF was evaporated to dryness to give 1.35 g of crude cyclic peptide. The crude product was analyzed by MALDI-TOF mass spectrometer.
Purification of crude cyclic peptide The above cyclized crude peptide was dissolved in an appropriate amount of DMSO (since the water solubility of the peptide was not good, the loading concentration was strictly controlled to less than 1 mg/ml), and the sample was filtered with a 5 μm filter paper. The preparative column was a Waters X-Bridge C18, 5 μm reverse phase column; the eluent: solution A was an aqueous solution of 0.1% TFA, and solution B was an aqueous solution of acetonitrile containing 0.1% TFA; the detection wavelength was 220 nm. The B solution was eluted by a linear gradient of 30% to 65% in 60 min, and the flow rate was 38 ml/min. Fractions with a purity greater than 95% were collected. Finally, lyophilization yielded 100 mg of the final product with a purity greater than 90%.
Analytical HPLC detection purity column is SepaxGP-C18 reverse phase column (4.6mm × 150mm, 5μm, 120); eluent: A solution is 0.1% TFA aqueous solution, B solution is 0.1% TFA acetonitrile aqueous solution; The detection wavelength was 220 nm. The B solution was eluted by a linear gradient of 60% to 80% in 20 min, and the flow rate was 1.0 ml/min.
MS Identification The molecular weight of the target product was determined by laser desorption ionization time-of-flight mass spectrometry.
(2) The synthesis route and purification of A1HS1 are slightly different from the synthesis of HS-1. In the synthesis of A1HS1, the synthesis methods mainly have the following changes: 1 resin is changed to Fmoc-Pro-Merrifield Resin; 2 amino acid is Boc protection ; 3 each time an amino acid residue is linked, the peptide chain is cleaved with 50% TFA/DCM, then deprotected, washed, activated, and then the next amino acid residue, and finally the peptide chain is cut from the resin. The solution was HF for 1.2 h; 4 the gradient elution conditions for the purification were B: 70% to 100%; 5 for HPLC analysis: B%: 61% to 81%; 6 synthesis order: Pro, Val, Tyr, Pro, Statine, Ile, Ile, Leu. The rest is synthesized with HS-1. After lyophilization, 250 mg of the final product having a purity greater than 93% was obtained. Since the solubility is better than HS-1, the yield is higher.
(3) The synthesis route and purification of A2HS1 is similar to the synthesis of A1HS1. Compared with the synthesis of HS-1, in the synthesis of A2HS1: 1 resin is Fmoc-Pro-CTC Resin; 2 amino acid is Fmoc protection; 3 the last cutting fluid is F solution, time 2.5h; 4 gradient elution conditions for purification B: 65% ~ 90%; 5 HPLC analysis B solution: 60% ~ 80%; 6 synthesis order: Pro, Ile, Ile, Leu, Pro , Val, Tyr, statine; 7 Because statine is protected with Boc, after linking statine, the peptide chain fragment is first cut from the resin, then deprotected, washed, resin activated, and then connected to the next residue. The rest is synthesized with HS-1. After lyophilization, 268 mg of a final product having a purity greater than 92% was obtained. Since the solubility is better than HS-1, the yield is higher.
(4) The synthesis route and purification of A3HS1 is similar to that of A1HS1. Compared with the synthesis of HS-1, in the synthesis of A3HS1: 1 resin is Fmoc-Val-Wang Resin; 2 amino acid is Fmoc protection; 3 cutting fluid is F Liquid, time 2.5h; 4 gradient elution conditions for purification is B solution: 43% ~ 85%; 5 HPLC analysis for liquid B: 50% ~ 70%; 6 synthesis order: Val, Tyr, Pro, Pro, Ile, Ile, Leu, statine; 7 Because statine is protected with Boc, after linking statine, the peptide chain fragment is first cleaved from the resin, then deprotected, washed, resin activated, and then the next residue. The rest is synthesized with HS-1. After lyophilization, 245 mg of a final product having a purity greater than 98% was obtained. Since the solubility is better than HS-1, the yield is higher.
(5) The synthesis route and purification of A4HS1 is similar to the synthesis of A1HS1. Compared with the synthesis of HS-1, in the synthesis of A4HS1: 1 resin is Fmoc-Pro-Merrifield Resin; 2 amino acids are Boc protection; 3 each amino acid is linked The residue was cleaved with 50% TFA/DCM, then washed, and then the next amino acid residue was added. Finally, the peptide chain was cleaved from the resin, and the cleavage liquid was HF for 1.2 h. Solution B: 62% to 80%; 5 HPLC analysis: liquid B: 62% to 82%; 6 synthesis order: Pro, Val, Tyr, statine, statine, Ile, Ile, Leu; 7 because statine is protected with Boc, Therefore, after the statine is attached, the peptide chain fragment is first cleaved from the resin, then deprotected, washed, activated by the resin, and then attached to the next residue. The rest is synthesized with HS-1. After lyophilization, 215 mg of a final product having a purity greater than 90% was obtained. Because the solubility is better than HS-1, the yield is higher.
1.1 Instruments and reagents Polypeptide synthesizer (CS536, CSBio, USA), semi-preparative high performance liquid chromatography (Waters Delta Prep4000, Waters, USA), analytical high performance liquid chromatography (Agilent 1100, Agilent, USA), frozen Dryer (Christ Alpha, Christ, Germany), laser desorption ionization time-of-flight mass spectrometer (LCQ Deca, Thermo-Finnigan, USA), UV spectrophotometer (Beckman DU7400, Beckman, USA), C18 reverse phase analytical column (Sepax GP) C18, 5μm, 120, 4.6mm×150mm).