Cyclophase synthesis: A productive technology
for liquid phase peptide synthesis







One Cycle of Synthetic Process:

(1) Dissolve the reactants in solution by using one volume of solvent(s).

(2) React to completion by checking TLC or other analytical methods.

(3) Precipitate for separation by mixing 4 to 5 fold volumes of poor solvent.

(4) Filter to collect the solid for next cycle of reaction.



Practical guidance for liquid phase peptide synthesis using Cyclover-amine





BASIC INSTRUCTIONS



Reactions are monitored by thin-layer chromatography (TLC) carried out on silica gel plates, with detection by UV absorption (254 nm). The solvents used for TLC development begin with ethyl acetate:hexane (1:3). Polarity changes incrementally with ethyl acetate along the progress of the peptide synthesis. The increase of the ratios (ethyl acetate:hexane) is recommended as follows: 1:2, 2:3, 3:4, 1:1, and so on depending on the polarity of the amino acids during elongation.





GENERAL HPLC ANALYSIS



For Cyclover-tagged-peptide: C8 column was used with a linear gradient of tetrahydrofuran (THF) from 70% to100% with water containing 0.1% trifluoroacetic acid (TFA) from 30% to 0% in the range of 10 min, followed by 100% THF for 5 min at 1.0 mL/min.

For Tag-free-Peptide: C18 column was used with a linear gradient of 10-60% acetonitrile in water containing 0.1% trifluoroacetic acid at 0.20 mL/min.





GENERAL PROCEDURE FOR INSTALLATION OF FUNCTIONAL LINKERS ON CYCLOVER-AMINE



To a solution of Cyclover-Amine (MW: 1205.14) in dichloromethane (DCM) (40 mM), Linker-COOH (1.5 mol equiv), N,N’-diisopropylcarbodiimide (DIC) (1.1 mol equiv.) were added. The reaction mixture was stirred at room temperature until the reaction was completed (30 min). After completion, methanol (5 fold volume of the solution) was used to dilute the reaction mixture. The resulting precipitates were recovered by vacuum filtration and washed with methanol (5 fold volume of the solution x 3) to give the amide of Cyclover-tagged functional linker.





GENERAL PROCEDURE FOR PREPARATION OF THE ESTER OF CYCLOVER-TAGGED-HYDROXYL LINKER WITH FIRST AMINO ACID



To a solution of Cyclover-tagged-hydroxyl linker (40 mM) in dichloromethane (DCM), Fmoc-AA-OH (1.5-2.0 mol equiv.), N,N’-diisopropylcarbodiimide (DIC) (1.5 - 4.0 mol equiv.), and N,N-dimethylaminopyridine (DMAP) (0.05 mol. equiv.) were added. The resulting reaction mixture was stirred at room temperature for 1 hour, followed by dilution with methanol (5 fold volume of the solution). The resulting precipitates were recovered by vacuum filtration and washed with methanol (5 fold volume of the solution x 3) to give the ester of Cyclover-tagged first amino acid.





GENERAL PROCEDURE FOR DEPROTECTION FROM FMOC GROUP OF CYCLOVER-TAGGED-PEPTIDE



To a solution of Cyclover-tagged-peptide in THF (40 mM), 1% DBU and 1% piperidine (in volumes) were added. The resulting reaction mixture was stirred at room temperature for 10 min, followed by quenching with 1N aqueous HCl at 5-15 °C and dilution with methanol (5 fold volume of the solution). The resulting precipitates were recovered by vacuum filtration and washed with methanol (5 fold volume of the solution x 3) to give Fmoc-deprotected Cyclover-tagged-peptide.





GENERAL PROCEDURE FOR ELONGATING OF AMINO ACID TO CYCLOVER-TAGGED-PEPTIDE



To a solution of Fmoc-deprotected Cyclover-tagged-peptide in THF (40 mM), Fmoc-AA-OH (1.5 mol equiv), HBTU (1.2 mol equiv.), HOBt (1.2 mol equiv.), and N,N-diisopropylethylamine (DIPEA) (2.0 mol equiv.) were added. The resulting reaction mixture was stirred at room temperature for 1-3 hours, followed by dilution with acetonitrile (5 fold volume of the solution). The resulting precipitates were recovered by vacuum filtration and washed with acetonitrile (5 fold volume of the solution x 3) to give Cyclover-tagged-elongated-peptide.





GENERAL PROCEDURE FOR ONE-POT SEQUENTIAL COUPLINGS AND DEPROTECTIONS (ELONGATION AND SUBSEQUENT FMOC-DEPROTECTION IN ONE-POT)



To a solution of Fmoc-deprotected Cyclover-tagged peptide in THF (40 mM) stirred at room temperature were added Fmoc-AA-OH (1.5 eq.), DMT-MM (1.45 eq.), and N,N-diisopropylethylamine (DIPEA) (5.0 eq.). After stirring at room temperature for 1-3 hours (checked by TLC and HPLC), propylamine (1.8 mol equiv.) was added. The resulting reaction mixture was stirred at room temperature for an additional 30 min, and then piperidine (1.5 mol equiv.) and DBU (7.0 mol equiv.) were added. After stirring at room temperature for 10 min (also checked by TLC and HPLC), the resulting reaction mixture was neutralized by 1N aqueous. HCl at 5-15 °C, followed by dilution with acetonitrile. The resulting precipitate was recovered by vacuum filtration and washed with acetonitrile (2 times) to give the Cyclover-tagged elongated peptide with Fmoc-deprotected ready for further elongation.





GENERAL PROCEDURE FOR GLOBAL DEPROTECTION OF FINAL CYCLOVER-TAGGED-PEPTIDE CONTAINING WANG-LINKER



To a solution of the Cyclover-tagged peptide in trifluoroacetic acid (TFA), triisopropylsilane (TIS, 2.5% in volume) and water (2.5% in volume) were added. The reaction mixture was stirred at room temperature until the reaction was completed. After completion, the solution was diluted with ethanol and the precipitate was removed by filtration, and diisopropyl ether was added to the filtrate to give the peptide as a precipitate which was obtained as a solid after filtration.


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