PotM: Cyclover-Amine for Peptide and Oligonucleotide Synthesis

To produce peptides on a commercial scale, e.g., for therapeutic and cosmetic applications, synthesis protocols need to be scalable and efficient regarding cost and time. Despite its drawbacks, namely high solvent consumption and excess-reagent use, to date, solid-phase synthesis (SPS) has been the major manufacturing process in this context.

Herein, we are presenting Cyclover-Amine (CC-1000), a novel soluble tag for the synthesis of peptides and oligonucleotides combining the advantages of both: liquid and solid-phase synthesis.

Structure of Cyclover amine: Four long hydrophobic saturated C18 alkyl chains are attached to an amino functionalized triazine core and determine the solubility characteristics of Cyclover. The third amino function of the triazine is modified with piperazine, where a secondary amine is available for further derivatization with a suitable linker.

The special properties of Cyclover amine come from its four hydrophobic octadecyl residues which largely define its solubility. For (peptide) synthesis, Cyclover-Amine [N2,N2,N4,N4-tetraoctadecyl-6-(1-piperazinyl)1,3,5-triazine-2,4-diamine] and the Fmoc amino acids are dissolved in a defined organic reaction medium, e.g., dichloromethane, or THF (20 mL per gram Cyclover), allowing for an overall uniform distribution of the reactants. After completion of the reaction, e.g., coupling of one amino acid in solution, the product can easily be precipitated by changing the polarity of the solvent, e.g., by adding the 5-fold volume of methanol. Notably, excess reagents and side-products remain dissolved and are washed away in the filtration step. After collection of the precipitate (= the peptide-carrying Cyclover-Amine) by filtration, the next reaction cycle of dissolution and precipitation can be initiated to grow the peptide chain. Alternatively, unwanted molecules also may be removed by liquid-liquid extraction.

The progress of each reaction step may be monitored in process by drawing samples and analyzing them directly by thin layer chromatography or HPLC, without prior isolation/cleavage of the peptide, as required for resin-based protocols.

Cyclover-Amine is robust against acids and oxidants and especially well-suited for the preparation of short peptides. Unreacted activated building blocks may be scavenged with a soluble amine, e.g., N-propylamine, thus isolating and cleaning the peptide before removing Fmoc is not necessary.

After cleaving the completed peptide from the Cyclover-Amine “carrier tag”, the product may be isolated by ion exchange chromatography.

Cyclic workflow for peptide synthesis with Cyclover. Reactions are performed in solution (hydrophobic solvent, e.g., THF). For purification, Cyclover with the attached growing peptide chain is precipitated by adding a miscible hydrophilic solvent like acetonitrile or methanol or recovered by a liquid-liquid extraction. When unreacted building blocks are trapped with a scavenger like, e.g., N-propylamine, the number of steps in a synthetic cycle may be reduced.

This synthetic cycle may be carried out in a simple vessel equipped with a fritted bottom and a stirrer. In contrast to traditional LPPS, no time- and solvent-consuming chromatographic purification steps between two couplings are required. In addition, contrary to SPS, typically only a slight excess of the building blocks is required for this application and the use of DMF can be avoided.

Schematic representation of a synthetic heptapeptide built on Cyclover-Amine: A Rink amide linker (RL‑1027) has been attached to the secondary amino group of the piperazine moiety to grow the peptide chain.

One gram of Cyclover-Amine (M_r = 1205 g/mol) corresponds to about 0.83 mmol reactive amino groups and has a similar loading capacity per weight as our Rink amide AM Resins (0.4 - 1.0 mmol/g) or HypoGel® resins (0.5 to 0.9 mmol/g) and a higher capacity than our TentaGel® resins (S: 0.2 to 0.53 mmol/g; H: 0.4 to 0.6 mmol/g).

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References:

Improved Tag-Assisted Liquid-Phase Peptide Synthesis: Application to the Synthesis of the Bradykinin Receptor Antagonist Icatibant Acetate; Y. Okada, R. Takasawa, D. Kubo, N. Iwanaga, S. Fujita, K. Suzuki, H. Suzuki, H. Kamiya, K. Chiba; Org. Process Res. and Devel. 2019; 23(11): 2576-2581. https://doi.org/10.1021/acs.oprd.9b00397

Peptide synthesis beyond DMF: THF and ACN as excellent and friendlier alternatives; Y. E. Jad, G. A. Acosta, S. N. Khattab, B. G. de la Torre, T. Govender, H. G Kruger, A. El-Faham, F. Albericio; Org. Biomol. Chem. 2015; 13(8): 2398-2398. https://doi.org/10.1039/c4ob02046d

Total synthesis of α-conotoxin MII using a soluble-tag-assisted method; S. Kitada, S. Fujita, Y. Okada, S. Kim, K. Chiba; Tetrahedron 2013; 69(12): 2555-2559. http://dx.doi.org/10.1016/j.tet.2013.01.068

Liquid-Phase RNA Synthesis by Using Alkyl-Chain-Soluble Support; S. Kim, M. Matsumoto, K. Chiba; Chem. Europe 2013; 19(26): 8615-8620. https://doi.org/10.1002/chem.201300655

Composition of a lipophilic agent for solution phase synthesis of biomolecules; Y. Li; 2023; US2023/0373936A1 and WO2023/244274A2.

Morpholino oligonucleotide manufacturing method; T. Torii, D. Takahashi, S. Katayama; 2019; US10415036.

Morpholino oligonucleotide manufacturing method; D. Takahashi, T. Torii; 2021; US11028386.