(44h) Soluble Tag-Assisted Liquid-Phase Peptide Synthesis Using Hydrophobic Benzyl Alcohols

[Introduction]
Peptides have attracted much attention as promising medicinal candidates. As compared with traditional small molecules and antibody medicines, peptides have medium-sized molecular structures. Therefore, they are expected to have both advantages of small molecules and antibody medicines, for example, chemical synthetic accessibility and highly selective pharmacological activity. Current chemical synthesis of peptides is mainly carried out through solid-phase methods, which use insoluble resins as supports to realize easy separation processes and also enable fully automated synthesis. However, soluble tags assisted liquid-phase approach has also been studied intensively since large scale preparations are still challenging in solid-phase method. In this context, polyethylene glycols(PEGs) are the first option as soluble tag and they are central to many applications in the field. Since PEGs are soluble in polar solvents and insoluble in less-polar solvents, a reaction of polymer-attached reactant or catalyst can be performed under homogeneous conditions in a polar solvent. After the reaction completion, the polymer is precipitated by the addition of a less-polar solvent. However, as for peptide synthesis, because both amino acids and coupling reagents are polar, the opposite solubility is preferable for soluble tag to rinse away excess chemicals by washing with polar solvents.
We have been working on the use of hydrophobic benzyl alcohols as soluble tags in liquid-phase method. In this method, reactions take place in homogeneous conditions in less-polar solvents and after the reaction completion, polar solvents are added to precipitate the tag-attached product. Since excess chemicals can be rinsed away efficiently by washing with polar solvents, this approach allows a production of peptide with high quality in relatively larger scale. For this strategy to be successful, high control of the tag-attached product solubility and the physical property must be addressed. In this study, the liquid-phase chemical synthesis of several therapeutic peptides and the investigation on poor-solvents-addition-induced precipitation mechanism will be presented.

[Experimental]
The hydrophobic benzyl alcohols synthesized from the corresponding hydroxyl benzaldehyde in 2 steps, including alkylation by respective bromide and reduction by sodium borohydride. At each step, the desired products were washed with polar solvents repeatedly to give pure forms, which were characterized by NMR. The chemical synthesis of peptides was carried out in less-polar solvents using N-protected amino acids and COMU as a coupling reagent. After the reaction completion, polar solvents were used as poor solvents to induce precipitations of the tag-attached products. The physical property of the recovered precipitates were characterized by scanning electron microscopy, particle size distribution measurement, and X-ray diffraction.

[Results and discussion]
High control of the solubility property was achieved for the hydrophobic benzyl alcohols. The tag-attached products were able to be precipitated quantitatively at most steps. Although dichloromethane was a good solvents for all chemicals used, including the tags, N-protected amino acids, and a coupling reagent, we found that tetrahydrofuran was also effectives as a reaction solvent. This is preferable for the large scale production of peptides from the viewpoint of environmental aspect. As for the poor solvent, methanol and acetonitrile were found to be effective to induce precipitation of the tag-attached products. By using this methodology, both couplings and deprotections were carried out in homogeneous reaction conditions to give several therapeutic peptides in gram-scales. In addition, the physical property of the precipitation was found to be dramatically different when cyclohexane was used as a reaction solvent instead of tetrahydrofuran. Especially, the biphasic system composed of cyclohexane and acetonitrile provided an ideal reaction condition, which solubilize the all chemicals used for the synthesis. The precipitations obtained from this reaction condition were fine powders that were able to be recovered readily through paper filtration.

[Reference]
(a) Okada, Y. et. al, Org. Lett. 2015, 17, 4264-4267. (b) Okada, Y. et. al. J. Org. Chem. 2013, 78, 320-327. (c) Tana, G. et. al. Chem. Commun. 2010, 46, 8219-8221