Due to the central roles that proteins play in biochemical processes, understanding protein structure and function is crucial to many aspects of biochemical research. The use of unnatural amino acids, such as the fluorotyrosines, enables atomic-level modifications to proteins. While the genetic incorporation of unnatural amino acids into proteins is a valuable approach, solid-phase peptide synthesis (SPPS) remains effective, provided that appropriate side chain protecting groups are available. The SPPS incorporation of the fluorotyrosines into peptides depends on the stability of protecting groups commonly used to protect the hydroxyl and phenol functional groups. The tert-butyldimethylsilyl (TBDMS) protecting group has been used to incorporate mono- and difluorotyrosines into peptides, but limitations remain with tri- and tetrafluorotyrosines. We hypothesized that the triisopropylsilyl (TIPS) protecting group, with greater stability due to an increase in steric bulk relative to TBDMS, would be generally applicable for the incorporation of fluorotyrosines during SPPS. To model fluorotyrosine protection, phenol and 2,3,5,6-tetrafluorophenol were evaluated, as they are analogous to the tyrosine and 2,3,5,6-tetrafluorotyrosine side chains and are more readily obtained. A series of silyl ethers were synthesized, purified, and characterized. Nuclear magnetic resonance spectra confirmed the presence of the silyl ethers, TBDMS- and TIPS-phenol, and TBDMS- and TIPS-2,3,5,6-tetrafluorophenol. To assess stability under common SPPS conditions, the silyl ethers were treated with TFA, 4-methylpiperidine, or HBTU and DIEA, and stability was quantified for two hours using high-performance liquid chromatography. Under all SPPS conditions, TIPS-phenol was more stable than TBDMS-phenol. The silyl ethers of 2,3,5,6-tetrafluorophenol were both intact after two hours in TFA, while both were 100% deprotected after two hours in either 4-methylpiperidine or HBTU and DIEA. The results lend support to the claim that TIPS is more stable than TBDMS; however, TIPS will likely be ineffective for the protection of 2,3,5,6-tetrafluorotyrosine during SPPS. These data warrant further assessments to evaluate the range of effectiveness of TIPS as a protecting group for other members of the fluorophenol series.

Evaluation of the triisopropylsilyl group for the protection of fluorophenols as a model for the protection of fluorotyrosines

ACS Fall 2021

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