Synthesis of 3-trifluoromethyl-3-pyrimidinyl diazirines as photoreactive amino acid analogs

Abstract

Photoaffinity labeling is a biochemical technique used for determining binding behavior of biomolecules. A labeled ligand and a target of interest are covalently crosslinked in response to a specific wavelength of light. These irreversibly bound complexes can then be analyzed by Western blot, mass spectrometry, or any method that can distinguish bound and unbound states. The most common photoactivatable groups used in photoaffinity labeling are aryl azides, benzophenones, and diazirines. Unlike diazirines, benzophenones are large and can disrupt binding interactions, while aryl azides require a short activation wavelength that can degrade proteins. Diazirines possess neither of these disadvantages, however some common diazirine-based photoaffinity labels, the 3-phenyl-3-trifluoromethyl diazirines, are limited in their utility in biologically relevant environments due to their poor stability in ambient light and poor solubility in aqueous media. In response to this limitation, 3-pyridyl- and 3-pyrimidinyl-3-trifluoromethyl diazirine photoaffinity labels that improve on these qualities have been previously characterized. Photoreactive amino acid analogs are used in photoaffinity labeling of targets that bind to amino acids. These compounds allow a photoactivatable group to be attached directly to a peptide of interest without further chemical conjugation. Photoreactive aromatic amino acid analogs are less common than their aliphatic counterparts, and those that are used, such as trifluoromethyldiazirinyl phenylalanine, lack adequate stability in ambient light (as most diazirines do). Therefore, expansion of the biochemical toolbox to include a photoreactive aromatic amino acid analog with improved ambient light stability is appealing. This work describes efforts towards the synthesis of a 3-pyrimidinyl-3-trifluoromethyl diazirine containing amino acid analog for use in photoaffinity labeling. Negishi coupling is used to form a carbon-carbon bond between an aryl halide and a β-iodo amino acid to form a novel amino acid analog. Since trifluoromethyl diazirines can be synthesized from trifluoromethyl ketones, significant efforts were made towards trifluoroacetylation of 5-halopyrimidines. However, these halides are not adequately reactive towards metal-halogen exchange. Finally, different experimentally explored and proposed synthetic routes towards these photoreactive amino acid analogs and potentially useful advanced intermediates are explored.