T. Shi, S. Brewer, E. E. Fenlon
Feb 16, 2016
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Journal
Biophysical Journal
Abstract
Unnatural amino acids (UAA) can be effective probes for studying protein structure, hydration, and dynamics. Azide UAA are especially good because the azide asymmetric stretch vibration provides a strong signal, which is sensitive to its local environment (hydration and electrostatic), and appears in a clear region of the infrared spectrum. Azide groups are small three-atom probes that should, depending on the site, cause minimal perturbations of the local protein environment. Among azide UAA, 4-(azidomethyl)-L-phenylalanine (1) is especially valuable because it is successfully incorporated into proteins in a site-specific manner with high yields and fidelity. UAA 1 is more stable than 4-azido-L-phenylalanine which photodegrades. Previous syntheses of 1 required 6-8 steps, were time consuming, labor intensive, and had a low overall yields of 10-30%.Work towards a more efficient and readily scalable synthesis of 1 will be presented. The key step converts 4-(aminomethyl)-L-phenylalanine into 1 by a diazotransfer reaction using imidazolesulfonylazide. The free base form of 4-(aminomethyl)-L-phenylalanine is commercially available and its hydrochloric salt form is readily synthesized in one step from L-phenylalanine without chromatography. The diazotransfer was conducted with both without protection of the alpha-amine or with protection through complexes with boron, copper, or zinc. Various deprotection strategies were tested. The new synthesis successfully provided 1 in 2-4 steps from phenylalanine and does not require flash chromatography. Future work will involve increasing the yields and purity of 1.