title: Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins.
abstract: An unnatural amino acid, 4-(2-azidoethoxy)-L-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the azide asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites by using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative.
authors.affiliation: Department of Chemistry Franklin & Marshall College, P.O. Box 3003, Lancaster, PA 17604 (USA).
authors.forename: Elise M
authors.initials: EM
authors.lastname: Tookmanian
authors.affiliation: Department of Chemistry Franklin & Marshall College, P.O. Box 3003, Lancaster, PA 17604 (USA). cpiro@fandm.edu.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Phillips-Piro
authors.affiliation: Department of Chemistry Franklin & Marshall College, P.O. Box 3003, Lancaster, PA 17604 (USA). efenlon@fandm.edu.
authors.forename: Edward E
authors.initials: EE
authors.lastname: Fenlon
authors.affiliation: Department of Chemistry Franklin & Marshall College, P.O. Box 3003, Lancaster, PA 17604 (USA). sbrewer@fandm.edu.
authors.forename: Scott H
authors.initials: SH
authors.lastname: Brewer
mesh_terms: D001386:Azides / Q000737:chemistry*; D057930:Click Chemistry; D061565:Cycloaddition Reaction; D034242:Cyclooctanes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D019265:Spectroscopy, Near-Infrared; D014732:Vibration
chemical_list: C000608653:4-(2-azidoethoxy)-L-phenylalanine; D001386:Azides; D034242:Cyclooctanes; D049452:Green Fluorescent Proteins; D010649:Phenylalanine
keywords: IR spectroscopy; azides; click chemistry; structural biology; vibrational reporters