Iminothioindoxyl Donors with Exceptionally High Cross Section for Protein Vibrational Energy Transfer

Various protein functions are related to vibrational energy transfer (VET) as an important mechanism. The underlying transfer pathways can be experimentally followed by ultrafast Vis-pump/IR-probe spectroscopy with a donor-sensor pair of non-canonical amino acids (ncAAs) incorporated in a protein. However, so far only one donor ncAA, azulenylalanine (AzAla), exists, which suffers from a comparably low Vis extinction coefficient. Here, we introduce two novel donor ncAAs based on an iminothioindoxyl (ITI) chromophore. The dimethylamino-ITI (DMA-ITI) and julolidine-ITI (J-ITI) moieties overcome the limitation of AzAla with a 50 times higher Vis extinction coefficient. While ITI moieties are known for ultrafast photoswitching, DMA-ITI and J-ITI exclusively form a hot ground state on the sub-ps timescale instead, which is essential for their usage as vibrational energy donor. In VET measurements of donor-sensor dipeptides we investigate the performance of the new donors. We observe 20 times larger signals compared to the established AzAla donor, which opens unprecedented possibilities for the study of VET in proteins. To observe site selective vibrational energy transfer (VET) in proteins in ultrafast pump probe experiments, an energy donor is required to inject vibrational energy. The only known donor amino acid azulenylalanine (AzAla) suffers from a low Vis extinction coefficient. Here, two novel iminothioindoxyl (ITI) based donor amino acids were introduced with a 50 times higher Vis extinction coefficient to reach superior VET signal intensities.+image