Synthesis, photophysics, and application of fluorescent protein chromophore analogs

The Green Fluorescent Protein has gained significant attention and the 2008 Nobel Prize as a non-invasive fluorescent marker that has revolutionized molecular biology. Within the 11-stranded β-barrel of the protein is protectively housed an arylideneimidazolidinone (AMI) chromophore formed via a post-translational cyclization that is responsible for the fluorescent properties of the protein. Following removal from the protective β-barrel, the chromophore exhibits remarkably different spectroscopic properties with a 104 diminished emission quantum yield. This observation has been attributed to torsional motions that allow for rapid internal conversion. These motions are a function of the structure of the AMI chromophore where cis/trans isomerization can occur about the benzylidene double bond as well as the formal single aryl bond. From this, the question arises: if these torsional motions are inhibited, can the fluorescence of the chromophore be restored?;In order to study these chromophores, AMI's were synthesized with a variety of functional groups. Synthesis of these chromophores is afforded through a 2+3 cycloaddition providing a combinatorial synthetic protocol contrary to the commonly cited synthetic procedure of the Erlenmeyer azalactone synthesis. The combinatorial cycloaddition provides tolerance for a myriad of functional groups and the relative synthetic ease has readily allowed for the synthesis of hundreds of AMIs to date. With an arsenal of AMI chromophores a number of studies can be accomplished researching the photophysical questions surrounding these chromophores.;It is rationalized that inhibition of conformational freedom should provide for the restoration of the emission quantum yield. This study was undertaken through the transformation of the AMI chromophore to a bidentate ligand where upon metal binding torsional motions are inhibited. This was further explored through the encapsulation of hydrophobic chromophores within octa acid cavitands that provide a restrictive environment similar to what is found within the β-barrel. Results from these studies show the topological effects experienced by these chromophores and how restrictive environments provide for an enhancement in the emission quantum yield.;Using the encapsulation studies as motivation additional studies were motivated to use these compounds as fluorescent probes. Collaborating with Dr. Young-Tae Chang, these chromophores can be screened against a myriad of analytes to determine interactions through emission response in an efficient fashion. This led to the use of these chromophores, and coupled with rational design, as a human serum albumin probe. Further collaborative studies show that these chromophores can be used as agonists for nuclear receptors where binding the ligand binding domain provides for a restrictive environment as well as a fluorescent probe.;In addition to these interesting applications, the photochemistry of specific chromophores shows an interesting photochemical reaction of dimerization resulting in mechanical motion. Synthetic modifications also allow for these chromophores to act as liquid crystalline materials. As shown from this summary the chromophore of GFP characterized as an AMI chromophore show interesting properties and applications as well as great potential in a number of fields.