59th Annual Report on Research 2014

The supported work focuses on using single-molecule fluorescence microscopy to elucidate fundamental mechanistic processes in individual, working catalyst molecules.  Last period’s successes included synthesizing a family of surface-supported catalyst molecules and building a fluorescence microscope capable of imaging single molecules.  We have built upon these successes by synthesizing a family of fluorescently labeled surface-supported catalyst molecules and imaging them under turnover conditions. 

1.        Synthesis of fluorescently labeled palladium complexes

We have focused on palladium catalysts due to their synthetic importance and wide applicability, and N-heterocyclic carbene (NHC) complexes in particular due to their high activity and chemically stable metal-heteroatom bond which can be used as a robust linker to a surface anchor.   Difficulties were previously encountered with the use of silver oxide transmetalation chemistry to make mono-ligated palladium-NHC compounds.  We solved this problem through the use of PEPPSI (Pyridine-Enhanced Precatalyst Preparation Stabilization and Initiation) catalysts which are extremely air and water sensitive, highly functional catalysts, and convenient to synthesize.  Instead of the usual pyridine ligand, we synthesized a family of fluorescently labeled ligands.  For a fluorophore, we chose a green (532 nm excitation) absorbing BODIPY fluorophore due to BODIPY’s high chemical tolerances and lack of coordinating heteroatoms.  BODIPY fluorophores were attached to pyridine ligands via Sonogashira coupling followed by hydrogenation to make a labelled ligand.  Typical PEPPSI forming reactions entail reflux in neat ligand, but we found alternate conditions to minimize waste of the synthetically precious labelled ligand.   We applied our PEPPSI complex-forming reaction conditions to an NHC precursor that contained a silane surface anchor functionality.   The result is the compound shown in FIGURE 1.    Realizing that spectroscopic observations are made substantially more facile with brighter and more photostable fluorescent labels, we synthesized a dendrimeric version of the catalyst, shown in FIGURE 2, where four BODIPY fluorophores are attached to a single palladium catalyst.  Such dendrimers have been previously shown to enable weak-coupling between fluorophores, such that photobleaching of one will not quench emission from the others.  We synthesized the dendrimeric ligand though a 4+2 cycloaddition followed by deprotection and Sonogashira coupling. 

2.        Single-molecule fluorescence imaging of palladium complexes

Armed with the catalysts described above, we used our new imaging setup to observe individual catalyst molecules, beginning with the mono-labeled complex, producing images like that shown in FIGURE 3.  Single catalyst molecules are observed as individual globules of light in the fluorescence image.  Single-step photobleaching confirms that individual fluorophores are present.  Unfortunately, single-molecule imaging of the dendrimeric labeled-catalyst also showed single-step photobleaching, suggesting that inter-fluorophore coupling within the dendrimer was stronger than anticipated.  In addition, the brightness of the dendrimeric fluorophore was slightly reduced from the single-fluorophore construct, indicating a small amount of quenching rather than enhancement, likely due to pi-stacking interactions. 

                In order to image catalysts under turnover conditions, we require sample cells that satisfy specific criteria, including that they be 1) constructed with a glass coverslip bottom for imaging, 2) not contain any glues that can act as reservoirs for fluorescent impurities due to solvent leaching, and 3) be of simple and inexpensive construction so as to be disposable.  We designed an all-glass sample cell that satisfies these conditions and is now in routine use in the lab.  We also built a chamber heater that can be wrapped around the sample cell while it is on the microscope and stably heat the solution while simultaneously monitoring the temperature of the microscope objective, which cannot be taken arbitrarily high. 

                Using this cell, we were able to take single-molecule measurements of individual catalyst molecules in the presence of solvent, other chemical reagents, and at temperatures reflective of typical synthetic conditions.    First, catalysts are diluted in carefully purified solvent and allowed to form covalent bonds with the silica surface via their silane surface-anchor functionality.   Second, residual solution-phase catalyst is thoroughly washed away.  In this manner, low-density catalyst surface coverages conducive to imaging can be conveniently prepared.  We have now begun investigating the fluorescence dynamics of the catalysts during Suzuki cross-coupling conditions in a variety of solvents.

                In summary, we have synthesized a class of functional, surface-supported palladium complexes, including catalysts with multiple fluorophores per metal center.  Individual catalysts can be imaged under turn-over conditions through a specially constructed sample chamber and microscope stage.  Correlating dynamics with reaction conditions is now ongoing.    

                The above research effort was mainly performed by a postdoctoral researcher.  The researcher was in charge of the synthetic preparations of the above molecules, but also participated in the single-molecule imaging experiments which were led by another graduate student.  The postdoctoral researcher continues to expand his experience in organometallic chemistry, dye chemistry, and microscopy. 

                The Doctoral New Investigator Grant has allowed us to build up core infrastructure and skillsets for a challenging multidisciplinary project.  The results obtained and foundation built will substantially help in procuring long-term research funding. 

Figure 1

Figure 2

Figure 3