62nd Annual Report on Research 2017

The overarching goal for this project has been to create a photolytic method to release metal cations on an ultrashort timescale permitting us to explore the very first metal ion binding by proteins associated with neurodegenerative diseases, such as Alzheimers. We applied ultrafast laser spectroscopy on two model metal cage complexes to explore the development of this method. We compare and contrast results for experiments in bulk aqueous solution and in the confined environment in reverse micelles. 

Initial experiments utilized the copper3G cage molecule designed and synthesized by the Franz group¹ and zinc-NTA complexes developed by the Burdette group² (structures shown in Fig. 1)   We performed ultrafast (10 fs-1 ns) and fast (>50 ns) transient absorption spectroscopy experiments to investigate dynamics for the release of Cu²⁺ and Zn²⁺ from the Cu3Gcage and Zn-NTA complexes. We photolyzed the complexes in bulk aqueous solution and in reverse micelles (RMs) in the presence and absence of a zincon "sensor" ligand^3,4 (structure shown in Fig. 1). The zincon ligand has a high binding constant for both Cu²⁺ and Zn²⁺. Performing photolysis in AOT (sodium bis-ethylhexylsulfosuccinate) RMs, we intended to minimize diffusion by placing the metal cage complex and the zincon in close proximity.

Although we have not observed evidence for ultrafast metal-ion bursts, we have observed interesting behavior of complexes, confirmed their ability to deliver metal cations both in bulk aqueous solution and confined in RMs, and demonstrated our ability to detect metal cations released into bulk aqueous solution and RMs. Our experiments showed that we successfully photolyzed the Zn(II)-NTA complex, detecting chelation of released Zn(II) ions in complex in aqueous solution (buffered, Fig. 3A) and in RMs (AOT w₀=30, Fig. 3B). Following Zn-NTA photolysis (l=355 nm, Opolette laser) the diminishing spectral feature associated with free zincon and growth of the peak associated with the Zn-zincon complex. Two clear isosbestic points show clean conversion of species as the photolysis progresses. The zincon absorption spectrum in RMs differs from the spectrum in bulk solution, Fig. 3B, shifting to longer wavelength and displaying structure absent in the bulk aqueous spectrum.

The differences in zincon absorption spectra in aqueous and RMs could indicate that zincon partitions into the RM interface. We utilized 1D and 2D NMR spectroscopy to explore the origin of zincon spectral changes. Fig. 3. 1D ¹H NMR of free zincon and Zn-zincon in bulk aqueous solution and in RMs show significant differences in peak positions and spectral bandwidth suggesting that zincon may embed in the interface.

We have shown that we can successfully photolyze the metal-cage complexes in aqueous solution and within reverse micelles. Photolysis in reverse micelles resembles the dynamics in aqueous solution. We are working to explain the differences in the two systems, why we think those differences exist and how they relate observed zincon dynamics in the two systems. To our knowledge, the metal-ion burst within reverse micelles before has not been measured. This could have biological implications of membrane associated proteins, small molecules and other structures which use metal ions or metal complexes which must diffuse through an ionic layer before finding their mate to either chelate the metal ion or exchange their metals with an awaiting component in a membrane.

Impact on student education:

The research reported here has served to train students with laboratory and critical thinking skills. Dr. Richard Cole, in the Levinger group has led time-resolved spectroscopy studies, training graduate student Cheryle Beuning and several undergraduate students. Ms. Beuning has worked closely with undergraduate students who presented 3 posters at the CSU Celebrate Undergraduate Research and Creativity poster session, spring 2017. Ms. Beuning presented results at the 2017 spring national ACS meeting.⁵ Two undergraduate students will co-author the manuscript Ms. Beuning is currently writing about project results. 

References

1.         Ciesienski, K. L.; Franz, K. J., 2011, Keys for Unlocking Photolabile Metal-Containing Cages. Angew. Chem.-Int. Edit. 50, 814-824.

2.         Basa, P. N.; Antala, S.; Dempski, R. E.; Burdette, S. C., 2015, A Zinc(II) Photocage Based on a Decarboxylation Metal Ion Release Mechanism for Investigating Homeostasis and Biological Signaling. Angew. Chem.-Int. Edit. 54, 13027-13031.

3.         Platte, J. A.; Marcy, V. M., 1959, Photometric determination of zinc with zincon - application to water containing heavy metals. Anal. Chem. 31, 1226-1228.

4.         Sabel, C. E.; Neureuther, J. M.; Siemann, S., 2010, A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon. Anal. Biochem. 397, 218-226.

5.         Beuning, C.; Paryani, T.; Barkley, N.; Basa, P.; Cole, R.; Levinger, N.; Burdette, S.; Crans, D., 2017, Photolysis of a Zn(II)-nitriliotriacetate cage in AOT/isooctane reverse micelles. Abstr. Pap. Am. Chem. Soc. 253, 1.