Investigating the Photochemistry of Provitamin D3 as a Function of Liposome Properties

Abstract

7-dehydrocholesterol (DHC, provitamin D3) is the starting material that undergoes photochemical processes to turn into vitamin D3 in mammalian skin membranes. The process starts when UV-B light penetrates through the outer layer of mammalian skin and converts DHC in the skin membrane into a previtamin D3 species. The previtamin species can undergo further processes where it can relax to form vitamin D3. Additionally, the Smith-Lemi-Opitz syndrome (SLOS), a condition fatal to developing embryos, is caused by the genetic inability to convert DHC into cholesterol, resulting in destabilization of the cell membrane. Thus, most lipid research has focused on the structural influences of DHC and cholesterol on synthetic lipids that closely mimic the cell membrane. The photochemical dynamics of DHC can also provide additional insight into the cell membrane environment, but has not been explored. By using membrane models to study the initial dynamics of vitamin D3 formation, information about the photochemical process in the lipid environment can possibly be used to address issues that arise from the SLOS. The ultrafast photochemical transformation of DHC, ergosterol (ergo, provitamin D2) and DHC-acetate to previtamin D3, D2 and D3-acetate occurs upon a ring-opening reaction in the excited state where a cyclohexadiene (CHD) chromophore embedded within the molecules opens to form a hexatriene previtamin D species. In order to capture the dynamics and conformational changes of the three provitamins, broadband transient absorption spectroscopy is used. This dissertation investigates the excited state ring-opening and ground state relaxation of DHC, ergo and DHC-acetate in isotropic solution and anisotropic liposome environments. The solution measurements provide a baseline to explore the excited state and ground dynamics of the three molecules in a complex liposome environment. The primary lipid that is used in this work, 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), is reported to closely mimic the human skin and therefore allows us to study the initial steps involved in vitamin D production in a simple skin membrane model. The excited state of DHC is well studied in isotropic solvents where the molecule ring opens to form a previtamin D3 species with dynamics that are completed within 1-2 ps. On the ground state, the conformer relaxation process forms a stable previtamin D3 species within 6-7 ps. In solution, ergo and DHC-acetate have similar spectra, photoproduct distributions, and excited state and ground state lifetimes as DHC. However, unlike in solution, the cell membrane environment is hypothesized to stabilize the previtamin D conformation responsible for thermal formation of vitamin D. In this work we find that when DHC is incorporated into the DPPC lipid bilayer, the longest time constant is significantly slower than in solution, ca. 11 ps and 25 ps in the excited and ground state, respectively. To understand the photochemistry that takes place in biological skin membranes, DHC, ergo and DHC-acetate are investigated as a function of membrane parameters, such as hydrogen bonding, van der Waals interactions and lipid tail length. Dynamics in the excited state and ground state appear to be more significantly dependent on the lipid tail length than on the other parameters. The ring-opening reaction of DHC in the shortest lipid studied, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) is completed within 6 ps. The photochemistry of DHC in DLPC suggests that the shorter tail length environment reduces the conversion efficiency of vitamin D3 formation.