| OCR Text |
Show 221 THE UNIVERSITY OF UTAH Background: Leber's congenital amaurosis (LCA) is a rare genetic eye disease. Those affected express visual impairment and blindness. The cause is a mutation in either lecithin-retinol transferase (LRAT) or retinal pig-ment epithelium isomerase 65 (RPE65). Light induced membrane potential in photo-transduction is based on a cycle of converting 11-cis-retinal to all-trans-retinal then recycling the all-trans-retinal back to the cis form. This recycling is essential to maintain continuous vision. Once a photon is absorbed by 11-cis-retinal it undergoes a conformational change that activates rhodopsin and leads to the change in the rod cell membrane poten-tial causing the electrical signal that results in vision. All-trans-retinal is reduced and transported into RPE cells where LRAT esterifies all-trans-retinol to all-trans-retinyl esters.1 RPE65 then isomerizes the trans ester to 11-cis-retinol, which can be oxidized for absorption of a photon and repetition of the cycle.1 If a mutation in LRAT or RPE65 has taken place then it is plain to see that the recycling of all-trans-retinal cannot take place and mislocalization and aggregation of cone opsins is observed,2 reminiscent of the protein aggregation observed in diseases like Alzheimers.3,4 Professor Yingbin Fu in his lab at Moran Eye Center at the University of Utah has identified hydrophobic phenylalanine (Phe) residues (between residues 70-125) in cone opsins that are suscep-tible to the discussed aggregation phenomenon due to favorable π-π interactions.2 Nanoparticles (NP) have only recently begun to be explored in this context but have been repeatedly shown to be effective at dispens-ing their cargo in the eye.5 Purpose: The focus is to efficiently, with high yield and purity, synthesize naphthoquinone-amino acid conju-gates, specifically naphthoquinone-phenylalanine (NQ-Phe) derivatives, as therapeutic devices to prevent opsin protein aggregation. Hypothesis: NQ-Phe derivatives, in addition to Phe π electrons associating with opsin residues, will interact with the cone protein and inhibit aggregation. Methods: 10 mmol 2-naphthalene-1,4-dione was dissolved in 200 mL EtOH. Added to that mixture was 5 mmol L-amino acid, 10 mmol triethylamine, and 5 mmol water. The reaction was stirred at room temperature for a minimum of 12 hours but no longer than 24 hours in a 500-mL Erlenmeyer flask. Solvent was removed under re-duced pressure and the material was then dissolved in 50.0 mL CH2Cl2 and four scoops of silica gel were added. The solvent was removed under reduced pressure and the dried material was placed on a silica gel column. The column was eluted first with 4:1 hexane to EtOAc, then EtOAc, and finally MeOH. Solvent was removed under reduced pressure and then dissolved in 50.0 mL CH2Cl2 before washing four times with 4 M HCl (30.0 mL) and then washing twice with brine (30.0 mL). The organic layer was dried with anhydrous magnesium sulfate before removing solvent and obtaining a final mass. Spectrometry is then used to determine the structure (purity) of the final compound. The compound is then taken to mice trials to see the activity against cone protein aggre-gation. Results: Identity of the compounds was confirmed using mass spectrometry and NMR (300 MHz Varian NMR). Dr. Rainier synthesized NQ-Trp while James Robertson synthesized NQ-Phe with 25% yield. Data matched that reported by Katritsky et al.6 Jeremy Sanchez and Wesley Kerman synthesized further derivatives of NQ-Phe. NQ-Trp showed to have an IC50 ~ 5.2 μM while NQ-Phe was more active with an IC50 ~ 90 nM. Anticipated Results: The influence of NQ-Phe derivatives are being explored through synthesis with electron rich arenes, electron deficient arenes, sterics, and extended aromaticity substitions on both the NQ and Phe portions of the molecule. This is expected to expound upon the understanding of opsin protein aggregation and improve activity therefore resulting in a more effective NP delivered ocular drug. Implications: NPs can be used as effective drug delivery devices for diseases of the eye. The arsenal of quinone bearing peptides will increase due to their potential clinical significance in targeted chemotherapy. Finally the method of synthesizing naphthoquinone-amino acid conjugates will be refined and defined further. References 1. Travis, G. H.; Golczak, M.; Moise, A. R.; Palczewksi, K. Diseases Caused by Defects in the Visual Cycle: Retinoids as Potential Therapeutic Agents. Annu. Rev. Pharmocol. Toxicol. 2007, 47, 8.1-8.44 2. Zhang, T.; Zhang, N.; Baehr, W.; Fu, Y. Cone Opsin Determines the Time Course of Cone Photoreceptor Degeneration in Leber Congenital Amaurosis. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 8879-8884. 3. Frydman-Marom, A.; Rechter, M.; Shefler, I.; Bram, Y.; Shalev, D. E.; Gazit, E. Cognitive-Performance Recovery of Alzheimer's Disease Model Mice by Modulation of Early Soluble Amyloidal Assemblies. Angew. Chem. Int. Ed. 2009, 48,1981-1986. 4. Redmond, T.M,; Yu, S; Lee, E.; Bok, D.; Hamasaki, D.; Chen, N.; Goletz, P.; Ma, J.X.; Crouch, R.K.; Pfeifer, K. Rpe65 is Necessary for Production of 11-cis-vitamin A in the Retinal Visual Cycle. Nat Genet 1998, 20, 344-51. 5. Bourges, J.L.; Gautier, S.E.; Delie, F.; Bejjani, R.A.; Jeanny, J.C.; Gurny, R.; BenEzra, D.; Behar-Cohen, F.F. "Ocular Drug Delivery Targeting the Retina and Retinal Pigment Epithelium Using Polylactide Nanoparticles," Inv. Ophthal. Vis. Sci. 2003, 44, 3562- 3569. 6. Katritzky, A. R.; Huang, L.; Sakhuja, R. Efficient Syntheses of Naphthoquinone-Dipeptides. Synthesis 2010, 12, 2011-2016. SYNTHESIS AND OCULAR APPLICATION OF NAPHTHOQUINONE AMINO ACID CONJUGATES Jeremy Sanchez (Jon Rainier, Wesley Kerman) Department of Chemistry University of Utah health sciences leap program Jeremy Sanchez Jon Rainier Wesley Kerman |
