Synthesis of a Cargo-Linked Peroxynitrite Cleavable Monomer Shivani Avasarala, Andrea S. Carlini, and Nathan Gianneschi The Department of Chemistry & Biochemistry, University of California San Diego, 9500 Gilman drive, La Jolla, California ABSTRACT Background of ROS Responsive Materials Synthesis of Drug Screen of Buchwald-Hartwig Reaction Drug-Loaded Peroxynitrite Responsive Monomer Synthesis of Rhodamine References Conclusions and Future Directions Reactive oxygen species (ROS) responsive probes are of growing interest in the field of biomedicine. Because of its extreme reactivity and selectivity over other endogenous ROS, peroxynitrite is a promising target for the purpose of detecting and targeting the site of diseased tissue, such as a myocardial infarct. In this study, we worked towards the synthesis of a peroxynitrite-responsive cleavable monomer, which upon activation is expected to release a bound cargo; in our study, this cargo was a therapeutic small molecule drug or a fluorescent tag. The probe consists of a polymerizable moiety, a linker molecule, a peroxynitrite substrate, and a cargo which possesses a 2˚ amine. In the drug-linked monomer, it was determined that the 1,4-dihydropyridine amine was likely not nucleophilic enough for a Buchwald-Hartwig (C-N) amination. In switching gears to a monomer linked to a more nucleophilic fluorophore, 5(6)-carboxyrhodamine, initial protection and coupling steps were completed. Synthesis of the new monomer is ongoing. Given successful responsiveness to peroxynitrite, this probe can be polymerized and assembled into nanoparticles used for the effective detection of peroxynitrite production in diseased tissue, and may be altered to carry different cargos, such as drugs, labels, and targeting moieties. Cargo: drug molecule, fluorophore, contrast agent, targeting peptide Figure: General structure of peroxynitrite responsive cleavable monomer. N H O O O Figure: Amine drug molecule (left) and peroxynitrite-responsive cleavable monomer bound to drug molecule (right). Advantages of Covalently Bonded Cargos • High density packing • No leakage during storage of molecules • Tuned responsiveness and activated release Figure: Model demonstrates the efficiency of polymer with covalently bonded cargo (Right) as opposed to trapped free-floating cargo (Left) The Gianneschi Lab specializes in synthesizing responsive polymers and drug-loaded particles. Covalently bound cargos offer several advantages over encapsulated cargos in nanoparticle formulations. Conclusions: • Synthesized amine drug molecule with optimal purity and recrystallization • Completed one screen of Buchwald-Hartwig reaction in a vacuum-sealed glove box environment with the catalyst Pd(dba) 2 and four ligands: Xphos, XantPhos, RuPhos, (R)BINAP • Transitioned from using weak nucleophilic drug molecule to synthesizing a highly nucleophilic secondary amine, rhodamine Future: • Complete full screen of Buchwald-Hartwig reaction, incorporating several different reagents • Test responsivity of final probe in the presence of peroxynitrite Modified Reaction Scheme x L 2 Pd LPd Ar-X Pd L Ar X Pd Ar L X HNRR' N R R' H Pd L Ar X Base Base-HX Ar-NRR' N R R' Pd Ar L L Oxidative Addition Amine Binding Deprotonation Reductive Elimination Reagent Catalyst Ligand Aryl-X Amine Base Product Identity Pd(dba) 2 (R)-BINAP XantPhos Xphos RuPhos I-Phe Drug Cs 2 CO 3 Drug-Phe eq 0.05 0.1 0.1 0.2 0.2 1 1.2 1.4 1 # aliquots 4 1 1 1 1 4 4 4 4 E 85 °C 95 °C A B C D Figure: Screen of Buchwald-Hartwig Reactions (Top), color change in reaction viles indicates the activation of the catalyst by the ligand (Left), TLC and LCMS reveal consumption of Aryl-X but excess Amine drug remaining and no product material. (Right) Aryl-X Base Pd(dba) 2 Ligand Toluene/DMF CF 3 O O CF 3 O CF 3 OH O O O O ONOO- Cargo: drug or fluorophore 300 400 500 m/z 0 100 % 410.31 411.32 432.25 [M+H + ] 1+ [M+Na + ] 1+ Protection of Piperazine Figure: Transition from using the amine drug molecule to using a piperazine-taged rhodamine, a flourescent dye, for its higher nucleophilicity. N H O O O l k j i m h g f e d c b a n Figure: Mass spectrometry (Left) and H’NMR in chloroform-d (Right) of drug molecule NH HN N HN O O O O O Protection of Fmoc-(4-iodo)-Phe-OH H N O OH I O O H N O O I O O Figure: Tert-Butyl Protection (Top Left), Mass spectrometry of product (Top Right), C’NMR of product (Bottom) Figure: Mono-boc piperazine protection (Top Left), Mass spectrometry of product (Top Right), C’NMR of product (Left), H’NMR of product (Bottom) Figure: On-going reaction for the synthesis of rhodamine-piperazine, (Left), TLC Analysis under 365/254 nm UV light of the starting reagents and crude reaction solution (Middle and Right) Amine 408 m/z 339 m/z 637 m/z Reaction A Aryl-X 209.15 180 200 220 m/z 0 100 % 199.14 187.13 210.16 [M+H + ] + [M+Na + ] + a b c d e CH 2 Cl 2 85.1% yield a b c d [M+Na + ] + 580 600 620 m/z 0 100 % 592.23 593.19 594.23 1. Yang, D.; Sun, Z.-N.; Peng, T.; Wang, H.-L.; Shen, J.-G.; Chen, Y.; Tam, P. K.-H., Synthetic Fluorescent Probes for Imaging of Peroxynitrite and Hypochlorous Acid in Living Cells. In Live Cell Imaging: Methods and Protocols, Papkovsky, B. D., Ed. Humana Press: Totowa, NJ, 2010; pp 93-103. 2. 1. Peng, T.; Yang, D., HKGreen-3: A Rhodol-Based Fluorescent Probe for Peroxynitrite. Organic Letters 2010, 12 (21), 4932-4935. 3. 1. Surry, D. S.; Buchwald, S. L., Dialkylbiaryl phosphines in Pd-catalyzed amination: a user's guide. Chemical Science 2011, 2 (1), 27-50.