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Developing Molecular Probes for In Vivo Controlled Delivery of Chemical Agents Tan C. M. 1 and Yao S. Q. 2 Department of Chemistry, Faculty of Science, National University of Singapore 3 Science Drive 3, Singapore 117543 ABSTRACT Full knowledge of the human genome now allows us to inspect our proteome more closely. As enzymes are involved in many catalytic and pathogenic reactions in the body, they are attractive therapeutic targets. However, the intracellular location of many enzymes implies that any chemical delivery system must pass through barriers such as the cell membrane and endocytosis, which can severely limit uptake. Therefore, a delivery system into the cell overcoming these barriers and is only triggered by the target enzyme is needed. In this project, a trifunctional probe containing a fluorophore, the TEV protease recognition sequence and a protein transduction domain (PTD) was designed. The PTD allows mass­ and endocytosis­independent uptake of the probe, while the TEV sequence acts as a trigger when cleaved with non­endogenously expressed TEV protease, releasing the fluorophore. The use of solid phase peptide synthesis to generate the TEV sequence directly onto the probe allows fast and automated assembly of the peptide. 5 of the proposed 12 synthetic steps them were optimized and problems encountered were discussed. An alternative synthetic route which is both high yielding and cost­effective was proposed. This probe can be used for bioimaging and delivery of chemical agents such as probes or drugs. INTRODUCTION The holy grail of any chemical delivery system is to achieve full temporal, spatial and concentration control of the system within a cell. There is also a need to monitor enzyme activity in vivo as laboratory methods do not accurately reflect cellular conditions. Most delivery methods suffer from difficulties passing through the cell membrane due to reasons such as mass limitations (less than 500Da) or hydrophilicity. After passing into the cells, the probes must be activated only under controlled conditions by specifically reacting with an enzyme hence releasing our chemical agent of interest. In this project, the aim is to develop a molecular probe able to transduce into cells and through a specific enzymatic reaction with a non­endogenously expressed enzyme, release a fluorophore hence generating an observable readout. The applications of this probe are wide ranging: the location of a target enzyme can be found by the above trigger method. Once the enzyme’s location is determined, a localization sequence can be attached together with any chemical agent of interest (inhibitor, drugs etc), and the probe will be delivered to the specific area of the cell (e.g. organelles) hence achieving target­specific delivery. 1 2 Student Associate Professor Supervisor
PROJECT DESIGN Figure 1: Proposed Synthetic Scheme of Molecular Probe Facile uptake of the probe into the cell is conferred by the HIV1 Tat protein, with a sequence of YGRKKRRQRRR, which, when conjugated to any molecule, enables direct transduction of abnormally large molecules, reducing loss by rejection of entry or hydrolysis. This method allows efficient uptake of the probe into the cell, and versatility in coupling different kinds of chemical agents to be introduced into the cell. 4­ aminomandelic acid was chosen as the chemical adaptor as the α­hydroxyl group can be coupled to different drugs or probes (Figure 2). An ENLYFQ peptide sequence bound to the adaptor will be specifically cleaved by TEV protease at the Q­adaptor amide bond. This releases the coumarin fluorophore producing an observable fluorescent readout: Figure 2: (left) General concept of chemical adaptor system; (right) structure of target molecular probe
RESULTS AND DISCUSSION Figure 3: Completed synthetic steps for molecular probe synthesis A brief discussion of the major points of each individual synthetic step is laid out below: 1 à 2 : The addition of pyridine as proton scavenger is crucial to avoid acid­promoted elimination of t BuOH. Anhydrous conditions must be observed to reduce formation of H3PO4 by reaction with H2O. 2 à 3 :Di­brominated product occurs with prolonged reaction time hence the reaction was closely monitored by thin layer chromatography (every 15 – 30 min), and the reaction stopped just as side products form. 3à 4 : Reflux at 100 o C for 12h resulted in the deprotection of the t­Bu group, and room temperature resulting in very slow reaction. An optimal temperature of 50 o C provides a reasonable rate of reaction (5 hr for complete reaction) and with significantly less side products formed. 5 à 6 : Proper rinsing with MeOH must be done during filtration with Celite, due to crystallization of the product occurring. Losses could have been sustained during column purification, as 6 is quite polar which increases retention time in the column and band spreading hence elution of the product with side product. 6 à 7 : Proper addition of reagents resulted in this coupling reaction being high yielding. HATU is the most efficient coupling reagent due to its pyridine ring participating in intramolecular base catalysis. Pre­activation of the amino acid by mixing it with HATU and collidine (a base) to form the activated ester prevents side product formation. Use of distilled DMF (hence dimethylamine­free) prevents side reactions. FURTHER WORK A modification could be done to the existing scheme to make it less time consuming and ensure higher yield:
Figure 4: Alternative synthetic route Both the Yb(OTf)3­promoted Cannizzaro oxidation and the B(OH)3­catalysed carboxylic acid protection steps afford high yield when reported in the literature (99%). Furthermore, only simple filtration, extraction work­up and no purification by column chromatography was required. There was delay in shipping of Yb(OTf)3, hence the original scheme was adhered to. Little modification to the existing steps is required. The probe will be tested in vivo using cells with TEV gene introduced (hence expressing TEV protease), to determine transduction and localization of the probe using fluorescence microscopy. With success, this design will be an important first step in developing probes for use in mammalian cells, towards specifically targeting endogenously expressed enzymes. ACKNOWLEDGEMENTS I would like to thank my supervisor, Associate Professor Yao Shao Qin and all my lab mates for their advice and continued support throughout the course of my experiments. REFERENCES 1. Curini M., Epifano F., Genovese S., Marcotullio M. C., Rosati O. (2005) Ytterbium Triflate­Promoted Tandem One­Pot Oxidation – Cannizzaro Reaction of Methyl Aryl Ketones Org. Lett. Vol. 7, No. 7, 1331 – 1333 2. Gopin A., Pessah N., Shamis M., Rader C., Shabat D. (2003) A Chemical Adaptor System Designed To Link a Tumor­Targeting Device with a Prodrug and an Enzymatic Trigger Angew. Chem. Int. Ed. 42 No. 3 327­332 3. Lee M. R., Baek K. H., Jin H. J., Jung Y. G., Shin I. (2004) Targeted Enzyme­ Responsive Drug Carriers: Studies on the Delivery of a Combination of Drugs Angew. Chem. Int. Ed. 43, 1675 – 1678 4. Qing Z., Girish A., Chattopadhaya G., Yao S. Q (2004) Developing novel activity­ based fluorescent probes that target different classes of proteases Chem. Commun. 1512­1513