Brugia Malayi - Clark Science Center - Smith College

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DCats: Synthetic Tools for Selective Chemistry in Complex Settings Jiyeon Kim In response to a growing interest for chemical transformations in vivo and other complex environments, reactions that can selectively transform one target compound in a mixture are becoming high in demand. Currently, strategies for modifying a specific compound in the presence of similar compounds are mostly limited to proteins and have yet to be discovered for all other classes of molecules including small molecule metabolites, natural proteins, and sugars. 1 In the Gorin Lab, we synthesize and utilize DNA small molecule catalysts (DCats) to achieve site-selective chemistry. A DCat is modularly assembled from a DNA aptamer and a small molecule catalyst. An aptamer is an oligonucleotide that binds to a specific molecule with strong affinity through specific base coding and conformation. 2 Whereas in traditional chemistry a catalyst transforms all compounds with a susceptible functional group in a mixture, a DCat will speed up and direct a reaction to its target molecule while leaving other non-targets unchanged (Figure 1). This summer, I synthesized various DCats, optimized methods for product purification, and tested a DCat’s proof of concept. The first successful synthesis was a DCat for the hydrolysis of cholic acid nitrophenol ester. An aptamer for cholic acid with an amine at the 5’ end, previously evolved by SELEX, 3 went through a carbonyl substitution with an excess amount (>10 4 equivalents) of di(N-succinimidyl) glutarate (DSG) modified with histamine at one end (Figure 2A). DCats with non-catalysts were also synthesized in the same fashion (Figure 2B). The DCats were then purified by high-pressure liquid chromatography (HPLC) in the Center of Proteomics. Our initial method gave mixed fractions of the products; thus we optimized the method and were successful in purifying the desired DCats. The products were furthermore characterized by mass spectrometry also in the Center of Proteomics. DCats for the hydrolysis of ibuprofen nitrophenol ester were also synthesized and purified. With the DCats in-hand, I was equipped to test the selectivity of these special catalysts. The lab’s hypothesis, as illustrated in Figure 3, was that a DCat would speed up a reaction to its target molecule by bringing the target closer to the catalyst via the aptamer, increasing the probability of the two molecules reacting. Various reactions were prepared in necessary buffered salt solutions (Figure 3). The rate of reaction was quantified by checking the concentration of nitrophenol by UV-Vis at various times and comparing with a standard curve prepared. Initial results have been inconclusive. In the 2012-13 academic year, I will expound on the work I’ve accomplished during SURF through a senior honors thesis. First, I will follow-up on the results of our DCat reactions, drawing conclusions to develop the next steps for the project. For example, I will experiment with the linker between the aptamer and the catalyst to investigate if there is enough flexibility for the catalyst to reach the target. I will also develop other ways of monitoring the reactions. Additionally, I will investigate the effect of certain salts in the reaction buffer. These experiments will pave the road in validating DCats as synthetic tools for selective chemical reactions in complex environments. (Supported by the Trilink Biotechnology Research Rewards Program, the Cottrell College Scholar Award, and Smith College’s SURF Program) Advsior: David Gorin References: 1 Tsien, R. Y. “Constructing and exploiting the fluorescent protein paintbox.” Angew. Chem. Int. Ed. 2009, 48, 5612. 2 Wochner, A.; Menger, M.; Orgel, D.; Cech, B.; Rimmele, M.; Erdmann, V. A.; Glokler, J. “A DNA aptamer with high affinity and specificity for therapeutic anthracyclines.” Anal. Biochem. 2008, 373, 34. 3 Wilson, D. S.; Szostak, J. W. “In vitro selection of functional nucleic acids.” Ann. Rev. Biochem. 1999, 68, 611. 2012 77
O O A X A X B O X O DCat B O X linker N NH B O OH O C X C Figure 1. DCat-mediated selective hydrolysis to target molecule B X Figure 2A. Synthesis of cholic acid hydrolysis DCat Figure 2B. Control DCat with phenethylamine (left) and control DCat with glycyl glycine ethyl ester (right), synthesized in the same way Figure 3. Preliminary DCat reactions with predicted rate of hydrolysis. A) Cholic Acid DCat with cholic acid nitrophenol ester. B) Cholic acid DCat with ibuprofen nitrophenol ester. C) Imidazole with cholic acid nitrophenol ester. D) Cholic acid nitrophenol ester alone. E) Ibuprofen nitrophenol ester alone. 2012 78
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Brugia Malayi - Clark Science Center - Smith College

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