Synthesis of an Unnatural Fluorescent Amino Acid

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Synthesis of an Unnatural Fluorescent Amino Acid

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Synthesis of an Unnatural Fluorescent Amino Acid

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Organic Synthesis of an Unnatural Fluorescent Amino Acid

The long-term goal of this project is to chemically synthesize an unnatural fluorescent amino acid, 3-[7-nitro-2,1,3-benzoxadiazol-4-yl]-L-alanine. This amino acid can be used to build glow-in-the-dark proteins, allowing investigators to visualize a single protein in an otherwise transparent living cell. We have been unable to replicate the previously reported synthesis and are exploring different reaction conditions and workup procedures. To determine the efficiency of the new reaction conditions, reaction kinetics will be analyzed using 1H-NMR and LCMS, which follow the reactions over time

The Synthesis of an Unnatural Fluorescent Amino Acid

The long-term goal of this project is to develop a more cost-effective chemical synthesis of an unnatural fluorescent amino acid, 3-[7-nitro-2,1,3-benzoxadiazol-4-yl]-L-alanine. This molecule can be used to visualize a single “glow-in-the-dark” protein in an otherwise transparent living cell. We are investigating two synthetic routes: traditional organic chemistry and biocatalysis. The biocatalysis route utilizes the enzyme glutathione S-transferase from the thermophilic cyanobacterium, T. elongatus. For the organic reaction, nuclear magnetic resonance (1H NMR) spectroscopy is being used to monitor reaction kinetics with the goal of identifying the optimal solvent and reaction conditions. For the biocatalyzed reaction, a commercial vendor has synthesized the DNA that codes for the T. elongatus glutathione S-transferase gene and incorporated that gene into a suitable plasmid. We have used this plasmid to transform E. coli and have demonstrated expression of T. elongatus glutathione S-transferase. A plate reader has been used to monitor enzyme kinetics. We hope to develop a liquid chromatography-mass spectroscopy method that will allow us to monitor reaction kinetics for both the organic and biocatalysis routes

Synthesis of an Unnatural Fluorescent Amino Acid

The long-term goal of this project is to chemically synthesize an unnatural fluorescent amino acid (UFAA) that can later be used to build glow-in-the-dark proteins. UFAAs allow investigators to visualize a single protein in an otherwise transparent living cell. The specific UFAA target for this project is L-alanine, 3-[7-nitro-2,1,3-benzoxadiazol-4-yl], which is an analog of the natural amino acid tryptophan. This synthesis consists of a coupling reaction followed by a deprotection reaction. Products have been characterized using 1H and 13C NMR, and Liquid Chromatography-Mass Spectrometry (LC-MS). Future tasks could include optimizing the yield and purity of this UFAA

Biocatalytic Synthesis of an Unnatural Fluorescent Amino Acid

The long-term goal of this project is to chemically synthesize an unnatural fluorescent amino acid, 3-[7-nitro-2,1,3-benzoxadiazol-4-yl]-L-alanine, that can later allow researchers to visualize a single “glow-in-the-dark” protein in an otherwise transparent living cell. In addition to our attempted organic synthesis, here we report a biocatalytic synthesis. Specifically, we plan to use the enzyme glutathione S-transferase from the cyanobacterium T. elongatus to catalyze the key nucleophilic aromatic substitution reaction. As part of this effort, we are planning a new synthetic route to the non-natural amino acid, beta-amino alanine. In sum, we hope to synthesize these two unnatural amino acids in a more time- and cost-efficient manner

Biocatalytic Synthesis of an Unnatural Fluorescent Amino Acid

The long-term goal of this project is to chemically synthesize an unnatural fluorescent amino acid, 3-[7-nitro-2,1,3-benzoxadiazol-4-yl]-L-alanine, that can later allow researchers to visualize a single “glow-in-the-dark” protein in an otherwise transparent living cell. In addition to our attempted organic synthesis, here we report a biocatalytic synthesis. Specifically, we plan to use the enzyme glutathione S-transferase from the cyanobacterium T. elongatus to catalyze the key nucleophilic aromatic substitution reaction. As part of this effort, we are planning a new synthetic route to the non-natural amino acid, beta-amino alanine. In sum, we hope to synthesize these two unnatural amino acids in a more time- and cost-efficient manner

Synthesis of an Unnatural Fluorescent Amino Acid

The long-term goal of this project is to chemically synthesize an unnatural fluorescent amino acid (UFAA) that can later be used to build glow-in-the-dark proteins. UFAAs allow investigators to visualize a single protein in an otherwise transparent living cell. The specific UFAA target for this project is L-alanine, 3-[7-nitro-2,1,3-benzoxadiazol-4-yl], which is an analog of the natural amino acid tryptophan. This synthesis consists of a coupling reaction followed by a deprotection reaction. Products have been characterized using 1H and 13C NMR, and Liquid Chromatography-Mass Spectrometry (LC-MS). Future tasks could include optimizing the yield and purity of this UFAA