Passage
Fluorescent amino acids are useful for labeling proteins because they can be visualized spectroscopically, facilitating the study of protein structure and interactions. The noncanonical amino acid dansylalanine, a fluorescent amino acid that is sensitive to the hydrophobicity of its environment, is a good candidate to be incorporated into proteins because of its small size and fluorescent capabilities. Dansyl chloride and Compound 1 were used to synthesize dansylalanine, as shown in Reaction 1. Dansylalanine absorbs maximally at 340 nm and emits at approximately 540 nm.
Reaction 1 Synthesis of dansylalanineA leucyl-tRNA synthetase from Escherichia coli was engineered to aminoacylate bacterial tRNAs containing a CUA anticodon with dansylalanine. After verifying that the tRNA/synthetase pair does not interact with endogenous amino acids or tRNAs, it was transfected into the yeast Saccharomyces cerevisiae along with a plasmid encoding a mutated human superoxide dismutase (hSOD) . Using this system, dansylalanine was genetically incorporated into hSOD in place of either Gln-16 or Trp-33, and expressed in S. cerevisiae.Position 16 is located on the surface of hSOD at the N-terminus of a β-strand, and position 33 lies in the center of a strand within a β-barrel. To probe changes in the structure of hSOD, researchers exposed the purified protein to guanidinium chloride (GdmCl) to denature the protein at concentrations ranging 0-4.5 M. The experiment was conducted in a 35 mM sodium phosphate buffer at pH 7.2. The fluorescence of hSOD with dansylalanine at either position 16 or 33 was monitored at each GdmCl concentration, and the results of the unfolding experiment are shown in Figure 1.
Figure 1 Plot of dansylalanine fluorescence at different concentrations of GdmCl
Adapted from Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG. A genetically encoded fluorescent amino acid. Proc Natl Acad Sci USA. 2006.
-If researchers used the Gabriel synthesis to make the dansylalanine backbone, which of the following statements is true?

Question

Passage
Fluorescent amino acids are useful for labeling proteins because they can be visualized spectroscopically, facilitating the study of protein structure and interactions. The noncanonical amino acid dansylalanine, a fluorescent amino acid that is sensitive to the hydrophobicity of its environment, is a good candidate to be incorporated into proteins because of its small size and fluorescent capabilities. Dansyl chloride and Compound 1 were used to synthesize dansylalanine, as shown in Reaction 1. Dansylalanine absorbs maximally at 340 nm and emits at approximately 540 nm.

Reaction 1 Synthesis of dansylalanineA leucyl-tRNA synthetase from Escherichia coli was engineered to aminoacylate bacterial tRNAs containing a CUA anticodon with dansylalanine. After verifying that the tRNA/synthetase pair does not interact with endogenous amino acids or tRNAs, it was transfected into the yeast Saccharomyces cerevisiae along with a plasmid encoding a mutated human superoxide dismutase (hSOD) . Using this system, dansylalanine was genetically incorporated into hSOD in place of either Gln-16 or Trp-33, and expressed in S. cerevisiae.Position 16 is located on the surface of hSOD at the N-terminus of a β-strand, and position 33 lies in the center of a strand within a β-barrel. To probe changes in the structure of hSOD, researchers exposed the purified protein to guanidinium chloride (GdmCl) to denature the protein at concentrations ranging 0-4.5 M. The experiment was conducted in a 35 mM sodium phosphate buffer at pH 7.2. The fluorescence of hSOD with dansylalanine at either position 16 or 33 was monitored at each GdmCl concentration, and the results of the unfolding experiment are shown in Figure 1.

Figure 1 Plot of dansylalanine fluorescence at different concentrations of GdmCl
Adapted from Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG. A genetically encoded fluorescent amino acid. Proc Natl Acad Sci USA. 2006.
-If researchers used the Gabriel synthesis to make the dansylalanine backbone, which of the following statements is true?

Quizplus · Accepted Answer

11ecba2d_1274_50ef_a3bf_9125aeb7b0ed_MD0008_00 The Gabriel synthesis, also known as the malonic ester synthesis, is a method used to make primary amines, including α-amino acids, without overalkylation of the amine. The amine is generated from potassium phthalimide, a "protected" form of ammonia that prevents multiple alkylations due to the steric hindrance of the phthalimide group. The synthesis begins with an S_N2 reaction, where potassium phthalimide is the nucleophile that attacks an alkyl halide. The amine is then "deprotected" by removal of the phthalimide group. For the synthesis of amino acids, potassium phthalimide is the nucleophile that attacks the electrophilic carbon of diethyl bromomalonate, and bromide is the leaving group. The α-carbon is deprotonated with a base and attacks an alkyl halide to add the amino acid side chain via another S_N2 reaction. The phthalimide is removed through a basic hydrolysis reaction, yielding a dicarboxylic acid. Lastly, the diacid is decarboxylated with acid and heat. The resulting loss of CO₂ yields the final amino acid. The starting reagents for this synthesis are planar and do not have chiral centers; therefore, the amino acid product will be a mixture of L- and D-amino acids. (Choice B) Diethyl malonate contains the -CO₂ group that becomes the carboxylic acid in the final product; however, it does not have a leaving group on the -CH₂ between the two carbonyls necessary for the S_N2 reaction with potassium phthalimide. (Choices C and D) Potassium cyanide and an aldehyde are both used in the Strecker synthesis of an amino acid. Like the Gabriel synthesis, the Strecker synthesis provides a mixture of L- and D-amino acid products. Educational objective: The Gabriel synthesis is a method that uses potassium phthalimide and diethyl bromomalonate as starting materials to synthesize amino acids. The starting reagents of the Gabriel and Strecker syntheses are all planar; therefore, the product is a mixture of L- and D-amino acids.

Passage Fluorescent Amino Acids Are Useful for Labeling Proteins Because They