Passage II (Questions 1-8)

    When amino acids are synthesized in vitro, they form as a racemic mixture of the D and L enantiomers. Since the L enantiomer is desired, resolution is necessary. For resolving the mixture, one of three techniques can be applied.

Technique 1:
The racemate is first treated with benzoyl chloride to form an amide at the amino terminal. The racemate of N-benzoyl-DL-amino acid is treated with a brucine salt so that a salt of the D-amino acid precipitates from solution. The solid is filtered from solution and is then treated with acid to dissolve the brucine-amino acid salt. Upon treatment with aqueous hydroxide, the D-amino acid is isolated.

Technique 2:
The racemate is first treated with benzoyl chloride and then treated with a strychnine salt, so that a salt of the L-amino acid precipitates from solution. The solid is filtered from solution and is then treated with acid to dissolve the strychnine-amino acid salt. The solution is treated with aqueous hydroxide to generate the L-amino acid in relatively pure form.

Technique 3:
The racemate is treated with acetic anhydride to form N-acetyl-DL-amino acid. This racemic mixture of acylated amino acid is treated with hog renal acylase, which removes the acetyl group from the L enantiomer, forming the zwitterion. The D enantiomer remains acylated at the N-terminal, making it anionic (due to the C terminal). The two species are separated using ether-water extraction.

    The major drawback to the first two techniques is that only one enantiomer is isolated in pure form. The enantiomer that does not precipitate from solution is isolated after the solvent is evaporated. This procedure does not lead to a pure sample of the other isomer. The second drawback is that the success of the techniques varies with different amino acids. For instance, they work well with alanine, but are difficult to use with tryptophan. The third technique has the most universal application. Presented below is an experimental application of Technique 3:

Experiment:
A suspension of 12.9 grams of N-acetyl-DL-isoleucine in 0.80 liters of water is buffered to 7.0 in an aqueous solution, following which 0.010g hog renal acylase powder is added. The mixture is stirred for 16 hours at 37.0ūC (physiological temperature). The mixture is then acidified with 10.0 mL of acetic acid, filter through a frit, and evaporated under vacuum to a volume of roughly 50 mL. Ethanol is added to form L-isoleucine crystals. The crude product is recrystallized from an ethanol-water mixture to yield roughly 3.5 grams (77.1% yield) of optically pure L-isoleucine.