SYNTHESIS OF RESOLUTION OF ᴅⳑ-ALANINE

Benzoyl ᴅ alanine:

• Dissolve 100g (1.1 mol) of ᴅ-alanine in 400 ml of water containing 44.5g (1.1 mol) of sodium hydroxide and cool the solution in an ice bath. 

• Add 175g (1.2 mol) of benzoyl chloride and a solution of 49g (1.2 mol) of sodium hydroxide in 200 ml of water to the stirred, cooled, amino acid solution, alternately and in portions during 2 hours; continue to stir for a further 2-hour period. 

• Boil the reaction mixture with 10g of decolourising charcoal, filter, cool the clear yellow filtrate to °C and acidify carefully to Congo red with concentrated hydrochloric acid. 

• Triturate a portion of the oil which separates with water to induce crystallisation and then seed the bulk of the acidified solution with crystals and leave in an ice bath to complete the crystallisation process. 

• Filter off the product, wash the filter cake with 500 ml of ice-cold water and recrystallise from about 3.5 litres of boiling water. The yield of benzoyl-ᴅⳑ-alanine, m.p. 162-164 °C, is 194.5g (90%).

Benzoyl-l-alanine anilide:

• Use freshly boiled, cooled, distilled water throughout this stage. 

• Prepare an 0.1 m citrate buffer solution by dissolving 48g of anhydrous citric acid and 16.5g of sodium hydroxide in 2.5 litres of water. 

• Stir together 50g of technical powdered papain (1) and 4g of potassium cyanide in 500 ml of the buffer solution, adjust the pH to 5 (narrow range pH paper) with glacial acetic acid, and stir for a further 75 minutes. 

• Filter this enzyme extract through a Celite filter bed. 

• Dissolve 193g (1 mol) of benzoyl-ᴅ-alanine by warming it in 300 ml of the citrate buffer to which has been added 120 ml of 2.5 molar sodium hydroxide, 360 ml of 3 m sodium acetate solution and 93g (91 ml, 1 mol) of redistilled aniline; adjust the pH of this solution to 5 with sodium hydroxide solution. 

• Cool the solution to 45 °C, add the filtered enzyme extract and transfer the mixture to several conical flasks of suitable size such that each is filled to the neck and tightly stoppered with a rubber bung to exclude air. 

• Leave the flasks in an incubator held at 37 °C, shaking them occasionally during the early stages.

• Product begins to separate within 5 minutes and the contents of the flasks becomes almost immobile within 2 hours. 

• After 24 hours filter the mixture and return the filtrate to the incubator for a further 24 hours and remove the additional crop of solid which separates. 

• Wash the combined solids with 250 ml of water and recrystallise from 1 litre of 50 per cent aqueous ethanol with the aid of decolourising charcoal. The yield of benzoyl-l-alanine anilide is 122g (91%), m.p. 175-176 °C, [𝜶]ᴅ²⁰ -7.8° (c5 in MeᐧCO₂H).

l-Alanine:

• Heat a mixture of 50g (0.187 mol) of benzoyl-l-alanine anilide and 250 ml of 6 m hydrochloric acid under reflux for 5 hours. 

• Leave the mixture at room temperature overnight, remove the precipitated benzoic acid by filtration and evaporate the filtrate to dryness under reduced pressure (rotary evaporator). 

• Dissolve the brown oily residue in 100 ml of water and boil it with decolourising charcoal. 

• Filter and pass the filtrate through 450g of a weakly basic anion exchange resin, e.g. Amberlite IR4B (which has been washed free from soluble colour with dilute hydrochloric acid and regenerated with dilute aqueous ammonia) in the form of a column 60 cm long. 

• Collect the effluent (in all about 2 litres) until it gives no colour when boiled with ninhydrin. 

• Evaporate the effluent to dryness under reduced pressure (rotary evaporator) and boil the yellow solid residue with 20 ml of water and a little decolourising charcoal. 

• Add ethanol to the hot filtered solution until crystallisation begins and cool in ice to complete the separation of the L-alanine. The yield is 13.9g (76%), [𝜶]ᴅ²⁰ , + 12.0° (c4 in 1M HCl).

Notes to keep in mind:

1.  An inexpensive crude commercial product (dried papaya latex) was used; the activity was not determined. Purified highly active enzyme preparations may, however, be obtained 259 (e.g. Koch-Light, Sigma, etc.).