SYNTHESIS OF ⳑ-PROLINE (ⳑ-Pyrrolidine-2-carboxylic acid)

Ammonium rhodanilate:

• Heat a mixture of 250g (0.5 mol Cr 3 ®) of hydrated chromium(m) potassium sulphate (chrome alum), 290g (3.0 mol) of potassium thiocyanate and 250 ml of water in a 5-litre flask on a steam bath for 4 hours. 

• Cool, add 235g (230 ml, 2.53 mol) of redistilled aniline and heat in a water bath at 60 °C with stirring for 3 hours. 

• Cool again, add while still stirring 3 litres of water containing 300 ml of glacial acetic acid and leave at 0°C overnight. 

• Filter off the purple precipitate with suction, wash it with water and suck as dry as possible. 

• Extract the filter cake with 750 ml of methanol, filter and run into the filtrate, with stirring, 3 litres of water. 

• Cool at °C for 1 hour and filter off the crystalline purple mass of aniline rhodanilate. 

• Dissolve the damp product in 400 ml of methanol, add 200 ml of concentrated ammonia solution (d 0.88), cool to °C and then run in slowly, with stirring, 2 litres of water. 

• Collect the precipitate by filtration, and again treat it with 250 ml of methanol, 125 ml of ammonia solution and 1250 ml of water in the above manner. 

• Dry the resulting ammonium rhodanilate sesquihydrate in an oven at 50 °C; the yield is 130g (50.5%).

L-Proline:

• Place 150g of good quality sheet gelatin (cut into conveniently sized pieces) in a 1-litre flask and add 450 ml of concentrated hydrochloric acid. 

• Boil gently under reflux for 8 hours (fume cupboard), or boil under reflux for about 3 hours and leave on a steam bath overnight; complete hydrolysis is indicated by a negative biuret reaction (1). 

• Concentrate the hydrolysate to a syrup by distillation under reduced pressure (rotary evaporator), and remove excess hydrochloric acid by dissolving the syrup in water and evaporating twice more. 

• Dissolve the residual syrup finally in 500 ml of water, boil briefly with 3g of decolourising charcoal, filter, cool and dilute with water to 1200 ml. 

• Add this solution slowly with stirring to a filtered solution of 125g of ammonium rhodanilate in 750 ml of methanol and keep at °C for 2 hours to allow the complete separation of proline rhodanilate. 

• Filter off the latter, wash it with water and suck as dry as possible. 

• Dissolve the damp crude product in 400 ml of methanol, filter and add 800 ml of 0.5 m hydrochloric acid slowly with stirring. 

• Cool at °C for 2 hours, filter off the purified proline rhodanilate and wash it with 250 ml of cold water. 

• Dry the product in an oven at 50 °C; the yield is about 100g, m.p. c. 130°C, with preliminary softening and blackening.

• Suspend the purified salt in 850 ml of water in a stoppered bottle, add 25 ml of pure pyridine and shake the mixture for 4-5 hours. 

• Remove the insoluble pyridine rhodanilate by filtration and wash it with 100 ml of cold water (2). 

• Combine the pale pink filtrate and washings, and add glacial acetic acid dropwise until the formation of a small pink precipitate is complete. 

• Filter, evaporate the almost colourless filtrate to dryness (rotary evaporator) and suspend the residue in absolute ethanol and re-evaporate twice. 

• Dry the resulting faintly pink crude proline in a vacuum desiccator over silica gel; the yield is about 18g. 

• Recrystallise from the minimum volume of absolute ethanol to obtain 11g (7.3% based on gelatin) of ⳑ-proline, m.p. 218-219 °C (decomp.), [𝜶]ᴅ¹⁸-85.6° (c3.0 in H₂O). 

• Check the purity of the product by t.l.c. on silica gel using the solvent system butan-1-ol-acetic acid- water, 4:1:1; R𝑓 0.26 (yellow spot with ninhydrin).

Notes to keep in mind:

1. Remove about 0.25 ml of the hydrolysate, cool it and basify it with 5ᴍ sodium hydroxide solution. To a portion add a few drops of very dilute copper(u) sulphate solution, and note the absence of any colour change. As a control, prepare a specimen of biuret (NH₂⋅CO⋅NH⋅CO⋅NH₂) by heating about 10mg of urea just above its melting point for about 2 minutes. Add a little basified hydrolysate warm to dissolve, cool and add a trace of copper(u) sulphate. A deep pink colour superimposed upon the pale brown colour of the hydrolysate should be observed. If the hydrolysate gives a similar colour originally, it contains peptide material and hydrolysis should be continued until the biuret test is negative.

2. About 100g of pyridine rhodanilate is obtained. To convert this into the ammonium salt for re- use, suspend it in 175 ml of methanol and add 90 ml of concentrated ammonia solution. Stir at °C for 30 minutes and then dilute gradually with 900 ml of water. Filter off the precipitated ammonium salt, wash it with water and repeat the methanol-ammonia treatment once more. The final yield of dried ammonium rhodanilate is about 80g.