SYNTHESIS OF ᴅⳑ-SERINE (2-Amino-3-hydroxypropanoic acid)

• Prepare a suspension of 43.5g (0.2 mol) of diethyl acetamidomalonate in 25 ml of water and add in one portion 17g of neutral (1) 37-41 per cent w/v aqueous formaldehyde (0.21-0.23 mol). 

• Add 0.5 ml of 1 m sodium hydroxide solution as the catalyst, shake vigorously and leave at room temperature for 2 hours. 

• (Most of the solid goes into solution within 30 minutes; the mixture may require gentle warming on a steam bath to complete the dissolution of the solid.) Then add a solution of 18g (0.45 mol) of sodium hydroxide in 350 ml of water and leave at room temperature overnight. 

• Acidify the solution by adding 40g (38 ml, 0.67 mol) of glacial acetic acid and heat almost to boiling, when brisk decarboxylation sets in. 

• Continue to heat on a boiling water bath under reflux for 1 hour to complete the decarboxylation and then evaporate the solution to a syrup under reduced pressure on a rotary evaporator. 

• Dissolve the syrup in 120 ml of concentrated hydrochloric acid, boil under reflux (fume cupboard) for 1 hour and evaporate to dryness under reduced pressure. 

• Extract the dry residue with 200 ml of boiling absolute ethanol, filter off the sodium chloride and extract the latter with a further 100-ml portion of hot ethanol. 

• Evaporate the ethanol extracts on the rotary evaporator and boil the residue under reflux with 100 ml of concentrated hydrochloric acid for 1 hour (fume cupboard). 

• Evaporate again to a syrup, and dissolve the latter in water and re-evaporate twice more to remove most of the excess hydrochloric acid. 

• Finally dissolve the residual gum in about 200 ml of distilled water and pass the solution through a 50 x 2.75 cm column of a weakly basic anion exchange resin (e.g. Amberite IR 45, ᶿOH form, about 300 ml of moist granules). 

• Continue to elute the column with distilled water (about 1.5 litres) until the eluate gives no purple coloration when a portion is tested by boiling with a few mg of ninhydrin (2). 

• Combine all the ninhydrin-positive eluates, which should be free from chloride ion, and evaporate to dryness under reduced pressure (rotary evaporator). 

• Dissolve the straw-coloured residue in 150 ml of hot water, boil with a little decolourising charcoal, filter and add to the filtrate 750 ml of hot ethanol. 

• Cool in ice, filter off the purified ᴅⳑ-serine which crystallises, wash it with a little cold ethanol and dry in an oven at 50 °C. 

• The yield is 12g(57%), m.p.c. 235 °C (decomp.). The product thus obtained is homogeneous (R𝑓 0.57) on t.l.c. (silica gel, 15 cm run; butan-1-ol-formic acid-water, 6:3:1) (3).

Notes to keep in mind:

1. If necessary, the formaldehyde solution should be neutralised (narrow range pH paper) by the careful dropwise addition of 1 m sodium hydroxide solution.

2. After use, regenerate the resin in the following way. Firstly exhaust the column by passing 0.25 m hydrochloric acid through it until the pH of the eluate is about 2. Wash the column with 2 or 3 bed-volumes if distilled water, and then regenerate the resin by passing through it 0.25 m sodium hydroxide solution until the eluate is strongly alkaline. Finally wash the column thoroughly with much distilled water until the pH of the eluate is within the range 5.5-6.5 (narrow range indicator paper).

3. Using this solvent system, serine is not completely separable from glycine (R𝑓 0.58) which is a possible contaminant. The latter may be distinguished, however, by the characteristic brownish-pink spot which it gives on spraying with ninhydrin; that of serine is purple.