IDENTIFICATION OF THE ELUSIVE AMINO ACID 19 
contains two asymmetric carbon atoms, the synthetic product was obtained as a 
mixture of two diastereometic racemates. Separation of the two racemic modifications, 
and subsequent resolution of each racemate into its optical antipodes was achieved 
some 2 years later in our laboratory*® in Bethesda. Separation of the racemates was 
accomplished by exploiting the differential solubilities of the hydrochloride salts of 
the two racemates. Thus, after saturation of an aqueous solution of the diastereomeric 
mixture with hydrogen chloride gas, and subsequent chilling at —1o°, some half of 
the starting material deposited as a crystalline substance which analyzed for the 
lactone hydrochloride and to which the designation of A was assigned. The soluble 
racemate, which was designated as the B form, could subsequently be isolated in 
good yield from the mother liquors. 
The efficacy of this procedure for the separation of the two racemic diastereomers 
could be readily assessed with the aid of column chromatography, as shown in Fig. 6. 

ia 7 (as T 
A-FORM 
JS) {= = 
B-FORM 
= 
Ss 
= Loe 4 
O 
a 
: i 
2 
= 
ad 
OS i= = 
= 
=e 




0) —— J Sheer een 
07 0.8 09 1.6 ul 
EFFLUENT VOLUME (LITERS) 
Fig. 6. Separation of racemic diastereomers of y-hydroxyglutamic acid on Dowex-1-acetate, elution 
with 0.5 N acetic acid. A is allo-form, B is normal form. 
Use was made of the fact that the original epimeric mixture undergoes complete 
separation into two peaks upon passage through the Dowex-1 acetate system. As 
chromatography of a 15-mg sample of once recrystallized lactone A, or the once 
recrystallized B form, revealed but a single peak in each instance, the optical integrity 
of each racemate was demonstrated. Resolution of the A and B forms into their 
respective optical antipodes was then achieved by the conversion of each racemate 
into its corresponding N-chloroacetyl derivative, succeeded by stereospecific enzymatic 
hydrolysis of the L-form of each of these derivatives with hog renal acylase I. As this 
enzyme is L-directed, the resolution procedure permitted both the eventual isolation 
of the four optically pure stereoisomers and the assignment of an L- or D-configuration 
to the a-carbon atom of each. 
Now only the configuration of the a-asymmetric carbon atom of each of the four 
isomers of y-hydroxyglutamic acid remained to be determined. This was accomplished 
by the conversion of each stereoisomer to the corresponding dihydroxyglutaric acid 
isomer through deamination with nitrous acid, as shown in Fig. 7. Now kinetic studies 
References p. 22/24 
