10 M. WINITZ 
DETERMINATION OF OPTICAL CONFIGURATION 
Other methods are, of course, available for determining the optical configuration of 
a naturally occurring amino acid aside from synthesis, and subsequent resolution of 
the synthetic material with a biological agent of established optical specificity. For 
the present purposes, such methods may be roughly categorized as chemical, biological, 
physical and optical (cf. ref. 53). 
Chemical methods are concerned primarily with the transformation of the natural 
amino acid either into another amino acid of established optical configuration or 
into some other compound of known optical configuration, using organic chemical 
procedures that do not induce rupture of any of the bonds surrounding the asymmetric 
carbon atom. These chemical methods cannot be effected by any “set” or standard 
procedure but, rather, vary one from the other because of differences both in the struc- 
ture and in the nature of the functional groups of the side chains of the amino acids 
under consideration. They must therefore depend, for the most part, on the ingenuity 
of the chemist to devise a chemical route from one compound to another, on his 
ability and discretion to draw properly from the most pertinent of a vast wealth of 
available synthetic and degradative reactions, and on his skill as a technician to 
guide such reactions to their satisfactory culmination. When properly executed, this 
approach forms the most powerful, as well as the most unequivocal of all available 
methods for determining the optical configuration of an amino acid. The chemical 
method, however, generally necessitates the expenditure of a good deal more time 
and effort than most of us can reasonably afford to devote to it, so that recourse is 
more frequently made to biological and physical methods. 
Biological methods for the determination of configuration depend upon the remark- 
able ability of living organisms, as well as certain tissues and enzyme fractions derived 
therefrom, to preferentially metabolize, incorporate, or chemically alter one antipode 
of a racemic amino acid, or a suitable derivative thereof, while leaving the other anti- 
pode essentially intact. An amino acid of unknown optical structure can therefore 
be assigned a D- or an L-designation with a high degree of reliability if one or the other 
of its optical isomers is susceptible to the action of a biological agent whose specificity 
toward amino acids of known configuration has been previously established. Toward 
this end, use has been made of the ability of certain molds, yeasts, bacteria, and even 
mammals, to more or less stereospecifically metabolize the Lsomer of a given amino 
acid at an appreciably faster rate than its corresponding D-antipode. Much greater 
use, however, has been made of the stereospecific oxidative and decarboxylative 
action of amino acid oxidases and decarboxylases, respectively, the hydrolytic action 
of acylases, amidases and carboxypeptidases, and the ability of certain proteases 
to catalyze peptide bond formation®’. Probably the most convenient, and one of the 
most reliable of the enzymatic methods for the determination of optical configuration 
entails the use of amino acid oxidases. An amino acid oxidase generally exhibits an 
antipodal specificity which, depending on its origin, may be either L-directed or D- 
directed. In any case, the reaction, which may be conveniently followed in a Warburg 
respirometer, essentially involves an oxidative deamination of one molecule of the 
susceptible amino acid antipode, with the consumption of one molecule of oxygen, 
to yield one molecule each of the corresponding a-keto acid, ammonia and hydrogen 
peroxide, as follows: 
References p. 22/24 
