Biochemistry of Amino Acids - A. Majster 1961

Naturally Occurring Amino Acids
Production of Optical Isomers of Amino Acids

Amino Acids can be obtained from natural sources or prepared via chemical synthesis. The former approach typically yields the L-isomers, whereas amino acids produced by chemical synthesis (with the exception of Glycine, ß-Alanine, etc.) exist as racemates. The isolation Procedures for Amino acids are diverse, and a vast literature is dedicated to this subject. Certain Proteins serve as excellent raw Materials for specific amino acids: wheat gluten is the primary source for the commercial production of L-glutamic acid, corn gluten is a valuable source for isolating L-leucine and L-Tyrosine, and L-Arginine can be extracted from gelatin and Blood. Commercial preparations of L-asparagine are commonly obtained from asparagus shoots (cf. [14]).

A variety of methodological approaches are employed for the isolation of amino acids, including isoelectric precipitation, isolation as salts, esters, hydrochlorides, sulfonic acid salts, electrodialysis, and ionophoresis. Particularly widespread are the precipitation Methods used for arginine as a flavianate, glutamic acid as a hydrochloride, aspartic acid as a copper salt trihydrate, and Methionine as a mercury complex [116, 494–496].

There is no doubt that highly purified L-amino acids can be obtained from protein hydrolysates. Nevertheless, commercial amino acid preparations are frequently contaminated with Other Amino Acids, which can be a source of experimental error. Consequently, when preparing media for microbiological amino acid assays, researchers often prefer synthetic DL-amino acids over L-isomers isolated from protein hydrolysates. Examples of common contaminants include methionine in protein-derived preparations of leucine and glutamic acid, arginine and asparagine in glutamine preparations, tyrosine in Tryptophan preparations, and cystine in tyrosine preparations. Isoleucine isolated from hydrolysates typically contains leucine, and vice versa. The Development of modern chromatographic techniques has greatly simplified the isolation of amino acids, and the widespread adoption of these methods will undoubtedly improve the quality of commercial amino acid preparations.

Currently, the resolution of synthetic amino acid racemates is one of the primary methods for obtaining L-isomers and, especially, D-isomers. The wide variety of published synthesis methods attests to the remarkable ingenuity of organic chemists. A detailed Structure/133.html">Discussion of these techniques is beyond The Scope of this book, but comprehensive reviews on the subject are available [116, 497].

Several methods have been developed to obtain optical isomers of amino acids from their corresponding racemates. Schulze and Bosshard, utilizing the mold Penicillium glaucum, obtained certain D-isomers through the selective destruction of the corresponding L-isomers (p. 80). Later, the selective action of microorganisms on the L-isomers of corresponding racemates was employed to produce other D-amino acids (alanine [498], valine [499], glutamic acid [498], isoleucine [498], Histidine [498], tyrosine [499], phenylalanine [500], and Serine [500]). Administering racemates of Certain amino acids to animals occasionally results in The excretion of D-isomers in the urine [501]; D-histidine was obtained in this manner [502–503]. Somewhat later, Enzymes began to be applied for the same purpose. D-Arginine was prepared by treating DL-arginine with a crude arginase preparation [504]. Specific D- and L-amino acid oxidases were utilized to obtain the D-isomers of leucine, methionine, and phenylalanine [505], as well as the L-isomers of alanine [506, 507], methionine [507], and Proline [508]. D-Lysine [509] and D-glutamic acid [510] were prepared by treating racemates with bacterial Decarboxylases specific to the L-isomers.

Mechanical Separation of isomer crystals has very limited application. The pioneering work of Pasteur on the selective action of Penicillium glaucum and the differential solubility of diastereomeric mixtures ultimately led to The Use of Alkaloids and enzymes for resolving DL-amino acids into optically active forms. Isotope-labeled amino acid isomers have been prepared by diluting a small amount of a radiolabeled racemate with a large excess of the desired optically active form, followed by fractional recrystallization [511, 512].

Fischer [513] was the first to apply THE PRINCIPLE OF diastereomerism to resolve amino acids: an optically active alkaloid (brucine, strychnine, cinchonine, quinidine, or quinine) was added to the N-acyl derivative of a racemic amino acid, yielding two diastereomeric salts with differing solubilities. After separating these diastereomers, the alkaloid was regenerated, and the acyl group was removed by Hydrolysis. Fischer and his coworkers [513–515], and later other researchers [516], successfully resolved numerous DL-amino acids using this method. In some cases, this approach can be applied directly to free amino acids without prior acylation and subsequent Cleavage of the acyl group. However, all these methods, much like Fischer's original Procedure, suffer from several drawbacks, most notably their highly empirical nature.

At present, the most suitable and convenient Methods for Amino acid resolution are those based on stereospecific enzymes. Bergmann and his coworkers [517–520] discovered that in the presence of N-carbobenzoxy-DL-Amino Acids and aniline, Papain catalyzes the Synthesis of N-carbobenzoxy-L-amino acid anilides at a significantly faster rate than the corresponding D-anilides. The L-anilides precipitate first, enabling the separation of the isomers. Subsequently, this general approach for obtaining amino acid isomers was adopted by many researchers using various acyl derivatives and enzymes (cf. [521, 522]). Although this method yields pure amino acid isomers, it is not without limitations. In particular, the enzyme may synthesize anilides of both L- and D-isomers. The propensity to form D-anilides depends on The structure of The amino acid, The Nature of the acyl group, and other reaction conditions. Because the L-anilides generally form faster than the D-forms, arresting the reaction at the precise moment is critical. If the reaction is terminated too early, the D-isomer will be contaminated with the L-isomer; conversely, exceeding the optimal reaction time leads to the opposite problem. Nonetheless, by collecting precipitates at regular intervals throughout the reaction, fractions consisting of pure isomers can be obtained.

Numerous investigators have utilized the asymmetric hydrolysis of amino acid esters to resolve their optical isomers. In 1906, Warburg demonstrated that pancreatin (free of lipase) hydrolyzes DL-leucine ethyl ester asymmetrically, successfully isolating pure L-leucine from the reaction mixture [523]. A number of racemic amino acid esters have since been resolved using crude pancreatic preparations and crystalline Chymotrypsin [524–528]. The utility of this method is limited by the tendency of Certain amino acid esters to undergo spontaneous hydrolysis or polymerization during enzymatic Treatment, yielding polypeptide esters [529].

Today, Enzymatic hydrolysis of acylated amino acids is widely regarded as the most valuable general method for resolving amino acid stereoisomers. Although the stereospecificity of certain hydrolytic enzymes toward acylated amino acids has been known for some time [530, 531], practical procedures utilizing enzymes to prepare D- and L-amino acid isomers were developed only recently [532, 533]. The general enzymatic resolution of racemic amino acids developed by Greenstein and co-workers [534–536, 585] relies on the observation that renal acylase I and carboxypeptidase react exclusively with the L-isomer when acting upon racemic N-acylamino acids. The reaction goes to completion after the hydrolysis of 50% of the racemic product. Separating the resulting free L-amino acid from the acyl-D-amino acid requires only a single step, as the free L-amino acid is insoluble in nonpolar organic Solvents in which the acyl-D-amino acid remains soluble. A similar method utilizing purified amidase is employed for isolating certain amino acids (proline, tert-leucine, and α,ε-diaminopimelic acid). The separation of the acyl-D-amino acid (or D-amino acid amide) from the L-amino acid can also be achieved via Ion-exchange Chromatography [536]. Typically, acetyl and chloroacetyl derivatives of amino acids are employed; acid-catalyzed hydrolysis of the resulting acyl-D-amino acids upon boiling does not cause racemization. It is also noteworthy that acyl derivatives of L-aspartic acid are hydrolyzed by a specific acylase II, which is similarly sourced from Kidney tissue.

It has been established that under certain conditions, acylase I acts upon acetyl- and chloroacetyl-D-methionine, although The ratio of the hydrolysis rates for the L- and D-methionine acyl derivatives is on the order of 40,000. When trifluoroacetyl Amino Acid Derivatives are used, significant hydrolysis of certain D-isomers occurs. Consequently, this acyl group is unsuitable for isomer resolution via acylase I; however, trifluoroacetyl derivatives of aromatic DL-amino acids are asymmetrically hydrolyzed by carboxypeptidase from pancreatic juice [537, 538].

Several observations indicate the theoretical feasibility of chromatographic methods for resolving amino acid isomers. Under specific conditions, chromatograms of kynurenine [539, 540], 2,5-dihydroxyphenylalanine [540], 2,3-dihydroxyphenylalanine [540], 3,4-dihydroxy-2-methylphenylalanine [540], phenylglycine [541], and 3-sulfonyltyrosine [542] reveal two distinct spots. In experiments with the latter compound, the spots were eluted separately and shown to correspond to the two optical isomers [542]. Similarly, the two spots obtained upon chromatography of racemic kynurenine corresponded to the D- and L-isomers [539, 540]. It has recently been discovered that in a methanol–Water–pyridine solvent system, the DD- and LL-isomers of α,ε-diaminopimelic acid migrate at different rates on chromatograms, whereas the meso- and DD-forms exhibit identical mobility [543]. There are also reports on the resolution of optical isomers of DL-histidine monohydrochloride monohydrate [590] and DL-tryptophan [591] via paper chromatography. Furthermore, literature References [585] describe the spontaneous crystallization of one isomer from a solution of its racemate. While this technique is not yet widely used for amino acid resolution, future research into the underlying mechanisms may lead to a highly useful separation method.

Investigations into the optical configuration of amino acids span various directions; some are covered in this chapter, while others will be discussed in subsequent chapters dedicated to Amino acid METABOLISM and Nutrition. The Need for optically pure amino acid isomers in experimental research is self-evident. Although specific optical rotation measurements are useful for characterizing amino acid isomers, this technique generally lacks the high sensitivity characteristic of enzymatic methods. The purity of many amino acid isomers can be verified using stereospecific enzymes, such as D-amino acid oxidases (p. 184), L-amino acid oxidases (p. 187), or bacterial decarboxylases specific to L-isomers (p. 204). These methods make it possible to detect less than one molecule of an amino acid isomer in the presence of 1,000 or even 10,000 molecules of its antipode [544]. With further refinement, even higher sensitivities can likely be achieved.

Many chemical and Physical Properties of the D- and L-isomers of a given amino acid are identical; for example, they share the same solubility in optically inactive solvents, ultraviolet and infrared absorption spectra, melting or decomposition points, and reactivity toward optically inactive Reagents. Amino acid isomers can be distinguished by their optical rotation, reactions with specific optically active substances, behavior toward enzymatic action, and occasionally by chromatographic techniques. It has been demonstrated that the optical isomers of amino acids often differ significantly in taste (Table 8). Similar observations were made many years ago by Pasteur and other investigators, yet a complete understanding of this phenomenon remains elusive to this day.

Class="center">Table 8 Relationship between optical configuration and taste properties of amino acids *

* According to data from [545] and the author.

Although observations of this kind are subjective, it is nevertheless intriguing that D-isomers predominantly taste sweet, whereas the corresponding L-forms are generally described as tasteless or even bitter. L-Glutamic acid possesses a characteristic savory ("meat-like") taste and is widely used as a flavor enhancer.