Amino acid racemization method

The amino acid racemization method is based on the measurement of the degree of racemization acquired by a material expressed as the ratio dextro amino acid (D) enantiomeric form/levo amino acid (L) enantiomeric form, which is an indicator of the age of the object. This method has been applied in fossil shells, bones, teeth, wood, plant remains, and coral [68]. 

Amino acid racemization (Section 27.2) A method for dating archeological samples based on the rate at which the stereochemistry at the a carbon of amino acid components is randomized. It is useful for samples too old to be reliably dated by decay. 

FIGURE 88 Dating methods. Shortly after the discovery of radioactivity, at the beginning of the twentieth century, it was found that the decay of radioactive elements could be used to keep track of time. Many of the dating techniques developed since then are, therefore, based on radioactive decay phenomena, but others, such as the hydration of obsidian, amino acid racemization, and dendrochronology, are based on other physical, chemical, or biological phenomena. 

Rutter, N. W. and B. Blackwell (1995), Amino acid racemization dating, in Rutter, N. W. and N. R. Catto (eds.), Dating Methods for Quaternary Deposits, Vol. 2, Geological Association of Canada, Geotext, St. John s, Newfoundland, pp. 125-164. 

The enantioselective binding properties of certain chiral crown ethers have been employed in the resolution of amino acid racemates. The racemic amino ester is adsorbed onto silica gel as its ammonium salt and eluted by a chloroform solution of the chiral crown ether. An excellent separation of the two enantiomers is achieved by this method (74JA7100). 

Attempts to apply thermoluminescence (TL) and electron spin resonance (ESR) data to date soil horizons, and to use ESR to date bone are underway. As yet, there are no agreed-upon criteria on which to evaluate the overall reliability of age inferences based on ESR data (13). It has been suggested (14) that in some circumstances the obsidian hydration method can be used to infer chronological age over in excess of 100,000 years (14). However, obsidian is not a widespread natural resource and some of the hydration rate structures appear to yield problematic results. Amino acid racemization (AAR) values can be used under some conditions to infer accurate age values for bone, but seriously anomalous values can be obtained (15, 16). The conditions under which AAR values can be used to accurately infer age, particularly for bone samples, continue to be investigated. 

Amino acid racemization analyses on shell are carried out in two ways sampling all the amino acids extracted from the shell (called total) and sampling only the protein-bound amino acids (referred to as protein). The first method is essentially identical to the processing... 

With new coupling methods and/or reaction techniques, the extent of racemization has usually been determined u g one or more standardized evaluation methods. One of the earliest was due to Anderson where the condensation of Z-Gly-Phe with Gly-OEt was employed and specific rotation of the product measured. Also, racemate may be isolated by fractional crystallization. By this means down to 1-2% racemization may be detected. Next in 1963 appeared the Weygand (Z-Leu-Phe - - Val-OBu , partial hydrolysis and glc determination of D-Phe-Val, sensitive to 0.1-1% racemization) and Young (Bz-Leu - -Gly-OEt, specific rotation of crude material and isolation of racemate, can detect 1-2% racemization) procedures. In 1969 Izumiya s amino acid analyzer method was reported and details were available last year. With this tech-... 

In contrast to the above-mentioned amino acid resolution methods involving amino acid esters, -amides, or Af-acylamino acids where the natural L-enantiomer is preferably hydrolyzed from a racemic mixture, hydantoinases usually convert the opposite D-enantiomer [153-155], and L-hydantoinases are known to a lesser extent... 

The most dangerous contamination of the peptide fixed to the gel phase exists in the incorporation of the specific amino acid racemized to a small extent by the method of C-terminal activation. In several investigations it was demonstrated, however, that dicyclohexylcarbodiimid activation in dichloromethane of urethane-protected amino acids — with the exception of histidine — beyond room temperature level does not cause significant racemization [210], which is enhanced, however, in dimethylformamide as solvent. In cases of apprehended racemization, additives like N-hydroxysuccinimide [43] or - more recently explored - 1-hydroxy-benzotriazole [44, 139,140] are in use, which admixtured in two equivalents per one of the diimide, intermediately form the activated ester with a considerably diminished tendency to racemize. This technique will be described in Sect. 3.3.5, since it was shown to be a helpful method to activate even peptide fragments without racemization. 

Several methods are used for the synthesis of amino acids. A mqjor problem with any synthesis is preparing enantiopure products. Most of the syntheses shown here give racemic amino acids, but methods are known that produce amino acids highly enriched in one enantiomer (see Chapter 9). Enantioselective synthetic methods will not be discussed. A method used quite often to obtain an enantiopure amino acid prepares the racemic compound, followed by isolation of the 1-amino acid by resolution, as described in Chapter 9, Section 9.8. 

There are a number of chemical methods for the preparation of racemic p -amino acids. Selected methods are shown in Scheme 14.1, and include Michael addition to unsaturated carboxylic acids (a), homologation of a-amino acids (b), Thorpe reaction followed by reduction (c), nitrile reduction (d), and nitrile hydrolysis (e), among many others. Strategies for the stereoselective synthesis of p-amino acids are emerging, but there is not as yet a method that is generally appHcable [24, 25]. 

For resolution of amino acids, the method of ligand-exchange chromatography has received by far the most emphasis. The utility of this chromatographic technique in areas other than the resolution of race-mates has been reviewed (Davankov and Semechkin, 1977). Only results pertinent to the use of this chromatographic method in the resolution of racemates will be discussed here. 

Another important property of the protein amino acids is that they are all L-isomers, with the exception of glycine (glycine only has one configuration). The body cannot use the D-isomers of these amino acids, and, in fact, some o-isomers may be toxic. An exception is D-methionine, which the body can convert into L-methionine through transamination. Naturally produced amino acids are L-isomer however, manufactured amino acids can be a racemic mixture with half of the molecules being D-isomers. These can then be separated and only the L-isomer used to fortify foods. The amino acid analysis methods normally employed in the nutritional laboratory will not differentiate the d- and L-isomers. There are, however, chiral chromatography and enzymatic methods that can be used if the product is suspected of containing D-isomers. 

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