Racemization amino acid analyses

Bender, M. L., Reliability of Amino Acid Racemization Dating and Paleotemperature Analysis on Bones, Nature, 1974, 252, 378-379. 

Amino acid duplications, in sulfonamide resistance, 23 505 Amino acid inhibitors, 13 300-302 Amino acid racemization dating, 5 752 Amino acid residues, 9 494 26 376 Amino acids, 2 554-618 20 447 achiral derivatizing agents, 6 96t analysis, 2 596-600 analysis in green coffee, 7 253t analysis in roasted, brewed, and instant coffee, 7 255t... 

Amino acid racemization (AAR) dating and analysis in lacustrine environments. Bonnie A. B. Blackwell... 

Amino Acids publishes contributions from all fields of amino acid and protein research analysis, separation, synthesis, biosynthesis, cross linking amino acids, racemization/enantiomers, modification of amino adds as phosphorylation, methylatlon, acetylation, glycosylation and nonenzy-matic glycosylation, new roles for amino acids in physiology and pathophysiology, biology, amino acid analogues and derivatives, polyamines, radiated amino adds, peptides, stable isotopes and Isotopes of amino acids. 

Figure 2 Analysis of eight amino acid racemates. Chromatography conditions column MCI GEL CRS10W, eluent O.Smmoir copper (II) sulfate, flow rate 1.0 ml min , detection 254 nm, peaks 1 o-Ala, 2 i-Ala, 3 o-Pro, 4 o-Val, 5 L-Pro, 6 L-Val, 7 o-Leu, 8 o-NIe, 9 D-Tyr, lOi-Leu, 11 o-Eth, 12i-Tyr, 13L-Nle, 14o-Phe, 15L-Eth, 16L-Phe. (Reproduced with permission from Weston A and Brown Ph (1997) HPLC and CE - Principles and Practice. Academic Press, p. 61 Eisevier.)...

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

Derivatization of a racemic compound with an achiral group may play an important role in the analysis of a chiral compound (Fig. 7-15). In the case of substances with low or no UV-activity, the compounds can be rendered detectable by introducing an UV-absorbing or fluorescent group. If the racemate itself shows selectivity on a chiral stationary phase (CSP), this method can be applied to reduce the limit of detection. Examples have been reported in the literature, especially for the derivatization of amino acids which are difficult to detect using UV detection. Different derivatization strategies can be applied (Fig. 7-16). 

J Kovacs. Racemization and coupling rates of N -protected amino acids and peptide active esters predictive potential, in The Peptides Analysis, Synthesis, Biology, Vol. 2, pp 485-539. Academic Press, New York, 1979. 

Derivatives of FLEC are formed without any racemization of the sample and the derivatives of the D and L forms of the test molecules are eluted from reverse-phase columns in sequence. FLEC derivatives are fluorescent, having an excitation maximum at 260 nm and an emission maximum at 315 nm and are particularly useful in the analysis of amino acids. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

The absolute configurations of amino acids 198a (the racemic counterpart was designated 186c) and 198i on the basis of X-ray crystal structure analysis data of the a-azido esters 197a-Me and 197i-Bn were (R) and (S,S,S), respectively. 

In Section 7.3, the subject of amino acid analysis is covered including a brief description of hydrolytic techniques, column preparations, and data analysis 7,8 This is followed by a discussion of racemization assays (Section 7.4). These areas of analysis are critically important for researchers in the field of peptide synthesis 9-11 In Section 7.4, a systematic approach is outlined for the study of racemization and mechanisms are discussed for epi-merizations at asymmetric sites. A comparative study is presented for determining the intrinsic rates of racemization, based upon urethane-protected V-carboxy an hydrides (UN-CAs). These approaches are extremely important for understanding the tendencies of novel amino acids and other building blocks to racemize. 

Related to this is the use of amino acid derived reagents for resolution of racemic carbonyl compounds and determination of absolute configurations by X-ray analysis at the imine stage. This is exemplified by the L-valinol derived imine of tricarboxyl (l-formyl-2-methoxyphenyl) chromium (see p 417)88. 

Not mentioned in Table 2 (and often not in the original papers ) is the optical form (chirality) of the amino acids used. All the amino acids, except for glycine (R = H), contain an asymmetric carbon atom (the C atom). In the majority of cases the optical form used, whether l, d or racemic dl, makes little difference to the stability constants, but there are some notable exceptions (vide infra). Examination of the data in Table 2 reveals (i) that the order of stability constants for the divalent transition metal ions follows the Irving-Williams series (ii) that for the divalent transition metal ions, with excess amino acid present at neutral pH, the predominant spedes is the neutral chelated M(aa)2 complex (iii) that the species formed reflect the stereochemical preferences of the metal ions, e.g. for Cu 1 a 2 1 complex readily forms but not a 3 1 ligand metal complex (see Volume 5, Chapter 53). Confirmation of the species proposed from analysis of potentiometric data and information on the mode of bonding in solution has involved the use of an impressive array of spectroscopic techniques, e.g. UV/visible, IR, ESR, NMR, CD and MCD (magnetic circular dichroism). 

The Pirkle-type chiral stationary phases are quite stable and exhibit good chiral selectivities to a wide range of solute types. These CSPs are also popular for the separation of many drug enantiomers and for amino acid analysis. Primarily, direct chiral resolution of racemic compounds were achieved on these CSPs. However, in some cases, prederivatization of racemic compounds with achiral reagents is required. The applications of these phases are discussed considering re-acidic, re-basic, and re-acidic-basic types of CSP. These CSPs have also been found effective for the chiral resolution on a preparative scale. Generally, the normal phase mode was used for the chiral resolution on these phases. However, with the development of new and more stable phases, the reversed phase mode became popular. 

Restricted rotation, as surface conjestion increases, was postulated as being responsible for the observed optical rotations as the generations increase,1251 the possibility of racemization during amino acid acylation was also investigated. Amino acid isolation and high-performance liquid chromatography (HPLC) analysis following acidic hydrolysis of the asymmetric dendrimers revealed an enantiomeric excess > 96 %. 

Another example in which biocatalysis is combined with analysis is the system reported by Honda et al. [436]. A microreaction system, consisting of an enzyme-immobilized microreactor, for optical resolution of racemic amino acids was devel-... 

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