Racemisation, amino acids

In the following sections, common features of gas chromatographic procedures applied to proteinaceous material identification in paint are discussed such as sample pretreatments and data analysis. Finally, a section is devoted to the recognition of the amino acid racemisation in ancient proteins encountered mostly in archaeological contexts. 

L and the D/L ratio approaches zero. After the death of the living organism, proteins start to spontaneously break down. An inter-conversion of the amino acids occurs from one chiral form (L) to a mixture of D- and L- forms following protein degradation this process is called amino acid racemisation. The extent of racemisation is measured by the ratio of D/L isomers and increases as a function of time and temperature. The longer the racemisation process continues the closer to 1 the ratio between the D- and L-forms becomes. If the D/L ratio is <1 it may be possible to use it to estimate age. The D/L ratio of aspartic acid and isoleucine are the most widely used for this dating technique [104]. Dates have been obtained as old as 200 000 years. However, it has been used mainly to date samples in the 5000 100 000 year range. Recent studies [ 105] mention an estimation of the method accuracy to be around 20%. 

Considering all the above factors for amino acid racemisation rates, this technique cannot be used as an absolute dating technique. To overcome the problem of uncertainty, the amino acid racemisation method must be calibrated against other more reliable techniques. 

In paintings, amino acid racemisation depends upon the presence of albuminous binders, such as blood or egg white. The ability to apply this technique is based on the observation that the number of amino acids present in proteins decreases over time. Decay curves are constructed of paint samples of known age. Moreover, amino acid decay rates are also dependant upon micro-organisms (whose enzymes can digest various types of proteins) and environmental conditions [114]. 

Bada, J.L. and Shou, M-Y. (1980). Kinetics and mechanism of amino acid racemisation in aqueous solution and in bones. In Biogeochemistry of Amino Acids, ed. Hare P.E., Hoering T.C. and King K. Jr, John Wiley, New York, pp. 235-255. 

Masters, P.M. (1986a). Amino acid racemisation dating - a review. In Dating and Age Determination of Biological Materials, ed. Zimmerman M.R. and Angel J.L., Croom Helm, London, pp. 39-58. 

Chemically synthesised D,L-hydantoins prepared from the corresponding aldehydes via die Bucherer Berg reaction are converted by the bacterial cells (Bacillus brevis), containing a D-spedfic hydantoinase, to a mixture of D-N-carbamoyl amino acid and L-hydantoin. The latter compound undergoes rapid and spontaneous racemisation under the conditions of the reaction, therefore, in principle 100% of the hydantoin is converted into the D-N-carbamoyl compound. The D-amino add is obtained after treatment of the D-N-carbamoyl compound with nitrous add. This process is operated on an industrial scale by the Japanese firm Kanegafuchi. 

The processes occurring at hydrothermal systems in prebiotic periods were without doubt highly complex, as was the chemistry of such systems this is due to the different gradients, for example, of pH or temperature, present near hydrothermal vents. Studies of the behaviour of amino acids under simulated hydrothermal conditions showed that d- and L-alanine molecules were racemised at different rates the process was clearly concentration-dependent. L-Alanine showed a low enantiomeric excess (ee) over D-alanine at increasing alanine concentrations. The same effect was observed with metal ions such as Zn2+ in the amino acid solution. Thus, homochi-ral enrichment of biomolecules in the primeval ocean could have resulted under the conditions present in hydrothermal systems (Nemoto et al., 2005). 

Possible racemisation of imines, derivatives of amino acids and R(—)-myrtenal, has been examined by Dufrasne et al.1 After 72 h, no significant effect on chiral purity was observed. For imines being derivatives of chiral primary amines and the a-substituted 8-keto-aldehydes, no evidence of epimerisation has been indicated by the NMR measurements.3 For a series of imines, being derivatives of amino acids or amino acid esters and (R)-BINOL reagents, Chin et al.5 have tested the possibility of epimerization under experiment conditions. It was shown that R S ratio has changed only slightly, and after 24 h, the difference was lower than 10%. 

Racemisation is a chemical reaction, and its rate is different for each type of amino acid. An important fact is that this process is affected by many factors that influence the rate of change of the amino acids stereochemistry [106]. The main parameters affecting the racemisation process include the amino acid structure, the sequence of amino acids in peptides, the bound state versus the free state of the amino acids, the pH in the environment, the concentration of buffer compounds, the contact of the sample with clay surfaces... 

Fig. 3 Important 19F-labelled amino acids, (a) Compounds that are wo-steric to native amino acids can be incorporated into proteins biosynthetically, but they possess too many degrees of torsional freedom to be useful for ssNMR structure analysis, (b) In these artificial amino acids the 19F-reporter group is rigidly attached to the peptide backbone. They can be incorporated by solid-phase peptide synthesis, but some problems can arise due to racemisation (4F-Phg, 4CF3-Phg), steric hindrance of coupling (F3-Aib) or HF elimination (fluoro-Ala, F3-Ala). 4F-Phg is additionally problematic due to an ambiguity of the side-chain rotamer. The preferred 19F-labels for ssNMR structure analysis are CF3-Bpg and CF3-Phg (as suitable substitutes for Leu, lie, Met, Val and Ala), as well as F3-Aib and CF3-MePro...

The discovery of homochirality on a planet such as Mars could be an excellent biomarker and strengthen the argument for life on Mars. With an EE in the solar nebula there should be an EE on the surface of Mars of order 9 per cent but remains of ancient life on Mars would show a greater excess. The interchange of enantiomers occurs naturally in a process called racemisation and for the most labile amino acid, aspartic acid, the half-life for the racemisation is 800 years at 300 K in 800 years, half of the non-biotic aspartic acid would racemise and the EE would go to zero. In dry conditions, however, the half-life is much longer, perhaps as large as 5 x 104 years at 300 K. Extrapolation of the racemisation rate to 215 K, the equatorial temperature of Mars, extends the half-life further to 3 x 1012 years and to 1027 years at 150 K, Martian polar temperatures. Hence, discovery of a considerable EE in the Martian soil would be a strong indicator of ancient Martian life. 

I Antanovics, GT Young. Amino-acids and peptides. Part XXV. The mechanism of the base-catalysed racemisation of the p-nitrophenyl esters of acylpeptides. J Chem Soc C 595, 1967. 

F Weygand, A Prox, W Konig. Racemisation of the second last carboxyl-containing amino acid in peptide synthesis. Chem Ber 99, 1446, 1966. 

E Atherton, NL Benoiton, E Brown, RC Sheppard, B Williams. Racemisation of activated, urethane-protected amino-acids by jp-dimethylaminopyridine. Significance in solid-phase synthesis. J Chem Soc Chem Commun 336, 1981. 

JS Davies, AK Mohammed. Assessment of racemisation in A-alkylated amino-acid derivatives during peptide coupling in a model dipeptide system. J Chem Soc Perkin Trans 1 2982, 1981. 

I 2 Chemoenzymatic Routes to Enantiomerically Pure Amino Acids and Amines racemisation... 

Leucine.—The greater part of the leucine is contained in the ester fractions, which boil between 70° and 90° C. It generally occurs in considerable amounts in the protein, and is obtained by crystallisation from water, in which it is less soluble than the other amino acids which may be present. It is seldom present in its pure, optically active form, as this is easily racemised, and the various crops of crystals most probably also contain isoleucine. It is more easily isolated by completely racemis-ing the mixture of amino acids contained in this fraction by heating in an autoclave with baryta to 160-180° C., and then, after removal of the baryta, separating it by crystallisation. The difficulty of separating it from the other amino acids, especially valine and isoleucine, makes an exact quantitative estimation almost impossible. The values which have been found are therefore minimal ones, and they will also include in many cases the yield of isoleucine. 

Figure 2.6 By resolution of df-amino acid esters under conditions of dynamic resolution 100% of a single enantiomer may be produced. Using catalytic amounts of pyiidoxyl-5-phosphate, which forms a Schiff s base with the ester and not the acid, the unreacted D-ester may be racemised in situ and for instance L-tyrosin has been obtained in 97% ee and 95% yield.

Enzyme catalysed racemisation is an attractive method. The enzymes are known as racemases and they often need cofactois like pyrodoxyl phosphate (PEP) or bivalent metal ions to function properly. The snbstrates used in racemisation reactions have two features in common, i) the stereocentre carries a proton, ii) adjacent to the stereocentre is a carbonyl gronp or another function that make the proton at the stereocentre acidic. Most racemases work on amino acids and df-hydroxy acids. The principle of those needing PEP is formation of a Schiffs base between the aldehyde of PEP and the amino group of the amino acid (like in Figure 2.6). 

Chen, S.-T., Huang, W.-H. and Wang, K.-T. (1994) Resolution of amino acids in a mixture of 2-Methyl-2-propanol/water (19 1) catalysed by alcalase via in situ racemisation of one antipode mediated by pyridoxal 5-phosphate. J. Org. Chem., 59, 7580-7581. 

Differences of <5 = 0.32-0.40 in the, 9F chemical shift for epimeric A -acylated Mosher derivatives of aliphatic a-amino acids, in particular alanine, as well as its amides and peptide derivatives, are sufficiently large to serve as a racemisation test23. However, high-field spectrometers are recommended to provide improved sensitivity (<0.5%). Table 19, representing analysis data taken at 376.5 MHz, can be found in Section 3.2.2.9. Analysis of absolute configuration of amino acids and amines can be more accurate using a modified Mosher method24. 

Kroepelin, H., "Racemisation of Amino Acids on Silicates, in "Advances... 

This is a very simple and short method for the deprotection of N-Cbz and N-Bn groups, which is also applicable for N-Cbz protected amino acids and is compatible with Fmoc protecting groups, which remain unaffected under these conditions. Furthermore, the microwave protocol is fully compatible with enantiomerically pure amino acids and peptides, as no racemisation was observed in the resulting free amines. 

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