Heated Debate Which Amino Acid Enantiomer Precipitates First

It is important to widen our view and to understand that not only minerals such as quartz and molecules such as 1,1'-binaphthyl can crystallize in chiral space groups. Amino acids themselves - white powders at room temperature and ambient pressure - can precipitate from aqueous solutions forming crystals. This precipitation occurs with nucleation and autocatalytic crystal growth processes having the potential to show noticeable enantioenriching properties (see Thiemann 1974).

As we have seen above, an equimolar mixture of two enantiomers in solution may crystallize either as a racemic compound with both enantiomers in equal quantities embedded in the crystal lattice or - less commonly - a spontaneous resolution separates each enantiomer in different enantiomeric crystals as in the case of Pasteur's tartaric acid precipitation followed by manual separation (see formation way number 3). Most amino acids, including aspartic and glutamic acid, belong to the most frequently observed first type and do not show spontaneous resolution by crystallization. Crystals of racemic amino acids are usually obtained as 50% d-enantiomers and 50% l-enantiomers.

In 2001, Cristobal Viedma (2001) from the University of Madrid, Spain, showed how the amino acids D,L-aspartic and D,L-glutamic acid as racemic compounds in aqueous solution crystallized at room temperature as distinguishable enantiomers with each enantiomeric molecule separate in its own enantiomorph crystal. In order to obtain such results, the author had added heterogeneous porous materials such as refractory porous bricks and porous paper sheets to influence the crystallization process allowing capillary action, concentration by evaporization and crystallization of the amino acids in a narrow upper zone of the porous materials. X-ray diffraction patterns were used for enantiomer identification verifying the spontaneous resolution of the enantiomers by this kind of crystallization. Viedma explained this result by assuming that the applied crystallization on a porous medium gives rise to high supersaturation levels. These levels might enable the system to go far from thermo-dynamic equilibrium where kinetic criteria dominate over thermodynamic criteria, and the formation of metastable and "anomalous" phases becomes possible. According to Viedma, this atypical behaviour might also be found in natural sedimentary environments and was thus proposed to be associated with the origin of life and its homochirality phenomenon. This is indeed an attractive model and we will wait for future experiments that will have to verify systematically whether other amino acids and chiral biomolecules such as sugars will show similar trends in atypical crystallization behaviour on porous media. Future research will also have to tackle the poorly understood crystallization behaviour in general: there are no firm rules to predict whether a particular pair of chiral partners will follow the behaviour of the vast majority of chiral molecules and crystallize together as racemic crystals, or as separate enantiomers (Fasel et al. 2006).

Since 2002, the research activities of Meir Shinitzky at the Weizmann Institute of Science in Rehovot, Israel, attracted tremendous attention in the field of the crystallization of an amino acid combined with the spontaneous appearance of an enan-tiomeric excess.8 Shinitzky et al. (2002) studied solubilities of D- and L-tyrosine in water applying several analytical techniques such as UV-absorption, radioactive labelling & tracing, and optical rotation, which were specifically adapted to the system. Surprisingly, the authors measured unexpected differences in the solubilities of

8 In this context, the spontaneous appearance of an enantiomeric excess is not necessarily the same thing as 'chiral symmetry breaking'. The situation can be compared with the non-stirred vs. stirred crystallization of NaClO3 in which in both cases a non-zero e.e. can be obtained, but only the stirred one represents chiral symmetry breaking. More deeply reflections on these semantics can be found in Avalos et al. (2004).

Fig. 4.5 Precipitation of d- and l-tyrosine enantiomers from 10 mM aqueous solution taken at 0, 6, and 24 h (Shinitzky et al. 2002). Illustration used with permission of the authors

the tyrosine enantiomers in water by all of these techniques. D-Tyrosine crystallized faster than L-tyrosine! The liquid phase above the precipitate became enriched in L-tyrosine! This effect is illustrated in Fig. 4.5.

Alain Schwartz from the University of Nijmegen, the editor of the journal 'Origins of Life and Evolution of Biospheres' in which these data were first printed, was equally surprised by these results and published a referee's comment simultaneously with the Shinitzky paper saying, "... in spite of the real prospect that future work will show this chiral discrimination to be an experimental artefact, I strongly encourage publication so that others may confirm, extend and amplify these results" (Schwartz 2002). And indeed, Wolfram Thiemann, Ute Jarzak, and I repeated the Shinitzky-precipitation experiment at the University of Bremen with various tyro-sine sources confirming the higher crystallization rate for D-tyrosine.9

The different crystallization rates of the tyrosine enantiomers disappeared in D2O. For the authors, it seemed possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules (see Chap. 5), could account for this observation. Shinitzky et al.

9 Shinitzky's original finding was further supported by radioactive tritium-labeling of tyrosine and post-crystallization radioactive tracing of 3[H]-l-tyrosine in the supernatant. The repetition of these experiments in the isotope laboratory at the University of Bremen did, however, not confirm the asymmetric counting rates obtained by Shinitzky et al. (2002).

concluded that the obtained results seem to violate the generally accepted axiom that chiral isomers are identical in their bulk energetic quantities.

Spectacular findings like this, however, require particularly robust and sophisticated data. Parity non-conserving energy differences between amino acid enan-tiomers had been calculated to be as small as 10~14 to 10~17 kT (see Chap. 5). Despite thorough research at the international level, an experimental proof of these energy differences has not yet been achieved. Shinitzky et al. (2002) investigated the amino acids D-tyrosine, L-tyrosine, and D,L-tyrosine from Fluka with >99% purity recrystallized in double distilled water. Subsequent to recrystallization, the purity of the amino acids was not controlled. Decomposition reactions of the amino acids were checked by thin layer chromatography only. Modern analytical techniques like enantioselective chromatography were not applied in order to detect and quantify possible contaminants.

In general, three fundamentally different routes (1-3) are used for the production of enantiomerically pure amino acids (see review of Breuer et al. 2004). 1) In extractive processes, amino acids are isolated as components of natural protein-containing material. 2) The alternative chemical method uses the Bucherer-Bergs variant of the Strecker-mechanism to produce racemic amino acids, followed by conversion into enantiomerically pure compounds by a number of biocatalytic methods. Finally, 3) biological methods such as biotransformations and fermentation methods allow the enantioselective synthesis of amino acids. We, therefore, have to assume that the supplier Fluka synthesized the different tyrosine enantiomers used in the Shinitzky precipitation experiment in different ways. L-Tyrosine can be isolated from biological protein-rich material, but also by one of the other ways, whereas D-tyrosine requires one of the above-mentioned chemical or biological synthesis pathways. As a consequence, the remaining pollutants - about 1% for each amino acid - are probably different in all products. These might be amino acids but also other organic and inorganic substances. As we know in physical chemistry, the growth of crystals (in this case, amino acids) and their surface properties are often dominated by impurities. Therefore, I assume, that the measured differences in the solubilities of the tyrosine enantiomers were caused by impurities (not necessarily amino acids!). Minute amounts of these impurities even in sub-trace abundances may have contaminated the L-tyrosine samples and worked as solubilizers.

In 2006, Scolnik et al., including Shinitzky, published that - indeed! - experimental tests of the hypothesis on parity non-conserving energy differences, where one enantiomer is compared to the other in solution, are hampered by the possible presence of "undetectable impurities" (Scolnik et al. 2006). Here, the authors correctly address the highly possible effect of impurities on their own previous experiment. Only the word "undetectable" might be incorrect. To my knowledge, however, the authors did not reject the above-mentioned paper published in the well-known Kluwer journal "Origins of Life and Evolution of Biospheres", which might have been a straightforward consequence.

Instead, Shinitzky et al. continue publishing (Scolnik et al. 2006) that they have now overcome the problem of possible impurities by measuring specific properties of synthetic polypeptides. Differences in structural transitions between poly-L- and poly-D-amino acids were reported. Surprisingly, the reader is informed that all of the four investigated polymers - each composed of 24 amino acids - show "purities >98%" only. This means, that out of 100 amino acids, two might be wrong. Consequently, in 4 polymers, two "wrong" amino acids might be implemented. In other words: two out of four polymers might be "wrong" (i.e. 50%!). Is it possible that these wrong polymers cause effects, which Scolnik et al. (2006) hurriedly interpret to possibly originate from parity non-conserving energy differences?

Recently, Meir Lahav who was employed at the same time as Meir Shinitzky by the world-famous Weizmann Institute of Science - ironically door to door working -challenged the original Shinitzky paper (Shinitzky et al. 2002) by reinvestigating the above mentioned tyrosine crystallization (Lahav et al. 2006) with D-Tyr, L-Tyr, and D,L-Tyr from different sources including samples directly from Shinitzky. Lahav et al. noted that samples provided by Shinitzky indeed displayed the effect he reported in his article, however, their results could not be reproduced with samples obtained from other sources. In contrast to the original work of Shinitzky et al., Lahav et al. focused on impurities in different samples of tyrosine enantiomers applying enantioselective chromatography in the well-known analytical lab of Volker Schurig at the University of Tübingen. Here, numerous other amino acids were identified in the tyrosine-samples and quantified as contaminants. The authors concluded that these contaminants - which were measured in quantities up to a few percent -caused the abnormal differential crystallization behaviour of the individual tyrosine enantiomers instead of any parity non-conserving energy difference.

Meir Shinitzky and his team replied to Lahav's criticism by insisting on the observation of different solubilities between D- and L-tyrosine. The new argument was that synthetic D,L-tyrosine was proven to show chiral enhancement in its supersaturated aqueous solutions. The precipitate and the supernatant were separated and their specific rotation indicated a highly significant chiral enhancement of approximately 60% L-tyrosine versus approximately 40% D-tyrosine in the supernatant (Deamer et al. 2007).

Most recently, Stanley Goldberg from the University of New Orleans entered into this scientific debate exposing another justification: he proved experimentally that a microbial spore which is often present in laboratory air is capable provoking different rates of the tyrosine enantiomers' crystallisation (Goldberg 2008).

It is not up to me to be the final judge of this scientific conflict. I would rather like to encourage experimentalists to study the chosen conditions of the opponents in more detail and to contribute to the solution of this divergence by supplying their own experimental data.

Very recently, Donna Blackmond and her colleagues at Imperial College London proposed a new approach in which crystallization could have played a part in selecting L-amino acids for proteins in living organisms. This approach is based on the equilibrium phase behaviour of amino acids (Klussmann et al. 2006). For some amino acids, such as serine, a tiny excess of one enantiomer can lead to that enan-tiomer's preferential precipitation in crystals, leaving the solution highly enriched in the other enantiomer. Blackmond et al. demonstrated that this enantiomer enrichment can be engineered for amino acids that don't display this behaviour by themselves. Small molecules such as achiral dicarboxylic acids added to the racemic mixture of amino acids cocrystallized and became thus incorporated into the amino acid's crystal. This cocrystallization promoted the extraction of one form of the amino acid from the solution (Klussmann et al. 2007).

A common characteristic of all these models is, however, that the resolution of enantiomers by spontaneous crystallization cannot be predicted at present (Koshima 2004). Furthermore, it is still a long way from optically active sodium chlorate or 1,1'-binaphthyl crystals to the distinct chirality of amino acids and other biomolecules. We will thus try to better understand the adsorption of organic molecules onto the surface of chiral crystals in the next paragraphs. Before doing so, we will have a closer look at the intriguing possibility that the direction of stirring might induce an enantiomeric enhancement in an appropriate chemical system.

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