This article is from
Journal of Creation 37(3):84–89, December 2023

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Enantiomeric amplification of L-amino acids: part 5—sublimation based on serine octamers

by Royal Truman and Stephen Grocott

This is part 5 of a series dealing with proposals on how a small enantiomer excess of L (e.e._L) amino acid (AA) might have been amplified in a putative prebiotic earth. Here We’ll discuss some experiments involving serine octamers, which are often referred to in the evolutionary literature.

Sublimation of serine at high temperature using zone refining

Klussmann et al. showed that some AAs can undergo enantiomeric amplification due to their equilibrium behaviour at the solid–liquid phase, with serine showing the largest effect.¹ This topic will be discussed in Part 8. Serine is unusual among the smaller AAs in that it has four functional groups able to form hydrogen bonds. This can lead to a much higher concentration of clusters upon sublimation, involving 2–8 serine molecules.² Serine octamer ions can form, and there is also evidence that neutral serine octamers (Ser₈) form, but such clustering does not occur for the other DNA-coding amino acids, except to a very small extent by threonine and proline.³

Following up on these insights, Perry et al. decided to see if an initial serine e.e. could be amplified, using purification by an established method for solids called ‘zone refining’.³ The experimental setup is shown in figure 1. About 100 mg of serine D, L mixture with a predetermined e.e._L was prepared and placed 3 cm from the end of a 22-cm Pyrex glass tube having an inner diameter of 16 mm. Enough acetone was added to make a paste, and a spatula was used to spread it along 11 mm of the inner surface while rotating the tube. The end coated with Ser was attached to two Vigreux columns, each 13 cm long, and a glass tube 11 cm long was attached to the other end. The Vigreux columns part of the end glass tube was surrounded by a Styrofoam container full of dry ice. N₂ gas was pumped continually at 45 cm³/mins from the coated end through the apparatus.

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A heating tape about twice the width of the coated region was placed over it and then the temperature was slowly increased from room temperature to the experimental setting and held constant.

Degradation thermolysis products, including D,L-alanine, dehydroserine, and ethanolamine were obtained in high concentration (see figure 2).

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Alanine and ethanolamine made up 18% and 34% of the sublimate when heating 205°C after only 2 hours (h), and with increasing temperature this proportion increased as shown by the red line in figure 3.³ Concurrently, the e.e._L of the sublimate serine decreased with temperature; see the blue line.

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Figure 3 shows that at 205°C, where the highest e.e._L was obtained, about 60% of the serine was destroyed after only 2 h of heating. Importantly, heat caused rapid loss of L-enantiomer excess in the residue. In the words of Perry et al.:³

“The effect of racemization is even more clearly observed in the residue at higher e.e. values of L-Ser. When a 98% e.e. L-Ser sample is heated for 2 h at temperatures ranging from 175–250°C in 15°C increments, the chiral purity of the residue decreases to 79% at 205°C and to 45% at 250°C.”

To emphasize, the experiment revealed a drop from e.e._L = 98% to 79% in only 2 h.

Another experiment revealed the same principle. When the residue of an initial 3% e.e. L-Ser sample was heated during the interval of 3–4 h the ~1 % sublimate produced had an e.e._L of 68%. However, the sublimate obtained during the heating period between 4–6 h dropped to 53%. This was attributed to racemization in the residue during the heating period.

The dramatic decrease in e.e._L with time in the heated region could not fail but have an effect on the e.e._L of the sublimate. In the temperature range of 190–210°C (which produced the highest amplification results), starting with 99% e.e._L serine led to a sublimate with an e.e._L of 74% after only 2 h of heating. This decrease was attributed to racemization via the thermolysis product dehydroserine.³

The rapid loss of e.e. explains why the N₂ flow was necessary: to decrease the time the sublimate would be exposed to high temperatures. These temperatures both racemized the serine and decomposed it.

A variant set of experiments confirmed the dependency of e.e. on temperature. A syringe pump was used to push the heating tape along the tube for ~18 h. Each experiment started with 5% e.e._L serine, and figure 4 shows that the highest e.e. found (~65%) occurred at 205°C, with significantly lower values at slightly higher and lower temperatures.

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The authors believe that enantioselective sublimation of serine occurred by forming homochiral octamers, a phenomenon displayed almost only by serine among the biological AAs. For the next best alternative, solid threonine with an initial 7% e.e._L at 208°C produced a sublimate having the much lower e.e._L of 1.2%.³

Critique of these studies

The experimental conditions are not expected to exist naturally in a theoretical prebiotic setting. Perry et al. claimed:³

“The combination of chiral enrichment and physical separation (transport of the purified enantiomer), if it occurs repeatedly in a region with a modest temperature gradient, can readily be imagined to be a source of chirally pure serine.”

To be ‘readily imagined’ is no substitute for plausibility. A surface would have had to be densely coated with serine and with an excess of L-enantiomer. This would later be exposed to random blasts of heat at sublimating temperatures, like from a volcano. This does not produce an ideal smooth gradient, but would expose the serine to long and varying temperature fluctuations. Definitely not for only a few hours.

Here are some points to consider.

• The enantiomeric enrichment applied only to the AA serine. At the high temperature needed, all other AAs nearby would have racemized rapidly, especially if even a small amount of moisture was present.⁴
• The zone refining experiments were optimized after much preceding experimentation. To illustrate
• Direct exposure to the necessary high temperature was minimized by pumping N₂ through the apparatus, and dry ice was used to rapidly freeze the sublimate.
• Exposure to heat was usually limited to 2 h in one set of experiments and 18 h in another set. This was only long enough for 100 mg to produce about 1 mg sublimate.
• Vigreux columns were used to optimize enantiomer separation.
• Enantiomer enrichment was negligible—only ±20°C from the optimal temperature of 205°C.
• Serine was destroyed and e.e. was negligible if serine continued to be exposed to the optimal temperature for just a few hours. An analogous natural setting would merely provide the ideal means of destroying existing serine, and especially any temporary e.e., after a few days.
• The maximum e.e._L achieved was much too low for biological purposes and was obtained for only serine.⁵
• The starting point was unrealistic. Any serine available would have degraded and lost any e.e. instead of waiting to be heated suddenly to ~205°C. Therefore, the researchers began each experiment by placing pure serine on a pristine glass surface at room temperature, then rapidly heated it on the timescale of minutes to the final temperature.
• The enrichment obtained relied on preferential L-L, D-D, and/or L-D molecular interactions. This required a very high concentration of serine in the sublimate, which was achieved by deliberate laboratory manipulations (this would not occur naturally).
• Instead of pure Ser, many other substances would have diluted the concentration, including other AAs.
• Solid enantiomers were made as homogeneous as possible by grinding with a mortar and pestle, mixing with a spatula, and dissolving in acetone.
• The entire Ser sample was heated at the same time, ensuring an enormous amount of sublimate would form during a short time interval.
• The volume was severely constrained with a clean, smooth glass surface only centimetres away, at hundreds of degrees lower temperature, to trap the sublimate formed.

Formation of homochiral serine octamers using a soldering gun

Electrospray ionization has been used with serine to form stable clusters of eight serine molecules under special conditions.^6-8 The latest reports showed that these octamers were formed by rapid sublimation.^2,8 Yang et al. achieved this in a series of experiments using a soldering gun with a flat tip as a small hot plate upon which usually 0.02 g of sample was placed. The temperature was increased at a rate of 2°C/s from 30 to 300°C. The individual sublimation experiments lasted c. 90 min. The sublimation products were ionized at 5 kV and then analysed by a mass spectrometer.²

The flat tip of the soldering gun was located 1 cm below the bottom of a metal cooling funnel set up to collect products on its inner surface. A gas flow adapter was used, with dry ice (~–195°C), to cool the funnel.² An octamer began to form at ~180°C and reached a maximum concentration at ~220°C. At such temperatures, water vapour partially isolated the liquid droplet and the hot surface, decreasing the effectivity of heat transfer.⁸ However, the decomposition point of serine is ~222°C according to Sigma-Aldrich, but Yablokov et al. found the temperature range of decomposition for serine to be 198–222°C.^9,10 Therefore, for these kinds of experiments with serine, exposure to higher temperatures has to be limited to very short time durations.²

Yang et al. performed experiments using isotopically labelled D- and L-serines which were sublimed.² Analysis of the products indicated that clusters of 2–8 serines formed, attached preferentially to enantiomers of the same chirality, especially for the octamers. Octamers having 8/8, 7/8, 6/8, 5/8, and 4/8 L-serines were generated.

The overall pre- and post-sublimination proportion of Dand L-serine remained unchanged, only the location of the enantiomers was modified. The authors believe the octamer resulted from crystal disintegration into smaller clusters, with the most stable ones surviving the high temperature. Some pyrolysis products were also incorporated successfully into octamers by replacing one or more serines.²

Serine octamer exists in two forms

At temperatures around 150°C, solid serine sublimed mainly as monomers, but serine dimers and octamers appeared at higher temperatures.⁸ This explains why the researchers used such a rapid rate of heating in the sublimation experiments. It was necessary to quickly pass through the lower temperature stage. Formation of chiral octamers did not occur until at least 180°C and was most significant around 220°C. But at the much lower temperatures, serines present in a prebiotic earth would have sublimed and dissipated over time. A high concentration of pure serine was required to form octamers, and these had to be trapped by an extremely low-temperature dry surface. Therefore, in these optimized experiments, the serines were rapidly exposed to temperatures around +200°C and then cooled to about –200°C within a timescale of seconds!

To emphasize the point, under the carefully controlled laboratory conditions, serine octamers exist in two isomeric forms, A and B, but only the higher temperature conformer, A, displays the desired chiral effect.² In the sublimation experiments, conformer A only begins to appear in trace amounts at ~180°C.² The homochiral and heterochiral B-type octamers which formed first were equally stable and reached a maximum concentration at ~130°C.² This temperature is much closer to the boiling point of water and to temperatures amenable to life than is ~220°C. Conformer B was also more capable of forming octamers and larger 16-mers, 24-mers, and 32-mers metaclusters than was A.¹¹

Relevance to OoL speculation

Nanita et al. claimed that serine had been detected in experiments that simulate prebiotic conditions^8,12 and that serine can be formed from formaldehyde and glycine (see figure 5).¹³

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They then concluded, “Taken together, all the experimental evidence points towards serine as a likely prebiotic molecule.”⁸ All the experimental evidence? That is quite an exaggeration. In early experiments AAs were produced under allegedly relevant prebiotic conditions. The scientists had to assume the presence of gases leading to a reducing atmosphere. However, current geochemistry modelling denies such an atmosphere could have existed.^15,16

Therefore, Miller and other chemists repeated the original Miller and Urey experiments using realistic gas mixtures, but no amino acids could be obtained.^17-19 Plankensteiner et al. subjected a mixture of carbon dioxide, nitrogen, and water to continuously supplied sparks, and the only AAs obtained were glycine and alanine, the simplest amino acids.²⁰ Worse still, the latest view is that conditions in a putative primordial earth would have led to far fewer lightning strikes than commonly assumed.²¹

Serine could also allegedly result from the reaction between interstellar glycine and formaldehyde (see figure 5).^22,23 However, serine has not been detected in outer space spectra. In the rare cases when serine was found in meteorites, it was only in trace amounts. In the extensively studied Murchison meteorite, serine was found in sub part-per-million concentration, most or all of which was considered due to terrestrial contamination.²⁴

Assuming serine would have been available, e.e._L of serine could have been accomplished in theory by starting with an excess of L-enantiomer and running cycles of octamer formation and dissociation.^8,25 The n-mers formed would not have been all L, however, and no net overall e.e. would have resulted throughout nature, since only the microenvironment of the D and L serines has been modified.

In one set of experiments, the initial serine mixture contained an e.e._L of 20%, which was concentrated into octamers having an e.e._L of about 50%.⁸ Of course there would have been no 20% excess when life was supposed to have arisen in the extremely hot aqueous conditions following a putative Late Heavy Bombardment, between c. 4.0 and 3.8 billion years ago.²⁶ AA racemization during these millions of years would have been ensured. Suppose that somehow a 2% excess (and not the laboratory 20%) existed somewhere. The maximum excess in the octamers generated would now be irrelevant for any OoL discussion. A coincidental L-e.e. could plausibly be accompanied by a nearby D-e.e. and subsequent mixing of the products.

Experiments which used AAs such as threonine having an initial small e.e. were not reported, but, given the much lower enantioselectivities in their clusters, they would have resulted in virtually no change in e.e. in these mixed octamers.⁸ Therefore, chiral transmission to other amino acids through substitution of serines would have a minimal and irrelevant effect, even when carried out under ideally controlled conditions.

Incorporation of L-amino acids into serine octamers

Evolutionists have claimed that octamers might have played a role in the origin of L-AAs used in primitive proteins. For example, aerosols may have formed in hot springs or waterfalls.⁸ In a series of other experiments, Yang et al. examined pure L-enantiomers of each of the biological L-AAs to determine which incorporated into serine octamers.² Six L-amino acids (leucine, isoleucine, tryptophan, threonine, cysteine, and lysine) showed a preference to replace one or more L-serines in the octamer compared to the D-serines.² If D-amino acids were used with D-serine, the authors predict the mirror effect would occur. These experiments showed that octamers having mostly D or L will form. However, under alleged prebiotic ‘natural’ conditions, these octamers would be intimately mixed, leading to no spatial enantiomeric resolution.²

Since the incorporated AAs were already pure L, nothing useful has been demonstrated for OoL purposes. The results from beginning with racemic AAs or having a small e.e._L only would need to be evaluated.

Incorporation of other organic substances in serine clusters

Experiments reported by Takats and Nanita et al. showed that L-serine can undergo a condensation reaction with D-glyceraldehyde, the simplest aldose, in water at ~75°C after about 14 h.¹⁴ This dehydration product can substitute one or two serines in the L-octamer (about 8% abundance in the case of one Ser substitution, and 3% for two substitutions). The product of D-serine with D-glyceraldehyde was not incorporated into L-serine octamers.

There was also a chiral preference for L-serine to combine with D-glucose and of course also the opposite, D-serine with L-glucose, compared to the homoclusters.¹⁴ In these examples of molecule incorporation into serine clusters, the mirror effects ensure that no net e.e. would result on average.

Although Nanita and his collaborators have seriously oversold the relevance of serine octamers for origin of life (OoL) purposes in their publications, in a key paper their final comments did state matters in more realistic terms, writing that “The presence of homochiral serine octamers in the solution and solid phases remains to be demonstrated directly, as does the existence of stable neutral octamers” and “conclusive evidence for mirror-symmetry breaking does not yet exist for any system, and serine is no exception.”⁸

Critique of these studies

• Serine would be produced in only trace or no amounts under realistic, proposed prebiotic conditions. Being highly soluble in very hot water, it would readily racemize and therefore be entirely racemic and hence would display no tendency to concentrate together at some very hot location.
• If somehow highly concentrated under hot conditions, pure serines would first form octamers with a racemic content. If temperature then increased, the serines would sublime almost entirely as monomers. There is no reason why a large amount of pure serine would be enclosed in a small volume for all of it to be almost instantly exposed to a temperature ~200°C to produce octamers. Only in this highly contrived manner were the necessary high concentrations of serine generated. In a natural setting, individual serine molecules would racemize, decompose, and sublime individually over long time periods.
• There is no reason any octamers formed would not remain exposed to the high temperatures for a long period of time, decomposing the serines, instead of the wished-for sublimate being almost instantly cooled hundreds of degrees soon after being formed.
• Any minor e.e. generated would have been very short-lived. These would have been eventually exposed to water and dissolved back to individual serines.
• Threonine, proline, or other amino acids replacing serines formed very weak octamers with far weaker preference to form homochiral clusters.⁸ Non-biological sugars, AAs, and other substances could also contaminate the octamers, hindering formation of homochiral crystals.⁸

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