This article is from
Journal of Creation 37(3):79–83, December 2023

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Enantiomeric amplification of L-amino acids: part 4—based on subliming valine

by Royal Truman

I continue here the series dealing with proposals on how a small excess of an amino acid (AA) enantiomer might be amplified under natural conditions. The L-enantiomers of nineteen biogenetic AAs are coded for by the genetic code and used to construct proteins. The source of pure L-enantiomers in the absence of pre-existing enzymes, themselves built from only L-AAs, has been an enduring source of discomfort for evolutionists. This discomfort implies that the origin of cells involved more than natural chemical processes.

Sublimation of valine at very high temperature

Viedma and colleagues reported that when an equal proportion of D- and L-enantiomer of valine (Val) is rapidly sublimed at 430°C, the condensate on the wall of the glass vessel forms racemic conglomerates (i.e. pure D- and L-enantiomers crystallize into different crystals which are mixed together).¹

Obtaining conglomerate crystals was noteworthy since Val, like almost all proteinogenic AAs, forms racemic crystals under milder conditions (i.e. the D- and L-enantiomers are intimately mixed in the same proportion within the same crystal).² The observed switch from an initial heated racemic mixture to a conglomerate was studied using Powder X-ray (PXR) diffraction.

Very specific laboratory conditions were necessary to obtain this effect.¹ A hotplate stirrer was set to 430ºC, and a 1000-mL flask placed on top for 3 mins, creating an optimal temperature gradient. A Val mixture having 40% e.e._L (enantiomeric excess of L) was then placed on the bottom of the flask and stirred for a few seconds to maximize contact with the hot surface. Sublimation occurred almost instantly, and a dense cloud of condensing microcrystals was seen for ~2 mins on the much colder higher levels of the flask.

Chiral HPLC (High Pressure Liquid Chromatography) analysis did not reveal any enantioenrichment in the initial deposit, but the enantiomeric enrichment of the sublimate continued to ascend for about 10 mins. After completion, the system was cooled to room temperature. When using a closed flask (see figure 1A), a gradual vertical separation of Val enantiomers was found. The e.e._L ranged between 46%, at the top, to 80%, at the bottom, for an average of 56%. The experiment was repeated using an open flask, leading to lower e.e._L values having an average e.e._L of 47% (see figure 1B).¹ The latter represents a net increase of 7% in e.e. since the initial e.e._L was already 40%.

© CMI

The authors did not know the cause for this spatial enantiomer separation. One possibility is that, analogous to attrition-enhanced deracemizations, tiny crystal fragments with more L members may have interacted favourably in the sublimate, thereby offering a greater surface area for further growth.¹

As mentioned above, starting with a 40% e.e._L Val sample, the sublimate had an average e.e._L of 56% using a closed flask. When the sublimed crystalline material was isolated and used for a second sublimation experiment, the new crystals had an average of 69% e.e. Repeating a third time led to a further improvement, with sublimate now having an average of about 80% e.e.³

We can use these three data pairs for the closed-system experiments and assume that an initial 0% e.e. would produce a sublimate having 0% e.e. to construct an empirical first-order polynomial curve. This will allow calculating the results for other initial values of e.e._L (see figure 2, blue line).

© CMI

Repeated cycles to amplify the e.e._L were not reported in the open system. This is the environment which would occur naturally.⁴ A single data point is available from the open flask experiment which also started with 40% e.e._L, which produced a sublimate having an e.e._L of 47%.¹ Beginning a new sublimating cycle with 47% (instead of 56% as before), and using the empirical equation shown in figure 2 derived for the closed flask predicts an e.e._L of 62%:

e.e._L = 0.0926 + 1.6933 × (47) ‒ 0.0079 × (47) = 62

However, starting with 40% e.e._L led to 9% (56% – 47%) less enhancement in the closed flask than the open flask experiment, so we subtract 62% – 9% = 53%, shown as a red square in figure 2. Since the curve for closed flask behaviour increases less rapidly (see figure 2) the correction should be even more than 9%.

Remarkably, in his attempt to find a way to amplify an initial e.e., Viedma has documented an effective manner to decrease the initial e.e. throughout nature! From figure 1B, for the open flask experiments we see that near the intense heat source the e.e._L was 70%, and towards the top it was down to exactly the starting value of 40%. Since mathematically the total amount of L- + D-AA is constant (or lower if racemization occurred at such high temperatures) and all the AA sublimed, then beyond the 40% layer the rest of the AA must have an e.e._L <40%!

Furthermore, the process of freezing sublimate in nature would have been spread out over far greater distances than a few centimetres as in the cold flask. Therefore, there would have been a continuum of e.e._L values whereby the material closest to heat sources hundreds of degrees C would have been preferentially thermally destroyed. The surviving AA could only average to a lower e.e._L. than initially present. Future cycles would have continually been enriched in the ‘wrong’, i.e. D-enantiomer.

Using sublimed amino acid mixtures at very high temperature

Further experiments were reported by Viedma et al. using the hot stirring plate set to 430°C and a 1000-mL flask setup.^3,5

Experiments were conducted using four AAs which were known to crystallize from aqueous solutions as racemic compounds (i.e. the D- and L-enantiomers are mixed in equal proportions within the same crystal). Viedma et al. discovered that Val and Ileu recrystallized after sublimation into conglomerates (i.e. the individual crystals were composed of pure D- or L-enantiomers), whereas leucine and alanine recrystallized as usual into racemic compounds, as shown in figure 3.³

© CMI

Since enantiomeric separation via sublimation requires an AA to crystallize as a conglomerate, the researchers decided to combine an AA which conglomerated with one which did not. The results of sublimation experiments at 430°C conducted in closed flasks are shown in table 1.

A mixture of 40% e.e._L Val with 40% Leu led to a sublimate containing 51% e.e. Leu, see the first row in table 1.

The results using racemic Ala instead of racemic Leu were disappointing. The authors wrote:

“Alanine (the simplest chiral amino acid) was quite reluctant to undergo this sort of resolution, either with valine or isoleucine, and a poor increase in ee (ca. 4% on average) could be measured in the presence of scalemic isoleucine.”

This is the last row shown in table 1. The results of mixing 40% e.e._L Val with 40% Ala were not reported.

Note that experiments using Val and Ala were also conducted, but why were the exact values not reported? Also, an estimate of experimental reproducibility would have been helpful instead of only reporting an average value. No mention was made in the paper nor the Electronic Supplementary Information about experiments being repeated, so what was the basis for choosing which results went into the on average of ca. 4% when Ileu was used instead of Val? Considerable experimental error can be surmised, since derivatization of the AAs using thiol isobutyryl-L-cysteine and o-phthaldialdehyde followed by rehydration were carried out to facilitate the HPLC analysis.³ Therefore, one should conclude provisionally that amplification was demonstrated for only one AA (Leu), but for Ala this is doubtful.

Thus, amplification was achieved for one biological AA, Leu, which doesn’t form conglomerates. This was achieved by both Val and Ileu; see the data in table 1. Interestingly, the trend in amplification contradicted what Tarasevych et al. found, namely that partial sublimation of L-asparagine with various racemic AAs led to a sublimate enriched with the D-enantiomer (discussed in Part 6 of this series)!⁶ Whereas Viedma et al. would argue for the sublimate being a source of L enrichment, Tarasevych et al. would have to use the residue for this purpose. Whatever the phase selected, some mixtures of AAs might have increased the e.e._L of some AAs but concurrently decreased the e.e._L of other AAs.

The authors were not sure what the basis for these effects was. In the case of Val and Ileu crystals, the sublimate might be deposited on pre-existing crystals, moving upwards, away from the heat source. The experimental observation was that the highest e.e. was produced at the front sublimation line and decreased gradually upwards further from the intense heat source, the same effect reported above in figure 1.

High temperature diffusion would decrease enantiomeric excess

As mentioned before, no e.e. was found in the initial deposit, but this developed over about 10 mins as the sublimate continued to ascend the inner flask surface. The much cooler outer flask surface served to artificially concentrate the sublimate. We can make some very rough calculations to illustrate this.

From experience, we know that if a gas like HCl is released we will soon smell it a long distance away. We can envision a large open natural hot setting with a thermal gradient maximizing at the bottom at ~430°C into which an AA is suddenly placed. Some of the hot gaseous AA would soon diffuse, say one to 10 metres, representing a volume ≈ 10⁶–10⁹ cm³. We don’t know the thickness of sublimate formed on the flask surface in the experiments, but based on the miniscule size of the AAs involved let us assume this would be <0.01 cm, spread over a glass surface of ~10² cm². This would imply that the sublimate had been compacted into an effective volume of <1 cm³, i.e. by a factor of millions to billions. Furthermore, the concentration of AA and e.e._L used were considerably higher than naturally plausible. Clearly, the laboratory conditions had artificially facilitated L-L and L-D equilibration interactions. To further amplify this, the authors manually dissolved the sublimate from the surface, benefitting strongly from the highest e.e. obtained, which was just a few centimetres from the heat source. Figure 1 shows how rapidly e.e. decreased with distance, and sublimate located one to ten metres away would almost certainly be racemic. Therefore, the e.e._L values shown in figure 2 are not representative of what would occur under natural conditions.

We can use the red curve in figure 2 for the open flask and assume Val is initiated with a very generous e.e._L of 10% instead of the completely unrealistic 40%. Of course, the flask surface would still constrain the volume since sublimate can only escape from the distant narrow opening after being exposed to the cooler glass surface.

Even retaining all the unrealistic optimizations used by the researchers for the open-flask experiment (red curve in figure 2), the e.e._L of the sublimate would only increase by about 2%, while simultaneously lowering the e.e._L in the residue left behind. But flash sublimation would be a rare event, and, realistically, most of the sample containing an initial assumed 10% L excess would have thermally decomposed before being exposed to a temperature near 430°C.⁸

It is instructive to recall from figure 1B that only a few centimetres from the heat source the sublimate e.e._L was 40%, exactly the same as the initial excess. Zero enrichment was now present.

Without deliberate researcher intervention, the true net overall effect of this scenario would be to decrease any local e.e._L initially present in nature. Repeating the cycles through renewed exposure to very high temperatures would ensure that the original e.e._L would eventually be destroyed. There is no reason why nearly pure L sublimates would never again be subjected to the high temperatures.

Summary critiques of these studies

There are several objections to using these results to claim amplification of L-AAs would have occurred under prebiotic conditions.

• The results relied on preferential L-L vs L-D interactions, which required very concentrated AAs to have a measurable effect. Finding 0.05 gm of pure AA sublimate on a few cm² of surface by chance is unrealistic by many orders of magnitude.¹ Under natural conditions, sublimed AAs would be significantly diluted by other substances and probably often be bound in large chemical complexes (as observed in meteorites.⁷) An initial e.e._L of 40% is considerably higher than anyone has claimed could have formed naturally. These two factors alone suggest that the necessary inter-AA interactions would not naturally occur to a detectable level. Val has a decomposition point of 295–300°C.⁸ In an important report, Weiss et al. monitored the thermal decomposition of all 20 proteinogenic AAs, using calorimetry, thermogravimetry, and mass spectrometry. For the 8 AAs for which they reported in detail, the decomposition temperatures ranged from 185 to 280°C, with an average of 235°C. They concluded that the decomposition analyses “… put constraints on hypotheses of the origin, state and stability of amino acids in the range between 200°C and 300°C.”⁹

Viedma et al.’s results required all the AAs to be present and sublime quickly at ~430°C, more than 100°C above their decomposition point! These AAs would have been destroyed instead of being highly concentrated in a natural setting at such temperatures. Therefore, the researchers had to manually transfer the AAs from room temperature to the bottom of a preheated flask at 430°C within seconds. They also provided a much cooler, smooth, clean glass surface only a few centimetres away to capture the sublimate. Only through such manipulations was the destruction of the AAs avoided.

Besides decomposing, all AAs would sublime in free nature as soon as a sufficiently high temperature was reached, thereby rapidly dissipating over time. Without a high density of AA having a large e.e._L, the requisite L-L interactions would have been absent.

• As pointed out above, beyond some distance from where sublimation had initiated the e.e._L would have been lower than the initial value. The portion with highest e.e._L would have been located nearest the fierce heat source, and been the portion preferentially destroyed. Viedma’s experiments have revealed an effective way to decrease an initial e.e._L throughout nature!
• There would have been no plausible terrestrial environment which satisfies all the prerequisites after the putative Late Heavy Bombardment is supposed to have racemized any surviving AAs.¹⁰ Perhaps AAs could have been dissolved in hydrothermal vents, and some became isolated in a crevasse and desiccated. However, Sato et al. showed that AAs are destroyed under hydrothermal conditions. They experimented at 20 MPa pressure and all the AAs they examined decomposed at under 300°C.¹¹
  Viedma et al. alluded to sublimation in environments such as frozen planets with low or no atmospheric pressure.³ This is not credible for many reasons:
• Frozen planets don’t provide high concentrations of AAs at temperatures ~430°C.
• If AAs were located at such a temperature, they would not then be conveniently cooled about 200°C within seconds after sublimation to avoid obliteration.
• Recycling frozen enriched AA back into an environment hundreds of degrees hotter without being destroyed for additional amplification cycles is not plausible.
• These frozen planets would not be closed systems, so an excess of L-enantiomer would have dissipated slowly over millions of years at low temperatures and pressures instead of equilibrating on top of microcrystals.
• Concentrations of L-L interactions at any location would have been very low.
• If a suitable environment had existed on Earth, temperatures ~430°C would have heated any water nearby, in which AAs would eventually find themselves, ensuring loss of any e.e._L through enantiomer racemization. Considerable loss of e.e._L would be very fast in aqueous solutions near the boiling point of water, on the timescale of mere hours or days.¹²
• Racemization would be faster the higher the e.e._L attained, since the back reaction L → D would be favoured by the higher concentration of L-enantiomer. Repeating the sublimation cycles of transferring cooled crystals back to a new very hot, dry surface somewhere by chance would require much time, during which racemization would occur. The higher the starting e.e., the more difficult to further enrich it.
• Beginning with an e.e._L on the order of 40% with all the optimized parameters used for the two AAs able to form conglomerate crystals will not occur naturally.
• Many parameters were optimized to shorten the exposure time to the intense heat and inevitable decomposition.
• L- and D-enantiomers were ground in a mortar to increase their effective surface and to homogenize them to facilitate L-L interactions.
• The AA mixture was stirred to ensure maximum contact with the hot plate and to maximize the number of L-L interactions.
• The hot plate stirrer was previously heated so that sublimation was almost instant.
• A cool surface was provided mere centimetres from where sublimation occurred.
• The flask was prewarmed for 3 mins at 430°C to generate an appropriate thermal gradient.
• The solid AAs were rapidly transferred manually from room temperature to the bottom of the hot flask.
• A high concentration of AAs will not penetrate a closed container which also happens to be over a hundred degrees above their decomposition temperature.
• Only an open system or a huge, closed volume is plausible as a natural environment, considering also that an e.e._L must subsequently become available for further chemical processing, such as forming peptides. But in this environment, highly concentrated sublimates will not form or equilibrate; instead, they will dissipate as soon as formed.
• The highest enrichment was produced in the sublimate closest to the heat source, where thermal decomposition was most likely to occur. In addition, this portion of the sublimate would be proximate to the remaining residue which had just preferentially lost L-enantiomer. Future remixing, especially with the help of moisture, would preferentially combine the most enriched L sublimate with the D-enriched residue, decreasing or even reverting the average e.e._L. Without this contribution from the most L-enriched AA, the remaining sublimate in an open system would have very little or no L excess; see figure 1B.
• Very few biogenic AAs form conglomerate sublimates, and only a single credible example of enrichment through mixing was reported, for Leu. This sublimation scenario cannot explain the source of the wide variety of L-AAs needed to form proteins. The results of sublimation experiments under vacuum pressures and lower temperature reported by Tarasevych et al. need to be taken into account. Combining L-asparagine with several racemic AAs led to the opposite effect, namely a sublimate enriched with the D-enantiomer!⁵ This illustrates how multiple random effects tend to maximize entropy, in this case racemization of amino acids.

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