This article is from
Journal of Creation 37(3):128–134, December 2023

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Amyloids having sequence-, regio-, and stereo-selective properties don’t form under plausible prebiotic conditions

by Royal Truman

The Amyloid world theory for abiogenesis has been gaining popularity at the expense of the RNA world theory.¹ Many allegedly life-relevant properties have been attributed to synthetically produced amyloids. In a 2018 review article, Maury wrote:

“Rout et al. [2] showed that an amyloid can direct the sequence-, regio-, and stereo-selective condensation of amino acid synthesis.”³

These are three topics widely discussed in origin of life (OoL) circles. Correct sequences are necessary to form suitable peptides. Regiospecificity is necessary or nucleotides and amino acids (aa) would undergo the wrong chemical reactions. And stereoselectivity is a major problem since the key biochemicals to support life require that specific stereoisomers only be used.

The significance attributed to this paper is that allegedly,

“… it demonstrates that an amyloid formed from short peptides can direct the synthesis of its own constituent peptides under plausible prebiotic Earth conditions.”²

Examination of the paper revealed that this is not supported by the experiments reported. The constituent amino acids and peptides were already provided in pure, concentrated, and activated forms. We decided to examine the other claims attributed to this publication.

Biological proteins and amyloids

Cells manufacture proteins and amyloids having the three important properties mentioned above, thanks to the genetic code and pre-existing homochiral enzymes. It is also well known in molecular biology that special secondary structures in proteins, such as α-helices and β-sheets, only work for a restricted variety of aa residues at each position. The structures must not include side-chain reaction products, and only L-aa must be used.⁴ The secondary structures benefit from many H-bonds and an arrangement where hydrophilic side changes are oriented towards stabilizing interactions with water and the hydrophobic side chains are buried away from water.

Figure 1 shows part of a β-sheet, illustrating the large number of stabilizing H-bonds (shown in green). Only three types of aa were used, as in the experiments by Rout et al., with the most used phenylalanine (F) shown in red.² Stable β-sheets are a prerequisite to form the amyloid fibrils they reported.

© CMI

Decades of research have provided a set of rules and techniques chemists can use to synthesize these peptide secondary structures. Typically, aa from a biological source are isolated, and the end carboxyl and/or amino protons are activated by substituents, enabling them to react in water, or more often, in an anhydrous solvent. The question of interest here is whether various reported laboratory experiments reflect plausible prebiotic conditions.

Amyloids from simple synthesized β-sheets

The manner in which existing β-sheets constrain which residue is added can be illustrated by a puzzle, whereby correctly placed pieces determine which piece would fit in a new position. Figure 2 illustrates the strategy apparently used by Rout et al.

Addition of aa with suitable properties to extend an existing β-sheet would be favoured, since this results in a thermodynamically lower free-energy state. Suitable properties include having the correct chirality, steric interactions, and hydrophobic or hydrophilic nature to complement the counterpart residues. The principle can be visualized using figure 2, where a specific puzzle piece (aa F) would fit well at position I.

© CMI

Substrates and templates mixed to form amyloid substances

Rout et al. designed peptide substrates and templates having an alternating hydrophilic and hydrophobic L-aa residue in order to produce amphipathic β-strands.² The peptides to which activated aa could add were called substrates. In almost all the experiments, the peptides consisted of the aa R = arginine and F = phenylalanine. The amino end of the substrates contained a free -NH₂ group able to react with an activated aa carboxyl group.

Template peptides almost always had the structure (FE)₄, where F = phenylalanine, and E = glutamic acid. These were called template peptides, since they organized the substrates into β-sheets. These were designed so that the R and F groups would be chemically complementary in order to produce amyloid-like substances.

Both substrate and template peptides were modified chemically before being used, as mentioned in the section “Experimental details” below, and shown in table 1. This already eliminated any claim to plausibility for OoL purposes.

The three aa used to create these peptides were chosen so that both the template and substrate would be soluble at neutral pH and would also form an amyloid when mixed in high concentration. Their chemical structure is shown in figure 3.

© CMI

© CMI

Experimental details

The substrates and template were modified chemically, as shown in figure 4A. In addition, all aa were first activated with the condensing agent CDI (1,1’-carbonyldiimidazole, (C₃H₃N₂)₂CO). This process alone also eliminated any claim to plausibility for OoL purposes (see below).

The experiments based on four template/substrate pairs will be summarized next. The template and substrates used in the first three experiments are shown in table 1, aligned to show the longer template ‘overhang’, which helped select aa which added to the substrate.

Experiment 1

In the R(FR)₃/(FE)₄ substrate/template experiment, the template contained one more residue than the substrate. An insoluble aggregate (fibril) was produced.

Five activated aa (phenylalanine (F), aspartic acid (D), glycine (G), valine (V), and arginine (R)) were added individually, first to R(FR)₃ alone and then to the R(FR)₃/(FE)₄ amyloid. The yields of single aa addition are shown in table 2.

In spite of having used activated aa in high concentrations with rapid stirring, the single addition yields for these amyloids ranged from 3.5% to 50%, with the highest for phenylalanine, the optimal aa to extend the β-sheet. The key observations are:

• The reaction of five activated aa added with very different preferences to the arginine (R) end of the amyloid. Only a single residue was added.
• In this experimental setup, the amyloid was stereoselective for the L-enantiomer.
• When using the substrate alone, more D-enantiomers were selected—the opposite of what life needs!

Experiment 2

In the (FR)₃/(FE)₄ substrate/template experiment, the template contained two more residues than its partner. An insoluble aggregate (amyloid) was produced.

The activated aa F, D, G, V, and R were added to (FR)₃ alone and then to the (FR)₃/(FE)₄ amyloid in stoichiometric proportions. Now the amyloid no longer favoured single addition of the hydrophobic phenylalanine and valine, contra the results from experiment 1, shown in table 2. This is not surprising, since the aa added now aligned with a different position with respect to the template, see figure 2, position II. Instead, the yield of the now shorter amyloid was higher vs. for substrate (FR)₃ but only for arginine. Remarkably, for the four other aa the single addition yield was now much higher when using only the substrate.

Unfortunately, preferences for L- vs D-enantiomer (stereoselectivity) were not reported. We expect the substrate acting alone to favour addition of the D-enantiomers as before.

The key observation is:

• In the presence of the same template, substrates having different lengths produce contradictory yield patterns of single addition of the aa.

Experiment 3

In the R(FR)₂/(FE)₄ substrate/template experiment, the intention was to test whether a short substrate could provide three end-residue templating aa addition positions. However, the R(FR)₂ + (FE)₄ mixture remained soluble and did not form an amyloid.

Since experiment 2 (using (FR)₃/(FE)₄) did form an amyloid, if the end R group in the R(FR)₂ + (FE)₄ new mixture would react with a phenylalanine, an amyloid could be produced. Therefore, the researchers added activated DL-phenylalanine and DL-arginine at the higher concentration of 200 μM to the mixed R(FR)₂ + (FE)₄.

Low yields of addition products were obtained, as shown in figure 4 of reference 2, including single and double additions having a mixture of D- and L-enantiomers. Remarkably, of the triple additions, two were generated by far in the highest relative proportion, namely L-(FR)₄ and L-FF(FR)₃ i.e. using only L-enantiomers. Apparently, the three end positions of the template had a small directing effect. However, taking the 2:1 molar stoichiometry into account indicated that each triple addition yield was only about 1% (estimated from figure 4 of reference 2).

Unfortunately, addition preferences for a variety of different aa were not reported.

The key observation is:

• A carefully crafted templating β-sheet was able to favour addition of three activated L-aa to a peptide containing a complementary β-sheet in a yield of about 1%.

Experiment 4

In a final experiment the substrate used was (OV)₄ = (OVOVOVOV-NH₂), and the template was V(DV)₄ = (AcVDVDVDVDV-NH₂). O refers to ornithine, whose structure is shown in figure 5.

© CMI

It is not clear why the non-biological ornithine was selected for study instead of lysine, which differs by having one more CH₂ on the side chain.

The (OV)₄ molecule contained four side-chain -NH₂ groups and one N-terminal which could react with an aa to form an amide bond. Activated valine and activated phenylalanine each added to the N-terminal amino about a fifth of the time, whereas valine added to the amyloid N-terminal amino 65% and phenylalanine 85% of the time, i.e. the reaction was regioselective when using the amyloid.

The substrate (OV)₄ underwent sizeable degradation after hydrolysis at 90°C for 6 h, whereas almost none was reported for the amyloid.

The key observations are:

• Large insoluble amyloid fibrils can sometimes hinder the functional groups on side chains from reacting.
• Large insoluble amyloid fibrils can be thermally stable.

Discussion

Carefully designed and executed laboratory experiments per se have little to say about prebiotic chemistry. In fact, none of the results above are surprising from a chemical point of view. The fundamental issues are: plausibility under nonplanned putative prebiotic conditions, and the interpretation of the laboratory results. Can they be generalized in any useful manner or are they uniquely designed laboratory artifacts? Therefore, careful thought is necessary about the key details of the experiments.

1. None of the substrate and template peptides were used in their natural form. All had their C termini protected (amidated).
2. The template peptides also had a protective acetylated N-terminus to protect them from degradation. These two synthetic methods to protect peptides are well known to chemists.⁵
3. The end-carboxyl groups of all the free aa were activated with the condensing 1,1’-carbonyldiimidazole (CDI). CDI is readily destroyed in water by the following reaction:

   (C₃H₃N₂)₂CO + H₂O → 2 C₃H₄N₂ + CO₂.

   This is a problem in general with condensing agents. By their very nature of removing water from a condensation polymerization, they are readily degraded in water itself. Therefore, standard practice is to conduct the reaction in an anhydrous solvent such as tetrahydrofuran, chloroform, benzene, or dimethylformamide, none of which would have been present as a solvent in a prebiotic earth.⁶ The authors don’t mention which solvent was used, only that two equivalents CDI per aa were used, added on ice.
4. To form amyloids, they purchased substrate peptides and template peptides which were extensively purified by reverse-phase HPLC and then dissolved in 50 mM sodium phosphate buffer, pH 7.4.
5. Next, 100 μM substrate and 120–130 μM template peptide were mixed in water and agitated in an Eppendorf thermomixer agitating at 800 rpm at 37°C. Formation of fibrils was monitored using circular dichroism spectroscopy and appeared to reach equilibrium within a few hours. (It would have been easy to do some experiments rotating at, for example, 1 or even 50 rpm and see if fibrils would be produced.)
6. Since this formed flocculent aggregates, they were “sonicated (10 s, 20% power, Bandelin Sonoplus HD 2070 with MS73 microtip) to improve liquid handling before performing the addition reactions.”²

Prebiotically absurd conditions

Although we are focusing on a specific influential paper, many recurring principles characteristic of the OoL literature will be identified. Those not familiar with OoL publications might be mystified as to what the present experiments could possibly have to do with putative prebiotic chemistry. Reviewing the paper by Rout et al. shows it to be replete with phrases like prebiotic conditions, prebiotic reaction, prebiotic setting, prebiotic composition, and prebiotic system.²

What aspect of these experiments might have been meant to be representative of prebiotic conditions? One could evaluate every laboratory parameter used which were indispensable to obtain the reported results:

• Use of L-only peptide substrates and templates in extraordinarily high concentrations and ideal stoichiometry.
• Perfectly alternating hydrophilic and hydrophobic amino-acid residues known to form amphipathic β-strands.
• Perfectly complementary sequences for the substrates and templates.
• Elaborate measures to purify the substrates and templates before combining them.
• Capping of the amino and carboxyl end groups of all the templates.
• Capping of the carboxyl end groups of all the substrates.
• Activation of all aa using CDI that would hydrolyze quickly.
• Agitation in special equipment at 800 rpm at 37°C for at least 18 hours.
• Use of a closed container to force the components to interact.
• Termination of the experiments after the highest yields have been attained.
• Use of sodium phosphate buffer solution to ensure a pH of 7.4.
• Sonication to improve liquid handling.

Perhaps at least the three aa used to create the substrates and templates (phenylalanine, arginine, and glutamic acid) were the major aa expected to be found prebiotically? The 2023 publication by Kobayashi et al. was consulted, arguably the most careful simulation of aa produced from all sources under the most realistic reducing atmosphere.⁷ Absolutely none of these three aa were obtained. As a second test, the best review of all substances extracted from the Murchison meteorites is probably the 2017 review paper by Koga et al.⁸ Of the three aa only trace amounts of DL-glutamic acid were found.

Perhaps the peptides were based on realistic L-vs-D enantiomer ratios? If R(FR)₃ from experiment 1 had formed naturally it would have been part of a grand mixture of many DL- substances, such as:

2 R; 4 RF; 8 RFR; 16 RFRF;
32 RFRFR; 64 RFRFRF; 128 RFRFRFR (1),

since each aa could have a D- or L-enantiomer at each position.

These would have been mixed with all the enantiomeric template (FE)₄ alternatives and their precursor DL-enantiomers:

2 F; 4 FE; 8 FEF; 16 FEFE; 32 FEFEF;
64 FEFEFE; 128 FEFEFEF; 256 FEFEFEFE (2)

The researchers combined only pure L-RFRFRFR with pure L-FEFEFEFE, although all the alternatives would also have been present. Astonishingly, the individual L-peptides were prepared and co-located in a vast number of copies:

100 μM R(FR)₃ = 10^–4 × 6 × 10²³ = 6 × 10¹⁹ (3)

and

130 μM (FE)₄ = 10^–4 × 6 × 10²³ = 8 × 10¹⁹ (4)

each per litre.

Clearly, these experiments cannot be used to claim that amyloid fibrils were produced under plausible prebiotic conditions and that these led to sequence-, regio-, and stereo-selective addition of amino acids.

Casual readers are seriously misled.

It is unfortunate that so many read OoL papers like these and simply quote the claims stated, blindly believing that the results would indeed have occurred with no intelligent guidance. A second universal practice in this genre of literature is to also publish quantitative values which were obtained after years of expert design and optimization. These two are then combined and widely distributed using wording such as, “was obtained in high yields under plausible prebiotic conditions.”

Of course, nobody objects to scientists entertaining themselves by exploring unusual artifacts in a laboratory. Since it is fundamental to a chemist’s training to optimize yields of a desired effect, many of us would have gladly collaborated with researchers like Rout et al. to find the best conditions possible. After all, one needs quantitative results to begin exploring the effect of changing parameter settings to realistic values.

Conclusions

Let us perform a Gedankenexperiment and accept the trends reported in the above experiments. After all, a variety of other substrate and template peptides might someday also be examined.

Irrespective of the aa involved, condensation reactions in water are highly endothermic, so for any aa X and Y the aqueous concentrations of a substrate would be dominated by the smaller peptides:

X, Y >> XX, YY, XY >> XXX, XXY,
XYX, XYY, YXX YXY, YYX, YYY >> … (5)

The same applies for a template candidate.

Now, the proportion of a suitable substrate being co-located with a suitable template, having the requisite residue sequence and length (‘overhang’), would be many orders of magnitude lower than of the substrate alone. The data in table 2 shows that D-aa are preferentially added to substrates alone, whereas L-aa are preferentially added to the amyloids. Since the former would be in vastly greater proportion long before any amyloids could form, the net outcome would be peptides preferentially having the wrong chirality. Therefore, the L-only peptides needed to produce amyloids could not form.

Conclusion on stereo-selectivity: The authors should have reported that if their experiments reflected prebiotic conditions, then far more peptides of the wrong chirality would have formed in the presence of amyloids.

In experiment 2, the yields of a single addition of activated F, D, G, or V were higher for the substrate than for the amyloid. Since far more substrate would have existed independent of co-located template, also for all the precursor peptides, this would be the dominant outcome. For the amyloid, the preference was dominant only for the aa R.

The opposite trends were reported for experiment 1, based on a substrate one aa shorter. As shown in table 2, the yield of the addition of R was about the same for the substrate and the amyloid. However, for the remaining F, D, G, and V, higher yields of addition occurred when the substrate was part of an amyloid. Consequently, mixtures of different substrates and templates would tend to cancel each other’s effects.

Note that preferential addition of one aa was demonstrated for only five carefully selected aa of the 20 biological aa. The selectivity involved the addition of a single aa; was only possible thanks to the preceding chemical modifications of the substrate and template; required chemical activation of the aa; and resulted in considerable mutual cancelling of trends. Since substrate and template concentrations would not have been present concurrently in concentrations anywhere near 10¹⁶ peptides/ml, this selectivity artifact would have been much too small to detect among a mixture of all condensation reactions occurring throughout nature. Virtually all would have involved very short peptides which would not form secondary structures.

The two L-only triple additions found in experiment 3 were only possible because an L-only substrate and template had been used. The precursor peptides would have already racemized, since kinetic and thermodynamic calculations show that the rate of racemization of pure L-peptide is faster than their rate of elongation in water under realistic conditions.⁹

In all these experiments one must not overlook that the stability of the amyloid fibrils was achieved only after huge, dense, insoluble materials had formed. This, in turn, was possible only thanks to the very high concentration of co-locate substrates and templates under rapid stirring. Any small precursors to the β-sheet would have been exposed to destructive hydrolysis over long geological times.

Conclusion on sequence-selectivity: Plausible prebiotic amyloids can provide neither measurable nor reliable sequence selectivity.

In experiment 4, valine and phenylalanine were found to add preferentially to the end amino group of the amyloid. Note that this required first forming an amyloid fibril under extremely high concentrations using the substrate (OV)₄ and the template V(DV)₄. The authors did not explain how the four ornithine molecules (O) managed to form about 10¹⁶/ml pure linear peptides, nor where all the pure templates with both ends chemically blocked could have come from.

During the substrate and template build-up process, several activated ornithine molecules would have added to the side chains, not being yet embedded in hindering amyloid. (This assumes both ends of the template had not been chemically blocked. Otherwise, the activated ornithine would only add to the side chains of the substrate). Each ornithine added to the side chain would now offer two new competing -NH₂ groups. Each elongation in the linear direction would only replace the amino group just ‘consumed’, while providing ever more opportunities for side-chain reactions.

Conclusion on regio-selectivity: Plausible prebiotic amyloids containing aa with side chains would not have formed, since this would have required a concentrated pool of only linear peptides ab initio.