rationalskepticism.org

Greyman wrote:

Dr. Robert Shapiro, a chemist at New York University, said the recipe “definitely does not meet my criteria for a plausible pathway to the RNA world.” He said that cyano-acetylene, one of Dr. Sutherland’s assumed starting materials, is quickly destroyed by other chemicals and its appearance in pure form on the early earth “could be considered a fantasy.”

So, criticised by a proponent of "metabolism first" theory in a newspaper interview, and criticism answered in the very next paragraph:

Dr. Sutherland replied that the chemical is consumed fastest in the reaction he proposes, and that since it has been detected on Titan there is no reason it should not have been present on the early earth.

Actually, in the original 2009 paper, this is what Sutherland actually said. First, the abstract:

Powner, Gerland & Sutherland, 2009 wrote:Abstract

At some stage in the origin of life, an informational polymer must have arisen by purely chemical means. According to one version of the ‘RNA world’ hypothesis1–3 this polymer was RNA, but attempts to provide experimental support for this have failed4,5. In particular, although there has been some success demonstrating that ‘activated’ ribonucleotides can polymerize to form RNA6,7, it is far from obvious how such ribonucleotides could have formed from their constituent parts (ribose and nucleobases). Ribose is difficult to form selectively8,9, and the addition of nucleobases to ribose is inefficient in the case of purines10 and does not occur at all in the case of the canonical pyrimidines11. Here we show that activated pyrimidine ribonucleotides can be formed in a short sequence that bypasses free ribose and the nucleobases, and instead proceeds through arabinose amino-oxazoline and anhydronucleoside intermediates. The starting materials for the synthesis—cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde and inorganic phosphate—are plausible prebiotic feedstock molecules12–15, and the conditions of the synthesis are consistent with potential early-Earth geochemical models. Although inorganic phosphate is only incorporated into the nucleotides at a late stage of the sequence, its presence from the start is essential as it controls three reactions in the earlier stages by acting as a general acid/base catalyst, a nucleophilic catalyst, a pH buffer and a chemical buffer. For prebiotic reaction sequences, our results highlight the importance of working with mixed chemical systems in which reactants for a particular reaction step can also control other steps.

However, Sutherland and the other authors then move on to the actual meat of their paper, thus:

Powner, Gerland & Sutherland, 2009 wrote:Because they comprise phosphate, ribose and nucleobases, it is tempting to assume that ribonucleotides must have prebiotically assembled from such building blocks. Thus, for example, it has previously been supposed that the activated ribonucleotide β-ribocytidine-2',3'-cyclic phosphate 1 must have been produced by phosphorylation of the ribonucleoside 2, with the latter deriving from the conjoining of the free pyrimidine nucleobase cytosine 3 and the furanose form of ribose 4 (Fig. 1, blue arrows). This mode of assembly is seemingly supported by the facts that cytosine 3 can be synthesized by condensation of cyanoacetaldehyde 5 and urea 6]16 (the hydration products of cyanoacetylene 717, and cyanamide 818, respectively) and pentoses including ribose can be produced by aldol reaction of glyceraldehyde 9 and glycolaldehyde 108,9. The insuperable problem with this approach, however, is that one of the presumed steps, the condensation of ribose 4 and cytosine 3, does not work11. The reasons for this are both kinetic (the N1 lone pair of 3 is unavailable owing to delocalization) and, in water, thermodynamic (the equilibrium constant is such that hydrolysis of 2 to 3 and 4 is favoured over condensation). The same is true for ribosylation of uracil, which has also not been demonstrated. We have considered a large number of alternative ribonucleotide assembly modes, including those that extend back to the same small-molecule precursors as the traditionally assumed route described above19. By systematic experimental investigation of these options, we have discovered a short, highly efficient route to activated pyrimidine ribonucleotides from these same precursors that proceeds by way of alternative intermediates (Fig. 1, green arrows). By contrast with previously investigated routes to ribonucleotides, ours bypasses ribose and the free pyrimidine nucleobases. Mixed nitrogenous–oxygenous chemistry first results in the reaction of cyanamide 8 and glycolaldehyde 10, giving 2-amino-oxazole 11, and this heterocycle then adds to glyceraldehyde 9 to give the pentose amino-oxazolines including the arabinose derivative 12. Reaction of 12 with cyanoacetylene 7 then gives the anhydroarabinonucleoside 13, which subsequently undergoes phosphorylation with rearrangement to furnish β-ribocytidine-2',3'-cyclic phosphate 1. In a subsequent photochemical step, 1 is partly converted to the corresponding uracil derivative, and synthetic co-products are largely destroyed.

So, the steps in the Sutherland et al synthesis consist of:

[1] H2N-CN + HOCH2-CHO → H2N-<-C=N-CH=CH-O-> (cyanamide + glycoaldehyde gives 2-aminooxazole);

In the above, I've denoted a ring by leaving the bonds open at the ends of the ring, starting with the carbon to which the amine is attached, and enclosing the whole in <> characters, to denote that the end bonds are in fact identical, making the whole enclosed structure a ring. At the moment, we don't have a chemistry formula editor embedded in the forum, so this will have to suffice for the moment.

[2] 2-aminooxazole + glyceraldehyde (HO-CH2-CH(OH)-CHO) gives a range of pentose amino-oxazolines;

[3] Reduction of arabinose amino-oxazoline by cyanoacetylene (C2H-CN) gives the anhydroarabinonucleoside;

[4] Phosphorylation of this product gives β-ribocytidine-2',3'-cyclic phosphate;

[5] Photochemical rearrangement gives the uracil ribonucleotide.

The authors then reveal this:

Powner, Gerland & Sutherland, 2009 wrote:We had previously shown that in unbuffered aqueous solution, 2-amino-oxazole 11 adds to glyceraldehyde 9 to give the pentose amino-oxazolines including 12 in excellent overall yield20. Our starting point in the present work was therefore to find a prebiotically plausible synthesis of 11. Constitutionally, 11 is the condensation product of 8 and 10, and although there exists, in the conventional chemical literature, a procedure to bring about this condensation, it requires strongly alkaline conditions21. Because we wanted to generate 11, and then allow it to react with 9, which is unstable to alkali, under the same conditions, neutral-pH reaction conditions had to be found.

So the authors had already demonstrated the plausibility of Step [2] above, and needed first to find how to achieve Step [1] without requiring strongly alkaline conditions. They proceeded thus:

Powner, Gerland & Sutherland, 2009 wrote:We initially investigated the reaction with 8 and 10 in a 1:1 ratio starting at neutral pH in unbuffered aqueous solution. Only a small amount of 11 was produced under these conditions, and 1H NMR spectra were indicative of the formation of a variety of carbonyl addition adducts and other intermediates, for example 14–18 (Fig. 2a, b). The carbonyl addition adducts 14 were presumably formed reversibly, and so did not represent material irretrievably committed to other products, but rather intermediates stalled en route to 11. At low concentrations of hydroxide, it appeared that two additional types of reaction needed to make 11 were very sluggish: intra-adduct attack of the glycolaldehyde-derived hydroxyl group on the cyanamidederived nitrile carbon (for example 14 (n=0) → 15), and C–H deprotonation leading to aromatization (17 → 11).

Denied the opportunity of using hydroxide as a specific base catalyst to accelerate these slow steps, we sought a general base catalyst that could provide the same acceleration, but at neutral pH. Inorganic phosphate seemed to be ideal in this regard because its second pKa value is close to neutrality. Furthermore, as phosphate is ultimately needed in some form to make activated nucleotides, we decided to include it from the start of the assembly sequence. We repeated the earlier reaction of cyanamide 8 and glycolaldehyde 10, but in the presence of 1M phosphate buffer at pH 7.0. 1H NMR analysis revealed that 2-amino-oxazole 11 was produced in >80% yield (75% isolated yield) (Fig. 2c). With an excess of 8 over 10, the synthesis of 11 still takes place in the presence of phosphate, but is followed by slower phosphate addition to residual 8 giving the intermediate adduct 19, which partitions to urea 6 and cyanoguanidine 20 (Fig. 2d).

Lo and behold, the phosphate ions needed for Step [4] above, could also act as a neutral pH catalyst for Step [1]!

Powner, Gerland & Sutherland, 2009 wrote:We then investigated whether the subsequent reaction of 11 with glyceraldehyde 9 would be tolerant to the residual presence of phosphate. In the absence of phosphate, the ribose and arabinose aminooxazolines 21 and 12 are the major products, and the xylose derivative 22 is a minor product (Fig. 3a)20. The lyxose amino-oxazoline 23 is formed in intermediate amounts as an equilibrating mixture of pyranose and furanose isomers. All of the pentose amino-oxazolines have the potential to be converted reversibly into one or other of the 5-substituted 2-amino-oxazoles 24 and 25 by phosphate catalysis (by chemistry similar to that underlying the conversion of 16 to 11), but to differing extents depending on their stability. After one day in the presence of phosphate, all of the amino-oxazolines showed some conversion to the corresponding 5-substituted 2-amino-oxazole (24 or 25), but the lyxose amino-oxazoline 23 proved the least stable and underwent the greatest conversion (Fig. 3b). We then took a crude sample of 11 that had just been prepared from cyanamide 8 and glycolaldehyde 10 in the presence of phosphate, and added glyceraldehyde 9 to it. After overnight incubation, 1H NMR analysis revealed that although all four amino-oxazolines were still formed, the lyxose derivative 23 was selectively depleted and was now a minor product along with the xylose derivative 22 (Fig. 3c). With two of its stereoisomeric relatives now minor products, the path from the arabinose aminooxazoline 12 to ribonucleotides looked clearer. Selective crystallization of ribose amino-oxazoline 21 offers a further means of enriching 12 such that it becomes the major product in solution20,22.

So, after finding the catalysis route needed to produce Step [1] under neutral pH conditions, the authors then checked to see if the catalysis choice would interfere with Step [2], and found, no doubt to their delight, that unwanted stereoisomers were virtually eliminated, leaving them with the desired arabinose aminooxazoline needed to facilitate Step [3].

Moving on, the authors then conducted more experiments, to find out how Step [3] might proceed, and under what conditions:

Powner, Gerland & Sutherland, 2009 wrote:We then proceeded to the second stage of pyrimidine nucleobase assembly. Although our focus was on the chemistry of the key arabinose amino-oxazoline 12, the corresponding chemistry of the ribose aminooxazoline 21 was also studied (Supplementary Information). It had earlier been shown that in unbuffered aqueous solution, 12 reacts with an excess of cyanoacetylene 7 giving β-arabinocytidine 26, (Fig. 4a)23. The yield of 26 was relatively low, however, and we used 1H NMR analysis to determine why. It transpires that the pH rises during the course of the reaction, resulting in hydrolysis of anhydronucleoside intermediates and causing hydroxyl groups to undergo reaction with cyanoacetylene 7 (Supplementary Information). To prevent the rise in pH during the reaction, inorganic phosphate was added as a buffer. When the buffering pH was 6.5, the reactions were extremely clean, with little evidence for anhydronucleoside hydrolysis. Furthermore, excess cyanoacetylene 7 that did not evaporate underwent reaction with phosphate at this pH, giving cyanovinyl phosphate 27 instead of cyanovinylating hydroxyl groups. Using phosphate as a dual-function pH and chemical buffer in this way, the arabinose anhydronucleoside 13 could be produced in extremely high yield from 12

So, those phosphate ions needed for Step 4, not only acted as a neutral catalyst for Step [1], but provided a pH buffer to stabilise Step [3], and prevent cyanoacetylene from engaging in unwanted side reactions!

Moving on, the authors then provide this:

Powner, Gerland & Sutherland, 2009 wrote:Our finding that the reaction of the amino-oxazoline 12 with cyanoacetylene 7 could be controlled, by the pH and chemical buffering action of phosphate, to produce the arabinose anhydronucleoside 13 in excellent yield opened up the possibility of a combined phosphorylation–rearrangement24,25 reaction to convert 13 to the activated ribonucleotide 1. Furthermore, the formation of cyanovinyl phosphate 27, as a co-product in the nucleobase assembly process, extended the range of potential phosphorylating agents for such a process because, in aqueous solution, 27 undergoes reaction with inorganic phosphate to give pyrophosphate17. Accordingly, we investigated the phosphorylation of the anhydronucleoside 13 using both inorganic phosphate and pyrophosphate.

So, even better still, the additional reactions of cyanoacetylene with phosphate ions, provided additional phosphorylating agents for Step [4]!

Powner, Gerland & Sutherland, 2009 wrote:Prebiotic phosphorylation of nucleosides has been demonstrated by heating either in the dry state with urea26 or in formamide solution27. We were particularly attracted by the possibility of using urea 6 in the phosphorylation of 13 because it is a co-product of the chemical system in which 2-amino-oxazole 11 is produced from glycolaldehyde 10 and cyanamide 8, if the latter is initially present in excess (Fig. 2d). After preliminary experiments, and through consideration of the phosphorylation mechanism (Supplementary Information), we found that when 13 was heated with 0.5 equiv. of pyrophosphate in urea containing ammonium salts, 1 was formed as the major product in addition to 28 and 29 (the 5'-phosphate derivatives of 13 and 1, respectively) and small amounts of the hydrolysis product β-arabinocytidine 26 and its 5'-phosphate derivative 30 (Fig. 4b, procedure A; Supplementary Information). Alternatively, 1 was formed in very good yield—along with 29, the hydrolysis products 26 and [/b]30[/b], and the nucleobases cytosine 3 and diaminopyrimidine 31—by heating 13 with inorganic phosphate and urea in formamide solution (Fig. 4b, procedure B; Supplementary Information).

So not only did they find their desired nucleotide popping out of Step [4] (via Step [5]), but they also ended up with additional nucleobases being synthesised alongside their desired nucleotide!

After some deliberation on the likely reaction mechanism, which I'll leave out here, as I've provided a link to the paper previously, the authors then move on to another neat step, as follows:

Powner, Gerland & Sutherland, 2009 wrote:If the products of the phosphorylation reaction in urea were subsequently to dissolve in aqueous medium at neutral pH and incubate for any significant length of time, then any residual anhydronucleoside/ anhydronucleotide would undergo hydrolysis. Assuming such a rehydration, after phosphorylation in urea or urea–formamide mixtures, the major nucleosides/nucleotides that would accompany 1 would be 26, 29 and 30 (in addition to any products ultimately deriving fromthe ribose amino-oxazoline 21 that was a by-product in the synthesis of arabinose amino-oxazoline 12; see Supplementary Information). It is apparent that although 1 would be one of the major products, these coproducts might interfere with any subsequent incorporation of 1 into RNA. Accordingly, we sought a means of selectively destroying these co-products. Furthermore, we also hoped to find a way of converting 1 partly to the corresponding activated uracil nucleotide, β-ribouridine-2',3'-cyclic phosphate 33. It transpires that irradiation achieves both of these goals.

Limited irradiation of aqueous solutions of cytosine nucleosides with ultraviolet light having an emission maximum at 254 nm results in the reversible formation of photohydrates and partial hydrolysis to the corresponding uracil nucleosides28. Prolonged irradiation causes additional chemistry to take place29, and results in the destruction of most pyrimidine nucleosides and nucleotides (for example 26, 30 and the major nucleoside/nucleotide products deriving from ribose amino-oxazoline 21; see Supplementary Information). By contrast, however, we found that prolonged irradiation of β-ribocytidine-29,39- cyclic phosphate 1 causes significant hydrolysis to β-ribouridine- 2',3;-cyclic phosphate 33, with very little destructive photochemistry other than slight nucleobase loss; cytosine 3 and uracil 34 were both detected (Fig. 5). This finding suggests that there must be some protective mechanism functioning with 1 and 33 that does not operate with other pyrimidine nucleosides and nucleotides. Whatever the mechanism (Supplementary Information), the protection against the destructive effects of irradiation provides a means whereby 1 and 33, the two activated pyrimidine ribonucleotides needed for RNA synthesis, can be enriched relative to other end products of the assembly process we have discovered.

Now how's that for a discovery! Apparently, ultraviolet light selectively destroys the unwanted nucleosides, and enhances the production of the ones required for RNA synthesis! Which means that Step [5] above, not only provides the uracil nucleotide sought, but also provides a path for the generation of the cytosine nucleotide as well!

It really doesn't get much better than this, does it? Science, it works - bitches!