Authors: Dougal J. Ritson, John D. Sutherland
Categories: Article, Organic chemistry, Origin of life
Source: Nature chemistry
Over the past few years evidence has accrued which demonstrates that terrestrial photochemical reactions could have provided numerous (proto)biomolecules with implications for the origin of life. This chemistry simply relies on UV light, inorganic sulfur species and hydrogen cyanide. More recently, we reported that under the same conditions, reduced phosphorus species, such as those delivered by meteorites, can be oxidised to orthophosphate, generating thiophosphate in the process. Here, we describe an investigation of the properties of thiophosphate, and additional possible means for its formation on primitive Earth. We show that several reported prebiotic reactions, including the photoreduction of thioamides, carbonyl groups and cyanohydrins, can be markedly improved, and that tetroses and pentoses can be accessed from HCN through a Kiliani-Fischer-type process without progressing to higher sugars. We also demonstrate that thiophosphate allows photochemical reductive aminations, and that thiophosphate chemistry allows a plausible prebiotic synthesis of the C5 moieties used in extant terpene and terpenoid biosynthesis, namely dimethylallyl alcohol and isopentenyl alcohol.
When contemplating the chemistry which gave rise to life, one of the fundamental questions to be addressed is that concerning the set of molecules which comprised the basis from which life could emerge. As this question cannot be answered by inference from biology alone, chemical experiments are required to identify reaction pathways which could have led from simple, environmentally available feedstock molecules to (proto)biomolecules. For productive coupling of the various precursors, it is reasonable to assume that the prebiotic synthesis of the basis set of molecules occurred in reasonably close proximity on primitive Earth, rather than in disparate and distanced environments, and consequently a common type of chemistry would be expected to give rise to numerous (proto)biomolecules. Where the chemistry was confined to, at least initially, must have been defined by geology and geochemistry, hence all the chemical steps must comport with a geochemical scenario and the boundaries it imposes. Once this preliminary identification has been made, refinement of the prebiotic pathway or geochemical scenario can be informed and refined by its counterpart. For example, cyanamide (NH2CN) is an important prebiotic reagent, and the thermal conversion of Ca2[Fe(CN)6] to CaNCN with ensuing hydrolysis has been suggested as a source of NH2CN.^1^ However, under CO2-rich atmospheres, CaCO3 would be expected to precipitate rather than Ca2[Fe(CN)6].^2^ Thus, if Ca2[Fe(CN)6] is required, a reduced atmosphere must have been present, which is the expected outcome from the impact of a large, reduced meteorite.^3^ Cycling between geochemistry and prebiotic chemistry in this way should aide the improvement and plausibility of reaction pathways and the discovery of new reactions and reagents, in effect, acting as a type of triangulation.^4^
Recent reports from this laboratory have described the prebiotic synthesis of purine and pyrimidine nucleosides, precursors to amino acids and acyl glycerol phosphates, the components of the Krebs cycle and a means of harnessing and supplying chemical energy to potentially drive this inanimate collection of molecules towards life.^1,5,6,7,8^ The photochemical reduction of nitrile groups and thioamides (which can be derived from nitriles) to aldehydes constitutes a key reaction in these syntheses, and is repeated multiple times (Supplementary Fig. 1).^1^ The resulting aldehydes can be employed for further prebiotic reactions such as the Strecker synthesis of amino acids. Importantly, there is a systems chemistry aspect to this network, which would have allowed flexibility and access to alternative products from common starting materials, depending upon the conditions at a particular time or in a particular location on primitive Earth (Supplementary Discussion 1).
The geochemical scenario we have proposed that supports this chemistry has received detailed description several times,^1,9,10,11^ but broadly speaking is envisaged to be land-based, occurring in an impact or post-impact environment with prebiotic chemistry taking place in small streams (or possibly pools) which occasionally mix. The reagents and reactions required for the whole prebiotic network (Supplementary Fig. 1) are derived from and conform to this scenario, and primarily involve UV light, cyanide, ferrous iron and inorganic sulfur species, such as HS^-^. For the prebiotic scheme to work most efficiently, some separation of the chemistries is desirable, thus an interconnected system of small streams or flowing water is invoked which could allow mixing of reactants at various stages e.g. at a confluence.^9,10,11^ During the course of our studies, we found that phosphite (HPO3^2-^) and hypophosphite (H2PO2^-^) – anoxic corrosion products of reduced Ni/Fe-P mineral species found in reduced meteorites^12,13^ – could be oxidised to orthophosphate (PO4^3-^) by UV light and HS^-^,^14^ thus providing one solution to the long standing ‘phosphate problem’ and also complying with our geochemical and prebiotic model. We observed that thiophosphate (PSO3^3-^) was formed as an inevitable intermediate during this oxidation chemistry, which was noteworthy as we had previously reported PSO3^3-^ to be an efficient reagent for the formation of thioamides from nitriles and for phosphorylation reactions.^15^ As the means of production of PSO3^3-^ matched our geochemical model, a full evaluation of its potential in the context of our prebiotic reaction network (Supplementary Fig. 1)^1^ was warranted.^4^ Additionally, further assessment of the prebiotic availability and stability of PSO3^3-^ was made, as well as its in situ production and use (Supplementary Discussion 2).
Initially, we wondered if PSO3^3-^ possessed similar photochemical properties to HS^-^, which could mean HS^-^ and PSO3^3-^ were interchangeable in our prebiotic syntheses (Supplementary Fig. 1) – this seemed an attractive possibility as PSO3^3-^ is not volatile and can be formed at geologically relevant concentrations of H2S/HS^-^.^14^ As a representative example, we irradiated glycolonitrile 1 (20 mM) with low-pressure Hg lamps (principal emission at 254-256 nm) in the presence of PSO3^3-^ (20 mM) at pH 6.5. Reduction of 1 was efficient, the major products being glycolaldehyde 2 ~ 19% and glyceronitrile 4 ~ 9%, with lesser amounts of acetaldehyde 3, EtOH and lactonitrile 5 also being formed (Fig. 1, Supplementary Figs. 2–4 and Supplementary Table 1). Under identical conditions using NaSH as the reductant, 2 and 4 were produced in ~ 6% and ~ 5% yield, respectively, and only a trace amount of 3 was present (Supplementary Fig. 2 and Supplementary Table 1). Increasing the amount of PSO3^3-^ (1.5 equiv.) gave slightly improved yields after 1 h irradiation (Supplementary Fig. 2 and Supplementary Table 1). Irradiating the reaction for longer did not really affect the yield of 2 as α-deoxygenation of 2 became competitive with reduction of 1, giving increased yields of 3 and subsequent overreduction of 3 to EtOH (Supplementary Figs. 2 and 5 and Supplementary Table 1).
Fig. 1 Photochemical reduction of glycolonitrile 1 or 1 and HCN by PSO
3^3-^ yields multiple products of prebiotic interest.The major products of the reduction of 1 (horizontal reaction arrow) in the absence of phosphate are glycolaldehyde 2 and glyceronitrile 4, with lesser amounts of acetaldehyde 3, lactonitrile 5 and ethylene glycol 6; the reaction is more efficient in the presence of phosphate, with an increased yield of 6. In the presence of HCN (vertical reaction arrow), serine nitrile 8 and ethanolamine 9 are observed.
During the reaction the pH had increased (pH ~ 9.2 after 1 h irradiation), so we repeated the reduction of 1 with the inclusion of phosphate (PO4^3-^, 20 mM, 1 equiv.) as a pH buffer (pH 6.5), which is consistent with the geochemical scenario. Interestingly, reduction turned out to be much more efficient in the presence of phosphate, with glycolaldehyde 2 and glyceronitrile 4 being formed in ~ 34% and ~ 4% yield, respectively, after 1 h, in addition to increased amounts of ethylene glycol 6 (Supplementary Fig. 6 and Supplementary Table 1). Clearly, PSO3^3-^ is a far more effective reductant than HS^-^ alone and at least as efficient as the CuCN/HS^-^ system we have previously reported.^1,16^
We then attempted the in situ generation and homologation of glycolaldehyde 2 to glyceraldehyde 7 starting from 1 (20 mM) and HCN (20 mM). After 1 h irradiation, the crude ^1^H NMR spectrum indicated 2, its cyanohydrin 4 and lactonitrile 5 had been formed in ~ 4%, ~ 24% and ~ 3% yield, respectively (Fig. 1, Supplementary Fig. 7 and Supplementary Tables 2.1 and 2.2). Serine nitrile 8 (~ 16%) was also present, suggesting the nitrogen contained in HCN could be directly fixed into amino acids without the need for addition of NH3. Additionally, a small amount of glycolaldehyde imine had been reduced to ethanolamine 9. Repeating the reaction in the presence of PO4^3-^ (60 mM) at pH 6.5, we detected 7(~ 5%) and its cyanohydrin 10 (~ 13%), 2 (~ 6%), 4 (~ 9%), glycerol 11 (~ 6%) and 6 (~ 6%) after 1 h of irradiation (Fig. 2, Supplementary Fig. 8 and Supplementary Tables 2.1 and 2.2). Evaporation of HCN from the crude reaction mixture gave 2 and 7 in ~ 13% and ~ 14% overall yield, respectively, and left 6 and 11 unchanged (Fig. 2 and Supplementary Fig. 9). This solution was then left for 2 weeks at room temperature, after which 7 had partially equilibrated to dihydroxyacetone 12 (giving a ratio of ~ 1 of 7:12, although the end point for equilibration will favour 12,^17^
Fig. 2, and Supplementary Fig. 9), thus allowing the prebiotic synthesis of valine and leucine as previously described.^1^ Although HCN could have been potentially concentrated through the intermediacy of ferrocyanide,^1,9,10^ and can undergo reductive homologation to 7 under the same conditions (Supplementary Fig. 10 and Supplementary Tables 2.1 and 2.2), glycolonitrile 1 may be an equally attractive starting material given its surprisingly high boiling point (102-104 °C at 16 mm Hg^18,19^), which may have allowed its concentration in groundwater to some degree (Supplementary Fig. 11).
Fig. 2 Photochemical reductive homologation of HCN or glycolonitrile 1 + HCN by PSO
3^3-^ yields various higher sugars depending upon slight differences in starting conditions.Unless stated, reactions were carried out at ambient temperature. For convenience, aldehydes are depicted as the carbonyl compounds, but would also exist as hydrates in water. Conditions*: (±)CH2CHCN, formamide, 70 °C; formamide, CH2CHCN; Fe(CN)6^3-^, formamide; hv, formamide (see Supplementary Table 7).
As the Kiliani-Fischer-like homologation of 1 to glyceraldehyde 7 using thiophosphate had proven so efficient, we then repeated the reaction to see if the reduction products of glyceraldehyde cyanohydrin 10 were formed. The tetroses 13 (threose 13-t and erythrose 13-e) were present in the crude reaction mixture (~ 19%), in addition to 2 (~ 8%) and 7 (~ 8%, Fig. 2, Supplementary Figs. 12 and 13 and Supplementary Tables 3.1, 3.2 and 4). Little, if any, diastereoselectivity was observed with 13-t:13-e ~ 0.9:1 (Supplementary Fig. 13). The total yield of identifiable products was ~ 72%, comprising ~ 4% C1, ~ 27% C2, ~ 22% C3 and ~ 19 % C4 compounds (Supplementary Table 4).
In an attempt to make higher sugars, we increased the amount of PSO3^3-^ (100 mM) and HCN (45 mM) in the starting mixture and lowered the amount of glycolonitrile 1 (7.5 mM). After 2.75 h irradiation, the total yield of identifiable products was ~ 60%, comprising ~ 1% C1, ~ 8% C2, ~ 11% C3, ~ 11 % C4 and ~ 29% C5 compounds (Supplementary Figs. 14 and 15 and Supplementary Tables 3.1, 3.2 and 5). Although the presence of a small amount of 1 improved the efficiency of the synthesis of the pentoses 14, its inclusion was not vital, with the Kiliani-Fischer-like process forming 14 in ~ 19% yield starting from HCN alone (Supplementary Fig. 16 and Supplementary Tables 3.1 and 3.2). This constitutes an alternative one-pot, prebiotically-plausible synthesis of the tetroses 13 and pentoses 14 from a C1 feedstock molecule to that reported by Butlerow in 1861.^20^ If it is assumed that at each step of HCN homologation imine hydrolysis and cyanohydrin formation proceed quantitatively (which is clearly not the case given the presence of 6, 9 and 11), the photochemical reduction of nitriles proceeds at an average of ~ 65% yield. Indeed, the similarity between a crude reaction mixture resulting from reduction of HCN, or 1 and HCN, and a roughly equimolar mixture of 2, 7, 13-t, 14, 6 and 11, is notable (Supplementary Fig. 17). Interestingly, the hexoses were not observed in the ^1^H NMR spectra (Supplementary Fig. 18), even though the concentration of 14 was comparable to that of the C2, C3 and C4 sugars, and must be ascribed to the fact that the pentoses 14 exist almost exclusively in furanosyl 14(f) and pyranosyl 14(p) forms (in aqueous solution at circumneutral pH), with only traces (~ 0.01-0.04%) of the carbonyl forms being present (Fig. 2, Supplementary Fig. 19).^21^ Thus, a regulatory mechanism exists; by the time the pentoses 14 are being formed, the concentration of HCN is too low to force the equilibria in favour of pentose cyanohydrins and so further homologation of 14 is inhibited.
We then wondered if the transient protection of the carbonyls of 14, afforded by the cyclic hemiacetals 14(f) and 14(p), could have assisted the accumulation of 14 on early Earth. Therefore, the crude reaction mixture resulting from the reduction of 1 and HCN by PSO3^3-^ was evaporated to the point of dryness and the solid dissolved in D2O. ^1^H NMR Spectroscopy revealed that the C2, C3 and C4 sugars had decomposed (potentially via polymerisation) but the pentoses 14 were still present (< 30% decomposition of 14 taking place, Supplementary Fig. 20).
Competition experiments were then performed to examine the stability of 14 to photoreduction and in both the presence and absence of PSO3^3-^, 14-x displayed excellent resistance to UV-promoted decomposition relative to 13-t and 2 (chosen as representative examples) with the partly ring-closed tetrose 13-t (~ 90% 13(f))^22^ being ca. twice as stable as 2 (Supplementary Figs. 21 and 22 and Supplementary Table 6). Consequently, the photochemical reduction of 1 and HCN was repeated and the irradiation extended to 5 h. After this time, 2 and 7 were effectively absent from the crude reaction mixture and 13 had undergone partial decomposition, yet the concentration of 14 had increased relative to the 2.5 h timepoint (Supplementary Fig. 23). Although this presents a second potential mechanism by which the pentoses 14 could have been selected, it is not clear if this enrichment is required, as the selective crystallisation of ribo-aminooxazoline 16-r from a mixture of sugars has already been demonstrated (the aminooxazolines being formed by reaction of the sugars with cyanamide),^23^ and the elaboration of 16-r into purine and pyrimidine nucleosides has recently been reported.^5^
In order to connect to these previous studies, 1 and HCN were subjected to photoreduction using PSO3^3-^, then excess HCN was evaporated from the solution and NH2CN added. After heating for 2 h at 60 °C, conversion of 14 to 16 was observed (Fig. 2, Supplementary Fig. 24. CaCN2 was found to work equally efficiently as NH2CN,^1^
Supplementary Fig. 25). The procedure was then repeated using HCN alone as the carbon feedstock, and ribo-aminooxazoline 16-r was formed in ~ 1% overall yield (Supplementary Fig. 26). Thus, telescoped (without work-up/purification/isolation of intermediates, but with addition of reagent at a later point), prebiotically-plausible syntheses of 16-r from feedstock molecules thought to have been available on early Earth have been achieved, in 12 steps from glycolonitrile 1 or 15 steps from HCN.
Glycerol 11, a key component of all cell membranes, had been formed in several of the previous reactions, and likely resulted from the reduction of glyceraldehyde 7. We therefore assumed that 11 could be formed from the reduction of dihydroxyacetone 12 or glyceraldehyde 7, and consequently attempted the photoreduction of 12 (15 mM) by 2 equivalents of PSO3^3-^. This yielded 11 in ~ 51% after 0.5 h – almost doubling the yield previously reported and produced approximately ten times faster, yet requiring less than half of the amount of reductant (Supplementary Fig. 27).^1^ Reduction of glyceraldehyde 7 under the same conditions gave ~ 63% of 11 after 45 min irradiation (Supplementary Fig. 28). The reductions of both 12 and 7 to 11 by PSO3^3-^ were accompanied by α-deoxygenation side-reactions leading to acetone 17, and its 1,2-reduction product isopropanol, and smaller amounts of propan-1,2-diol, from 12, and propan-1,3-diol from 7 (Supplementary Figs. 27 and 28). As we had found previously (Supplementary Fig. 6), inclusion of PO4^3-^ increased the amount of 1,2-reduction, forming 11 in ~ 64% yield from 12 after 0.5 h reaction, or ~ 71% yield starting from 7 after 45 min reaction (Supplementary Fig. 29). Evaporation of water also removed the bulk of the side-products and left glycerol 11 in a highly pure and concentrated form (Supplementary Fig. 30). The one-pot synthesis of 11 could be favoured by increasing the amount of reductant (PSO3^3-^, 75 mM) and subjecting glycolonitrile 1 (10 mM) and HCN (10 mM) to irradiation for 2.5 h. Glycerol 11 was formed in ~ 27% yield, alongside ethylene glycol 6 (~ 32% yield) and minor amounts of the α-deoxygenated products mentioned above (Supplementary Fig. 31).
The ultimate fate of PSO3^3-^ is to be converted into PO4^3-^, hence the two components required to make glycerol phosphates 18, 19 and 20 under standard prebiotic phosphorylating conditions are formed in the same location. However, given that PSO3^3-^ itself is an effective phosphorylating agent,^15^ we considered reactions of 11 with PSO3^3-^. Heating 11 with PSO3^3-^ and acrylonitrile at 70 °C in formamide for 3 h,^15^ or in the absence of acrylonitrile for 10 h, gave 19 and 20, although we found that phosphorylation also occurred at room temperature in the presence of acrylonitrile (Fig. 2, Supplementary Figs. 32 – 35 and Supplementary Table 7). Low temperature phosphorylations were also made possible by activation of PSO3^3-^ with ferricyanide, or by photolysis in formamide (Supplementary Figs. 36 and 37 and Supplementary Table 7). Glycerol-1-phosphate 19 and glycerol-2-phosphate 20 were obtained in 14% – 30% yield and 4% – 9% yield, respectively, depending upon the conditions employed (Supplementary Figs. 32 – 37 and Supplementary Table 7). Furthermore, as low temperature phosphorylations were possible, cyclic phosphates such as 18, generated under more conventional conditions,^1,24^ could be avoided if desired.
The production of ethanolamine 9 (Fig. 1 and Supplementary Fig. 7), alongside glycolaldehyde 2, interested us as Orgel found that 9 is an effective catalyst for the polymerisation of nucleoside-2’,3’-cyclic phosphates,^25^ and it has also been shown that 2 is a precursor for the prebiotic synthesis of ribo-nucleoside-2’,3’-cyclic phosphates via 2-aminooxazole 21.^26,27^ Initially, we irradiated glycolaldehyde 2 (25 mM) in the presence of NH3/NH4 (150 mM) and PSO3^3-^ (100 mM) at pH 9.2 and observed ~ 14% of ethanolamine 9 after 6 h irradiation, in addition to EtOH (~ 16%) and ethylene glycol 6 (~ 11%, Supplementary Fig. 38 and Supplementary Table 8). Such high concentrations of ammonia on primitive Earth may have occurred after dissolution of Mg3N2,^1,9^ for example, but would likely only have been short lived given the volatility of NH3, and so we repeated the reaction at pH 7.0 in the presence of phosphate buffer when the majority of ammonia is protonated. Gratifyingly 9 was still formed, albeit in reduced yield (~ 6% after 6 h) in addition to EtOH (~ 12%), but the most dramatic change was the yield of 6, ~ 82% after 6 h reaction (Supplementary Fig. 38 and Supplementary Table 8). Unsurprisingly, increasing the concentration of NH3/NH4 increased the yield of 9.
We then attempted the telescoped synthesis of ethanolamine 9 and 2-aminooxazole 21 starting from glycolonitrile 1 (25 mM) and NH4Cl (75 mM) at neutral pH. After irradiating the starting mixture for 1.5 h, 2 (and its cyanohydrin 4) and 9 were present in ~ 32% and ~ 1% yield, respectively (Fig. 3) Cyanamide (50 mM) was added to the crude reaction and the solution was heated to 50 °C for 20 h, after which time 2-aminooxazole 21 could be seen in the ^1^H NMR spectrum in ~ 8% yield (based on 1, Fig. 3, and Supplementary Fig. 39 and Supplementary Table 9). In addition to 21, we determined 2-aminoimidazole 22 and 2-aminothiazole 23 were also present and had been formed in ~ 2% and ~ 9% yield, respectively (ethanolamine 9 was unchanged after reaction with cyanamide, Fig. 3, Supplementary Fig. 39 and Supplementary Table 9). Omitting ammonia from the reaction yielded 21 and 23 in ~ 7% and ~ 8% yield (3 equiv. PSO3^3-^) or ~ 16% and ~ 2% yield (1 equiv. PSO3^3-^), respectively (Supplementary Fig. 40 and Supplementary Table 9). It would seem unlikely that mercaptoacetaldehyde would form under the reaction conditions, which may give some indication of the mechanism(s) which lead to azole formation from 2 and NH2CN (Supplementary Fig. 41).
Fig. 3 Prebiotic photochemical reductive aminations and telescoped synthesis of azoles 21, 22 and 23.Irradiation of glycolonitrile 1 and NH
4Cl in the presence of PSO3^3-^ generates glycolaldehyde 2 (and its cyanohydrin 4) and ethanolamine 9. Addition of cyanamide results in the generation of azoles 21, 22 and 23. Irradiation of 9 and formaldehyde in the presence of PSO3^3-^ affords N-methyl ethanolamine 28 and N,N-dimethyl ethanolamine 29.
Thus, ethanolamine 9, 2-aminooxazole 21, 2-aminoimidazole 22 and 2-aminothiazole 23 could have been localised on early Earth. This may have implications for the prebiotic synthesis of (oligo)nucleotides as dihydroxyacetone 12 (the thermodynamically preferred triose isomer) can be converted to glyceraldehyde 7 by 23^17^ ~ the triose isomer required for reaction with 21 to yield ribo-aminooxazoline 16-r.^26^ This in turn, can be converted to ribo-nucleoside-2’,3’-cyclic phosphates,^27^ and 9 is a catalyst for the polymerisation of ribo-nucleoside-2’,3’-cyclic phosphates, producing short oligonucleotides.^25^ Finally, nucleotides activated by 22 have been suggested as labile surrogates for nucleotide triphosphates, allowing both the non-enzymatic copying of oligonucleotides^28^ and ribozyme catalysed RNA ligation.^29^ Although the full ramifications of this result warrant more detailed investigation, it is beyond the scope of the current paper. We do note, however, that the reaction of 7 (50 mM) with 2-aminooxazole 21 (50 mM) to give the pentose aminooxazolines 16 in the presence of 9 (6 mM), 22 (19 mM) and 23 (60 mM, constituting the ratio in which 9, 21, 22 and 23 were formed, see Supplementary Fig. 39) was still possible, giving 16 in ~ 30% yield (cf. ~ 40% yield in the absence of 9, 21, 22 and 23, Fig. 3 and Supplementary Fig. 42). Thus, the synthesis of short oligonucleotides via ribo-nucleoside-2′,3′-cyclic phosphates is potentially still viable from this mixture.
Whilst nucleotide-5’-phosphoro-2-aminoimidazolides are good substrates for the abiotic copying of oligomeric nucleotides,^28^ a source of chemical activation is still required. We recently showed that MeNH2
24 can be converted to methyl isonitrile (MeNC) 25 under prebiotic conditions using ferrocyanide salts (nitroprusside), and that the intermediate isonitrile complex 26 is stable and can be concentrated by evaporation (Supplementary Fig. 43).^7^ Ultraviolet light causes 26 to release 25, which, in the presence of an aldehyde or under mildly acidic conditions, can activate 5’-nucleotide monophosphates (Supplementary Fig. 43).^7,8,30^ The activated nucleotides can be intercepted in a highly efficient manner by imidazoles, such as 22, which form nucleotide-5’-phosphoro-2-aminoimidazolides in up 76% yield (Supplementary Fig. 43).^7,8,30^ Consequently, we attempted the reductive amination of formaldehyde 27 (25 mM) with NH4Cl (150 mM) using PSO3^3-^ (75 mM) and UV light at either pH 9.2 or pH 7.0 in the presence of phosphate. We were pleased to observe MeNH2
24 was formed in ~ 11% yield after 2 h reaction at pH 9.2 and in ~ 26% yield at pH 7.0 (Supplementary Fig. 44 and Supplementary Table 10). Halving the concentration of NH4Cl returned ~ 10% of 24 at either pH 7.0 or 9.2 (Supplementary Table 10).
Given the success of the reductive methylation of ammonia using 27 and PSO3^3-^, we next considered the methylation of ethanolamine 9. Irradiation of 9 (30 mM) and 27 (90 mM) in the presence of PSO3^3-^ (120 mM) gave N-methyl ethanolamine 28 in ~ 46% yield and N,N-dimethyl ethanolamine 29 in ~ 3% yield after 4 h reaction (Supplementary Fig. 45). Addition of more reductant (40 mM) followed by two cycles of addition of 27 (20 mM) then irradiation resulted in further methylation of the amines, giving 28 in ~ 56% yield and 29 in ~ 25% yield with only ~ 19% of 9 remaining (Supplementary Fig. 45), a process which could presumably be repeated further. Phosphorylation of 9, 28 and 29 (50 mM each) was achieved using PSO3^3-^ (50 mM) and Fe(CN6)^3-^ (100 mM) in formamide, leading to O-phosphorylethanolamine 30, N-methylethanolamine phosphate 31 and N,N-dimethylethanolamine phosphate 32 in ~ 16%, ~ 12% and ~ 26% yield, respectively (Supplementary Figs. 46 and 47). These products are reminiscent of the intermediates used by phosphatidylethanolamine N-methyltransferase in bacteria,^31^ and so a facile transition to the biosynthesis of primitive versions of phosphatidylcholine can be imagined.
We then considered thioamides, functional groups which are integral to our protometabolic network (Supplementary Fig. 1).^1^ We had already shown that cyanohydrins can be cleanly converted to α-hydroxythioamides by incubation with PSO3^3-^,^15^ and it now appeared that the products of thiolysis could be reduced by the same reagent. As a representative example, glycolonitrile 1 (20 mM) was reacted with PSO3^3-^ (80 mM) at 65 °C and pH 6.5 for 20 h, which gave α-hydroxythioacetamide 33 in ~ 97% yield (Fig. 4 and Supplementary Fig. 48). Addition of a second portion of PSO3^3-^ (80 mM) followed by irradiation at 254 nm for 4 h gave the expected reduction products thioacetamide 34 (~ 23%) and acetaldehyde 3 (~ 17%), and unexpectedly glycolaldehyde 2 (~ 14%, Fig. 4 and Supplementary Fig. 48). Previously, when an α-hydroxythioamide e.g. 2,2-dimethyl-2-hydroxythioacetamide 35, was subjected to HS^-^/CuCN photoreduction, clean α-deoxygenation occurred to furnish the corresponding thioamide i.e. isobutyryl thioamide 36, and ensuing reduction then afforded the corresponding aldehyde i.e. isobutyraldehyde 37, – α-hydroxyaldehydes were not observed (Fig. 4).^1^ This unexpected mode of reactivity of thiophosphate presented an intriguing possibility. If the reduction of 35 yielded some 2-hydroxy-2-methylpropanal 38, homologation (via cyanide addition, thiolysis and reduction) could lead to 3-methyl-1,3-butanediol 39 (Fig. 4). We expected phosphorylation of 39 under heating conditions to temporarily form the cyclic phosphate 40, which should be primed for elimination of the phosphate dianion at the tertiary centre, thereby affording dimethylallyl phosphate 41 and isopentenyl phosphate 42. These structures are analogous to the biosynthetic precursors of terpenes (dimethylallyl pyrophosphate and isopentenyl pyrophosphate) which are generally used to make secondary metabolites. Archaea, however, are absolutely dependent on linear isoprenoids for cell membrane formation, constituting the hydrophobic moiety of their phospholipids.
Fig. 4 The unexpected synthesis of glycolaldehyde 2 from photochemical reduction of α-hydroxythioacetamide 33 by thiophosphate (top), and a possible route to potential isoprenoid precursors 41 and 42 (dotted arrows).Unless stated, reactions were carried out at ambient temperature. For convenience, unidirectional reaction arrows are used and aldehydes are depicted as carbonyl compounds, although they would also exist as hydrates in water.
Acetone 17 (50 mM), a by-product from glycerol 11 synthesis (see Fig. 2, Supplementary Fig. 27 and Reference 1), was incubated with HCN (100 mM) and PSO3^3-^ (250 mM) at 50 °C for 4 h, which afforded ~ 29% of 35 (Fig. 5 and Supplementary Fig. 49). Gentle sparging with N2 left 35 as the sole product (Supplementary Fig. 49). The remaining solution was then irradiated for 4 h, after which time all of 35 had been consumed (Supplementary Fig. 49). Addition of HCN (30 mM) resulted in the formation of cyanohydrin 43 in ~ 27% yield starting from 35, or ~ 8% overall yield from starting from acetone 17 (4 steps, Fig. 5 and Supplementary Fig. 49). Although some α-deoxygenation took place, none of the resulting thioamide 36 or its secondary reduction product (isobutyraldehyde 37) were observed in the ^1^H NMR spectrum, only the fully reduced product isobutanol was present (~ 20% yield, Supplementary Fig. 49).
Fig. 5 Prebiotic route to potential isoprenoid precursors.Reactions were carried out at ambient temperature unless stated otherwise. For convenience, unidirectional reaction arrows are used and aldehydes are depicted as carbonyl compounds, although they would also exist as hydrates in water.
We investigated the next sequence of reactions starting from a prepared sample of 43 (90 mM). Thiolysis of 43 by PSO3^3-^ (545 mM) proceeded more slowly than some cyanohydrins, but α-hydroxythioamide 44 was obtained in good yield (~ 64%) after 2 days (Supplementary Fig. 50 and Supplementary Table 11). Although lower concentrations of PSO3^3-^ could be used for the reaction, thiolysis was slower. Using PSO3^3-^ (200 mM) under analogous conditions, ~ 41% of 44 had formed after 3 d reaction (Supplementary Table 11). Addition of further PSO3^3-^ followed by UV irradiation, fortuitously, gave the α-deoxygenated, fully reduced compound 39 as the major product (~ 40% yield starting from 38, Fig. 5 and Supplementary Fig. 50. 3-Methyl-1,2,3-butanetriol was present, but it was only produced in ~ 16% yield, Supplementary Fig. 50). Whilst the steps in this synthesis are high yielding, sequential addition of cyanide would likely be required to form 39 most efficiently, and this could potentially have been achieved by the confluence of a cyanide-rich stream with the reaction stream.^9,10,11^
Recognising that forcing conditions would be needed to eliminate H2O from 3-methyl-1,3-butane diol 39, we dissolved 39 (100 mM) in formamide with ammonium phosphate (300 mM) and heated the reaction at 150 °C. After 22 h, isopentenyl alcohol 45 (~ 48% yield), isopentenyl phosphate 42 (~ 6% yield), and dimethylallyl alcohol 46 (~ 3% yield) could be observed by ^1^H NMR spectroscopy (Fig. 5, Supplementary Figs. 51 and 52). For laboratory convenience we heated the reactions to 150 °C, but the reaction gave comparable results when run for longer periods of time at lower temperatures (Supplementary Fig. 53 and Supplementary Table 12), and on the geologic timescale, even lower temperatures may have sufficed (whilst not explored, metals ions and acid catalysis may also lead to elimination of water from 39). It is not possible to determine if the elimination step occurs via cyclic phosphate 40, although we did note the presence of 40 in the reaction mixture. As thiophosphate can phosphorylate alcohols under much milder conditions, we mixed 46 (50 mM) with PSO3^3-^ (100mM) in formamide and activated PSO3^3-^ (either with ferricyanide or UV light) which gave dimethylallyl phosphate 41 in ~ 10% yield (Supplementary Figs. 54 and 55).
Evaluation of the first prebiotic synthesis of activated pyrimidine ribonucleotides,^26^ in terms of a geological/geochemical scenario which could satiate the requirements of that synthetic route, led us to consider meteoritic impacts.^1^ Whilst potentially providing a localised, abundant source of phosphorus species,^12,13,32^ HCN also would be generated by those same impacts.^33,34^ This implied that cyanometallates would be present in the same location, and we then showed that these compounds are effective catalysts for the photochemical reduction of HCN and nitriles using inorganic sulfur species as the stoichiometric reductant.^1,9,16,35,36^ Returning full circle, we reassessed the processing of meteoritie-derived phosphorus species under photochemical conditions in the presence of HS^-^/H2S to find that hypophosphite and phosphite could be oxidised to phosphate, and during which a new reagent was formed – PSO3^3-^.^14^ In a further iteration of this process, we have now examined PSO3^3-^ in the context of our cyanosulfidic network, which has led to marked improvements over existing prebiotic pathways and allows entirely different ones. Most notably, we demonstrate a one-pot, prebiotic synthesis of C2-C5 sugars from a C1 feedstock and prebiotic access to isopentenyl alcohol 45 and dimethylallyl alcohol 46, or phosphates thereof – the biological precursors of terpenes.
Although the mixture of C2-C5 sugars can be enriched in pentoses 14, several reported prebiotic routes to ribonucleotides requiring (D)-ribose^37,38,39^ will be confounded by the lack of stereoselectivity of the current synthesis of 14. Ribo-aminooxazoline 16-r, offers a potential solution to this problem given its diastereoselective, and even enantioselective, crystallisation, which can occur under the appropriate conditions.^23,40,41^ Although 16-r can be easily accessed from the C2-C5 sugar mixture (Fig. 2, Supplementary Figs. 24–26), it could also be derived after aldol reaction of glycolaldehyde 2 and glyceraldehyde 7 (Fig. 2),^42^ which can be formed in almost equimolar amounts from HCN or HCN and 1 using thiophosphate as the reductant (Supplementary Figs. 8–10). Still a third avenue for the synthesis of 16-r exists, and proceeds through 2-aminooxazole 21 by reaction with 7 (Fig. 3, Supplementary Fig. 42),^26^ possibly requiring 2-aminothiazole 23 (Supplementary Figs. 39 and 40) to separate 2 and 7.^17^ Studies can now be undertaken to determine which of these routes is the most promising for a telescoped synthesis of (deoxy)ribonucleosides from feedstock molecules.
All reactions were run at least twice, and NMR spectra shown in the Supplementary Information are representative examples. Reagents and solvents were bought from Sigma-Aldrich, Alfa Aesar and Santa Cruz Biotechnology and were used without further purification, apart from thiophosphate which had variable amounts of impurities and H2O present – see Supplementary Information p. 1. Reagents were weighed using a Sartorius AX124 M-Pact analytical balance and small volumes were measured using a Gilson™ Pipetman™. Photochemical reactions were carried out using a Rayonet RPR-200 photochemical reactor chamber, with cooling fans switched on (the internal temperature of the unit when operational was approximately 40 °C) and fitted with low pressure RPR-2537A Hg lamps purchased from Rayonet (principal emission 254 nm). Hellman QS Spectrosil 10.0 mm quartz cuvettes with 4 UV transparent windows were used for photochemical reactions. A Mettler Toledo SevenMulti pH/mv module fitted with a Thermo Scientific Orion 8103BN pH probe was used to measure pH, and deoxygenation of solvents and HCl/NaOH solutions was achieved by sparging with Ar for 20-30 min before use. Although deoxygenation of HCl and NaOH solutions, used to adjust the pH of reactions, may have altered the concentrations of these solutions, it was not deemed to be important as adjustment of the pH of the reaction was our only consideration. Although using solvents which had not been deoxygenated was not anticipated to cause a significant difference to the outcome of the reactions, we wished to ensure reaction of O2 with any sulfur species was kept to a minimum, whilst also comporting with the expected anoxic environment of early Earth. The removal of dissolved O2 from aqueous media by sparging with an inert gas has been shown to be effective, although trace amounts of dissolved O2 may remain.^43^ Rigorous exclusion of O2 from solutions after deoxygenation was not possible, particularly when checking/adjusting the pH where the solution would typically be exposed to the atmosphere for ~ 45 s. For comparison, a thiolysis and reduction reaction were run in ‘oxygenated’ solvents i.e. not deoxygenated before use, using thiophosphate (see Supplementary Figs. 75 and 76 (p. 91-92)). ^1^H, ^31^P And ^13^C NMR spectra were acquired using a Bruker Ultrashield™ 400 Plus (at 400.1, 162.0 and 100.6 MHz, respectively) or alternatively, ^3^H and ^31^P NMR spectra were recorded using a Bruker Ascend™ 400 (at 400.2 and 162.0 MHz, respectively) using solvent suppression to collect ^3^H NMR data if reactions were run in a D2O/H2O mixture. If spectra were unsatisfactory, a small amount of D2O was added to the NMR sample and the spectrum reacquired. Yields were determined by relative integration of signals in ^1^H or ^31^P NMR spectra, or by addition of a standard of known volume and concentration and relative integration to this signal. For quantitative integration of phosphorus NMR signals, we used a Bruker Avance-II 500 spectrometer with broadband detect cryogenic probe at a ^31^P frequency of 202.4 MHz. Quantitative integration of phosphorus NMR signals was achieved by determining the relaxation time (T1) for the nucleus which was slowest to relax (thiophosphate, 8.4 s). ^31^P Quantitative NMR (qNMR) spectra were acquired with 30° pulse flip angle, >7 x T1 relaxation delay (giving > 99% relaxation of ^31^P nuclei), 160 ppm spectral width, 128k acquisition data points and the spectrum offset close to the midpoint frequency of peaks being integrated. Spectra were processed and quantified using TopSpin version 3.2 software. Coupling constants (J) are given in Hertz and the notations d, t and q represent the multiplicities doublet, triplet and quartet. Chemical shifts (δ) are given in ppm. Mass spectra were recorded with an Agilent Technologies 6130 Quadrupole LCMS using positive and negative Electron Spray Ionisation.
We would like to thank S. J. Mojzsis for helpful discussions and we are grateful to T. Rutherford for invaluable assistance with NMR spectroscopy. This work was supported by the Medical Research Council, as part of United Kingdom Research and Innovation (also known as UK Research and Innovation) [Grant No. MC_UP_A024_1009 to J.D.S.] and a grant from the Simons Foundation [Grant No. 290362 to J.D.S]. For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising.
Dougal J. Ritson, Email: dritson@mrc-lmb.cam.ac.uk.
John D. Sutherland, Email: johns@mrc-lmb.cam.ac.uk.
All data associated with this study are available in the published article and its supplementary information.
All data associated with this study are available in the published article and its supplementary information.