# A scope of prebiotic neat reaction conditions and the mechanism of urea-assisted phosphorylations of alcohols

Anastasiia Shvetsova, Lynda Merzoud, Augustin Lopez, Elodie Fromentin, Anne Baudouin, Henry Chermette, Isabelle Daniel, Michele Fiore, Peter Strazewski

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## ► To cite this version:

Anastasiia Shvetsova, Lynda Merzoud, Augustin Lopez, Elodie Fromentin, Anne Baudouin, et al. A scope of prebiotic neat reaction conditions and the mechanism of urea-assisted phosphorylations of alcohols. Nature Communications, 2025, 16 (1), pp.8929. 10.1038/s41467-025-63307-3. hal-05380117

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Submitted on 24 Nov 2025

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**Title:**

A scope of prebiotic neat reaction conditions and the mechanism of urea-assisted phosphorylations of alcohols

**Author list:**

Anastasiia Shvetsova,¹,² Lynda Merzoud,³ Augustin Lopez,¹ Elodie Fromentin,¹ Anne Baudouin,¹ Henry Chermette,³ Isabelle Daniel,² Michele Fiore¹ & Peter Strazewski¹✉

**Affiliations:**

¹Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR5246, CNRS, Université Claude Bernard Lyon 1, Villeurbanne, F-69622, France. ²Laboratoire de Géologie de Lyon–Terre, Planètes et Environnement, UMR5276, CNRS, Université Claude Bernard Lyon 1, Villeurbanne, F-69622, France. ³Institut de Chimie Analytique, UMR5280, CNRS, Université Claude Bernard Lyon 1, Villeurbanne, F-69622, France. ✉email: strazewski@univ-lyon1.fr

**Abstract:**

Two theories of the origin of life on Earth, one located in the Hadean seafloor, the other on the surface of subaerial landmasses and basins, need reconcilement. Natural energy flows moulded seafloors to locally sustain chemical reaction networks reminiscent of metabolism. Subaerial hot milieus gathered organic phosphates to provide robust compartments from phospholipids and heredity from nucleic acid replication and translation. Here, we report on the efficiency and product distribution of the phosphorylation of twelve model alcohols reacting separately, and in selected combinations, all under the same chemically activated conditions, mostly as neat hot solid-liquid mixtures. We afford proof for the dominant reaction mechanism and indicate how prebiotic seafloor and subaerial systems could profit from one another through atmospheric and tidal exchange of organic material.

**Introduction**

Phosphate takes an indispensable role in all three kingdoms of life, Bacteria, Archaea and Eukaryota, and currently represents as much as five gigatons of phosphorous in the biosphere¹,². Before the advent of enzymatic catalysis and the emergence of primary metabolic reaction networks³, and also before organic phosphate esters could accumulate on the early Earth and serve as available phosphate sources themselves⁴, they must have been first formed under abiotic conditions from organic precursor molecules and dissolved phosphate minerals⁵⁻¹⁰. The thermodynamics in water are challenging, since the endergonic nature of the phosphorylation of typical organic precursors with soluble inorganic orthophosphate (Pᵢ) requires chemical energy¹¹,¹².

Out of many proposed suitable prebiotic activation conditions, relatively long-lasting non-volatile reservoirs of cyanate (NCO⁻) and inorganic carbamoyl phosphate (CPᵢ), from NCO⁻ + O₂P(OH)₂⁻ ⇌ H₂NCOOPO₃²⁻ being present in inorganic phosphate-urea blends, seem most attractive for their reactivity with amines as N-carbamoylation agents¹³,¹⁴ and as O-phosphorylation agents for hydroxyl-bearing organic precursors¹⁵. However, the lately growing number of different prebiotic dehydrating conditions¹⁶⁻¹⁹ proposed to have furnished crucial organic phosphate esters shortly before the first living cells have emerged in the late Hadean and early Archean eras²⁰,²¹, leave at present the scientific community with a very large range of possibilities that lack thus far appropriate experimental comparison.
The Earth's first solid subaerial locations amenable to accumulate on their surface significant amounts of organic material were likely recurrently wetted with rivulets from hot springs<sup>22</sup> or ocean seawater spawning higher waves<sup>20</sup> and tides more violently rising and retrieving with the rhythm of the moon that orbited closer to Earth and faster than today<sup>23,24</sup>. Rocky surfaces were thus provided with water containing mostly hydrogen- and oxygen-bearing organics. Such compounds could accumulate over longer periods of time, unless they were too volatile, together with a lower abundance of non-volatile N-heterocycles, amino acids and soluble inorganic phosphate sources<sup>25-28</sup>. Regarding reduced-nitrogen compounds, there is a faint possibility that solid ammonia-formaldehyde stockpiles might have been blended with inorganic phosphides corroding to phosphites and phosphates at locations where large deposits of hexamethylenetetramine (HMT, sublimation at 280 °C and 1 atm) would be wetted, that had been delivered from asteroid impact<sup>29</sup> together with schreibersite<sup>30,31</sup>. Usually however, at daytime, shallow basins were periodically drying out, some likely within a few hours, consequently, electron-rich N-volatiles such as ammonia, methyl amine or hydrolytically unstable N-methyl isocyanide could barely amass or be "fixed" in less volatile and highly energetic deposits, for instance, as inorganic mono- and diamido-phosphates<sup>13,32,33,34</sup>. With such a very early, amine-poor, mostly hot and dry scenario in mind, being limited in badly soluble inorganic phosphate, we wondered if the phosphorylation of important prebiotic organic precursors could have happened in the absence of liquid water, in neat reaction conditions.

Here, we show how the phosphorylation of the abiotic or biotic precursors **5-16** (Fig. 1) of membrane components, nucleotides and phosphoenol pyruvate occurs under mostly neat and hot conditions (minimum 60, mostly 115, maximum 130 °C) in the solid or mixed solid-liquid phase, without episodic or periodic wet-dry cycling<sup>17,35</sup>, in the presence of an equimolar quantity of a pre-mixed (as if evaporated from aqueous) inorganic phosphate source $P_i^{28}$, $SP_i^{19,36}$, $PP_i$ or cTMP<sup>37,38</sup> (Fig. 1) and one of the most simple, moderately volatile compounds **1-4** bearing reduced nitrogen that could have been very early, abiotically "fixed" from atmospheric $N_2^{21,39-44}$. Our results demonstrate that the quantified efficiency of alcohol phosphorylation in neat conditions is particularly high for triols and diols (**5**, **6** and **11-15**); it works best when assisted by urea (**2a**) or its hydrolytic precursor cyanamide (**1**), whereas mono-functional alcohols (**7-10**, **16a**) are hardly phosphorylated in the same neat conditions, or not at all. We show how the different phosphate sources compare with one another, and confirm that neat cTMP efficiently phosphorylates only **5** and **6** but not the nucleosides (**11-15**). We provide proof of the reaction mechanism of urea-assisted phosphorylations by quantum theoretical and experimental means; the latter, in a time-dependent fashion and with the help of reagents enriched with the stable isotopes <sup>15</sup>N (**2b**, **3b**, **4b**), <sup>13</sup>C (**2c**) and <sup>18</sup>O in inorganic orthophosphate [<sup>18</sup>O<sub>4</sub>]P<sub>i</sub>. We determine the molecular structure of a wide range of different phosphorylated organic products, identified in part by additional blending the mixtures with glyceryl phosphate (**5a**), an equimolar mixture of decanoic, undecanoic, dodecanoic and tridecanoic acids (**7abcd**), one of the nucleoside-5'-monophosphates (NMP **11a-14a**), phosphoenol pyruvate (**16b**), or one of the amino acids L-alanine, L-valine or D-valine (**22abc**); see Supplementary Table 1 for a complete list. We conclude on how the different starting alcohols and other compounds, that could have been present in dried-out complex prebiotic mixtures, must have competed for inorganic phosphate sources available on the subaerial landmasses of the late Hadean era. Finally, we indicate how important and useful such arid environments could have been for the emergence of the first living cells forming under water at the ocean seafloor.

(here Fig. 1)

## Results

*Scope of neat phosphorylation conditions*

Briefly, in a nut shell: out of all tested alcohols **5-16a** we have found that their blending with $P_i$ and cyanamide (**1**) or urea (**2**) in 'dry' mixtures, the latter containing initially about 4 to 15 weight %
residual water, were generally the most resourceful and robust neat phosphorylation conditions on the triols **5, 11-14** and diols **6** and **15** in providing some endurance by way of forming organic carbamates and cyclic carbonates in addition to phosphate esters; mostly acyclic primary phosphates for **5** and **6**, predominantly 2',3'-cyclic phosphates for **11-14**. Slightly acidic neat **2** + $P_i$ mixtures (better than **1** + $P_i$) with excess **2** were much more beneficial for **11-15** than alkaline neat mixtures, where $SP_i$, $PP_i$ and cTMP proved superior for **5** and **6** but did not phosphorylate **11-15** at all. The presence of L-valine suppressed somewhat the phosphorylation of **11-14** by **2** + $P_i$, and more so than D-valine, suggesting a tighter complexation between D-ribonucleosides and L-valine than D-valine. Heated neat blends of **2** + $P_i$ with **5, 7abcd** and **11-14** contained up to diglyceryl, dinucleosidyl and glyceryl-nucleosidyl phosphodiester derivatives (Supplementary Fig. 145). We were sometimes successful in observing vesicles upon rehydrating crude phosphorylation extracts containing MPG phosphates from heated neat **6** + **2** + $P_i$ (Supplementary Fig. 197). By contrast, all precursor molecules furnished with only one hydroxyl group (the monools **7, 8, 9, 10** and enol **16a**) were by far less efficiently phosphorylated in all tested conditions, or not at all (Fig. 2).

(here Fig. 2)

## Kinetics and mechanism of neat urea-assisted phosphorylation conditions

Given the importance of neat hot urea-phosphate blends, we investigated the kinetics of exemplary phosphorylation reactions taking place at 115 °C in the solid or mixed solid-liquid state. The transient presence of NCO⁻ in neat urea + $P_i$ was proven by the observation of [¹⁴N,¹⁵N]urea from the reaction time-dependent exchange between natural urea (**2a**) and [¹⁵N₂]urea (**2b**) in hot neat mixtures of **5** + **2a** + **2b** + $P_i$ and **6** + **2a** + **2b** + $P_i$ (red filled circles in Fig. 3, see also Supplementary Figs. 47-53). We determined the apparent kinetic rate constants (Fig. 3) of the consumption of **2ab**, $P_i$ and the formation of well quantified products in the first 4-8 hours (major carbamates and cyclic carbonates), and identified quite long-lasting acyclic carbonylated diglyceryl phosphodiester derivatives that began to appear after 4-8 hours at 115°C (Fig. 4C).

(here Fig. 3)

We have also elucidated the chemical mechanism of this reaction by catching all gasses in specific traps — in the form of benzamide and benzoic acid (white zones in Fig. 4) — that escaped from neat mixtures containing inorganic phosphate that was highly enriched in the ¹⁸O isotope. We could thus dismiss the urea-catalysed phosphate dehydration pathway⁴⁵ and confirmed this by density functional theory (DFT) calculations, where a dihydrogen phosphate anion was placed between three molecules of urea and, in a second calculation, dihydrogen phosphate was surrounded by three ureas and two assisting water molecules, and then a methanol molecule was added (Figs. 4A, 4B and Supplementary Figs. 13-15). The calculated rate-limiting step (TS-1, Fig. 4A) on the urea-catalysed phosphate dehydration pathway (Fig. 4A, elementary step sequence E-A<sub>N</sub>-E-A<sub>N</sub>) proved too high, both, in neat urea (denoted U3W0) and in urea containing water molecules in a ratio 3:2 per dihydrogenphosphate (U3W2). The dissociative phosphate activation pathway (Fig. 4B, elementary step sequence taut-E-taut-A<sub>N</sub>-A<sub>N</sub>-E-E), whereby dihydrogen phosphate first tautomerizes urea to its zitterionic form (Int-1, Fig. 4B), either in the absence of water (rate-limiting direct tautomerization shown in Fig. 4B for U3W0) or in its presence (U3W2) through a proton-relay by an assisting water molecule (blue TS-1 in Supplementary Fig. 15), was calculated to be faster, especially when relayed. This zitterionic urea tautomer is predisposed to eliminate ammonia and isocyanic acid thereby generating with $P_i$ inorganic carbamoylphosphate ($CP_i$) on a relatively smooth potential energy surface. In fact, the calculated trajectory-determining transition state (highlighted bright yellow in Fig. 4B) provides a good rationale for the time-dependent formation of carbamoylated and carbonylated organic products found experimentally (Fig. 4C).

(here Fig. 4)
Discussion

Heating time-dependent selectivity of the urea-assisted phosphorylation of glycerol (5) and MPG (6):
Unlike glycerol phosphorylations carried out at 120 °C in DMF with a 10-fold excess urea and 20
mol% ammonium dihydrogenphosphate¹⁶, the organophosphates obtained here were
predominantly acyclic phosphoric mono- and -diesters, where the primary alcohols were always
more abundantly phosphorylated than the secondary (usually ~ 5:1 RCH₂OPO₃²⁻/R₂CHOPO₃²⁻, cf.
Supplementary Fig. 19 and Fig. 21C), and cyclic phosphates eventually appeared too, both, 5-
membered rings (after 30 minutes) and 6-membered rings (after about 8 hours) accompanied by the
formation of primary carbamates and cyclic carbonates at the remaining alcohol groups (Fig. 3). We
have recurrently observed and characterised in the DMSO extracts the formation of diglyceryl
phosphodiester compounds starting to appear after 8 hours at 115 °C (Fig. 4C and Supplementary
Figs. 45 and 46), which has an important bearing on the prebiotic appearance of precursors of
phospholipids having phosphoglyceryl (PG) and phosphoglyceryl-(cyclic-)phosphate (PGP, PGcP)
polar headgroups widely used in extant biological membranes. Both, cyclisations and the formation
of phosphodiesters require another phosphate activation and the elimination of a second water
molecule. Very small amounts of PPᵢ could be sometimes detected but hardly any sign of inorganic
polyphosphates Pₙᵢ⁴⁶, no cyclic metaphosphates or branched ultraphosphates⁴⁷. In six experiments at
3, 6 and 30 mmol scales (Supplementary Table 11), where equimolar mixtures of urea, Pᵢ and
glycerol (5) were kept at 115 °C, 88-98 % Pᵢ consumption were measured.

MPG phosphorylation at the same scales, viz. in 15 experiments starting from equimolar neat
mixtures containing natural urea (2a) or [¹³C]urea (2c)/Pᵢ/6 (1:1:1), was sometimes less complete:
69-95 % Pᵢ consumption were seen after 5 days at 115 °C (Supplementary Table 19). More than half
of the MPG appeared depalmitoylated in these mixtures (Supplementary Figs. 30-32) but, on the
other hand, dipalmitoylglyceryl phosphates were also found (Supplementary Fig. 33). Comparable
amounts of carbamoylated 6 (when compared to carabamoylated 5) appeared somewhat delayed
(up to 16 % after 16 hours) and cyclic MPG carbonate (about 9 % after 24 hours, Supplementary Fig.
53). At the 0.5 mmol scale we detected that Pᵢ consumption reached an end after 4 hours showing
an exponential rate constant at 115 °C that was essentially the same as in the glycerol
phosphorylation reaction (Fig. 3). We compared this time-dependent Pᵢ consumption from ³¹P NMR
peak integration with the time-dependent decay of 6 from HPLC peak areas of the same crude
mixture but extracted with methanol and calibrated using pure 6. The exponential fitting on MPG
consumption showed a thirteen-fold slower apparent rate constant than that of the corresponding Pᵢ
consumption (Supplementary Table 18), which suggested the presence of some intermediate
inorganic compound that could have accumulated in the first 2-8 hours of heating (Supplementary
Fig. 53B). Nevertheless, owing to the necessary extraction procedure, we have never found direct
spectroscopic evidence for the unstable inorganic reaction intermediates [¹³C]cyanate (N¹³CO⁻) or
[¹³C]carbamoyl phosphate (CPᵢ), cf. section 4.4 in the Supplementary Information (SI).
Notwithstanding, the transient presence of CPᵢ in these neat blends has been proven by the
exchange of nitrogen isotopes on urea while heating the mixture in the actual reaction conditions
(Supplementary Tables 10 and 13).

Scope of neat glycerol (5) and MPG (6) phosphorylation comparing different dehydrating conditions and inorganic phosphate sources (summary from sections 7.1-7.4 in the SI): We summarise here the most important features shown in the Supplementary Fig. 57 (5 + Pᵢ), Fig. 61 (6 + Pᵢ at 115°C), Fig. 62 (6 + Pᵢ at 75°C), Fig. 68 (5 + cTMP), Fig. 71 (6 + cTMP), Fig. 77 (5 + SPᵢ at 115°C), Fig. 78 (5 + SPᵢ at 75°C) and Fig. 85 (5/6 + minerals containing Pᵢ or PPᵢ). 6 actually required the presence of 2a to become efficiently phosphorylated at 115 °C, without urea at most 10 % Pᵢ was converted to organophosphates in our neat, hot conditions. By contrast, 5 was phosphorylated even in the absence of 2a showing Pᵢ conversions 36-77 % after 4-5 days at 115 °C, presumably through the intermediate formation of PPᵢ. When Pᵢ and 5 or 6 were heated to a mere 75 °C for 5 days, 1:1 neat
and in the absence of **2a**, zero Pᵢ consumption was seen with **6** and at most 2-4 % Pᵢ consumption with **5**. In the equimolar presence of **1** or **2a**, however, both **5** and **6** contributed to 65-77 % Pᵢ consumption after 5 days at 75 °C.

This meant that evaporative conditions in arid environments with relatively strong temperature variations below and above the boiling point of water could lead to very efficient phosphorylations of both glycerol (5) and MPG (6) when urea (2a) was present. Such conditions may have been dominant on the first volcanic islands in the late Hadean gobal ocean, especially, under the influence of more intense and more rapid temperature variations during the short day-night rythm of that era.

With increasing molar excesses of urea, 6/Pᵢ/2a = 1:1:1 ; 1:1:2 ; 1:1:4 ; 1:1:10, we observed an increase in Pᵢ consumption up to 95 % at 115 °C. Higher urea excesses expectedly led to more 5- and 6-membered cyclic phosphates. When 2a was replaced with cyanamide (1), or Pᵢ was replaced with cTMP, 5 and 6 were efficiently phosphorylated at both 115°C and 75°C in the dry state. With cTMP at 115°C, but not 75°C, 5 became partly carbonylated even in the absence of 2a or 1 (minimum 8.6 %, cf. Supplementary Table 30). This meant that CO₂ from the air could be activated by cTMP at 115 °C and transferred to 5, its glyceryl phosphate derivatives or 6, to appear as 1,2-cyclic carbonates (cf. Supplementary Fig. 69). However, when 1, 2a or 2c were replaced with equimolar amounts of one of the carboxamides, either formamide (3a), acetamide (4a) or their N-methyl substituted derivatives (3c, 4c, Fig. 1), the phosphorylation of 6 (measured as Pᵢ consumption) dropped to less than 10 % unless 2-4-fold molar excesses of 3a were used at 115 °C, whereas the phosphorylation efficiency of 5 by Pᵢ could be maintained at 60-82 % in equimolar dry mixtures containing any of the carboxamides, but only at 115 °C. The use of 2 mol equivalents [¹⁵N]formamide (3b) or [¹⁵N]acetamide (4b) produced ¹⁵NH₃ at 115°C that was trapped by benzoyl chloride.

Altogether, these experiments showed that the carboxamides did not accelerate the phosphorylation of 5 or 6 nor make the reaction more efficient. Instead, when 3a partly degraded at high temperatures⁴³ and there was still more liquid formamide left to keep the 'dry' solid-liquid mixture reasonably mobile, as was most likely the case when 2-4 mol equivalents 3a were initially added — then only 5, not 6 (nor any other alcohol), could be phosphorylated to a significant degree. When one of three minerals (or their analogue, cf. Fig. 1) were used as the phosphate source, that is, struvite (ammonium-magnesium Pᵢ), vivianite (ferrous Pᵢ) and the canaphite analog calcium PPᵢ, we measured up to 74 % Pᵢ consumption in the presence of 5 (Fig. 2 and Supplementary Fig. 85), which did not come by surprise, except that our attempts to phosphorylate 6 were unsuccessful, and the spectroscopic results from using natural vivianite powder (Fe₃(Pᵢ)₂) should be taken with caution, both for analytical reasons.

We also compared mono-, di-, and trisodium phosphates (Pᵢ) showing experimental pH values of equimolar mixtures with 5 and 2a in water of 4.3, 8.9 and 12.8, respectively (Supplementary Table 29). The total conversion of Pᵢ into glyceryl phosphates in the absence and presence of 2a grew from 0 % (pH 12.8) through 36 % (pH 8.9) up to 98 % (pH 4.3) in accordance with the increase in protonation degree, i.e., tribasic < dibasic < monobasic Pᵢ. By contrast, when compared to urea-assisted reactions containing NaH₂PO₄ as the only phosphate source, tribasic sodium thiophosphate (SPᵢ at pH 11.6) led to the phosphorylation of 5 under 'dry' conditions when heated to 115°C for 5 days, both, in the absence of any nitrogenous compound (22 % glyceryl phosphates) and in the presence of equimolar 2a or 1 (28-30 % glyceryl phosphates) along with higher amounts of condensed inorganic and organic phosphates (9-35 % PPᵢ and 1 % glyceryl diphosphates). The phosphorylation selectivity of primary versus secondary hydroxyls was not much more than 3:1 using SPᵢ (Supplementary Tables 32 and 33), thus, diminished when compared to Pᵢ or cTMP (≈ 5:1). A mixture of mono- and tribasic inorganic (thio)phosphates in 'dry' 5/2a/NaH₂PO₄/Na₃SPO₃ = 1:1:0.5:0.5 (pH 7.5 in water) provided protons for both leaving groups, H₂S from SPᵢ and NH₃ from urea, which increased the total conversion to 36 % at 115°C, while the efficiency of an equimolar di-
and tribasic dry mixture of 5/2a/Na₂HPO₄/Na₃SPO₃ = 1:1:0.5:0.5 (pH 10.6 in water) remained the same as with SPᵢ only (22 % glyceryl phosphates) (Supplementary Fig. 77). The phosphorylation power of SPᵢ remained substantial at 75 °C (Supplementary Fig. 78). When we added water right from the start, the conversion to phosphate esters dropped in most such ‘wet and evaporating’ glycerol (5) mixtures to roughly one half after 5 days, when compared to those heated to 115 °C for the same period of time, but the phosphorylation of ‘wet and evaporating’ aqueous MPG in 6/2a/NaH₂PO₄/Na₃SPO₃ = 1:1:0.5:0.5 for 5 days at 75 °C remained beneficial (Supplementary Table 35). When a partly labelled equimolar neat mixture of NaH₂P[^18O₄] and Na₃SPO₃, being approximately 50 % ^18O-enriched as in (5 or 6)/2a/NaH₂P[^18O₄]/Na₃SPO₃ 1:1:0.5:0.5, was kept at 115 °C for 5 days, we detected a diminished 31-39 % abundance of ^18O isotopes in the major glyceryl and MPG phosphates (Supplementary Tables 34 and 36). This showed that, in a competitive situation without particular irradiation¹⁹ but when sufficiently protonated, SPᵢ kinetically outperformed the 2a + Pᵢ couple by some 25-40 % under the tested neat reaction conditions.

Neat nucleoside phosphorylation in the absence and presence of glycerol and alkanoic or amino acids (summary from sections 7.5-7.6 in the Supplementary Information): The results obtained from all our experiments on nucleoside phosphorylation in neat conditions in a mixed solid-liquid phase (Fig. 2), where only very small amounts of formamide (3a) have been added as liquidiser (we use this word to distinguish physically mobilising from dissolving), are comparable to previous works carried out in aqueous or formamide solutions. The relative proportions and amounts of phosphorylated ribonucleosides obtained at 115 °C are similar to those obtained by others from reaction mixtures heated up to 85 °C for several days in a urea-ammonium formate-water eutectic, liquid sulfur dioxide or subjected to dry-wet thermal cycling using large excesses of both urea and Pᵢ (10-16 mol equivalents), also more reactive phosphate sources richer in protons from ammonium ions¹⁶,⁴⁸, and those in the presence of minerals, catalytic metal-doped clays, photoredox-active cerium phosphate, organo-catalytic 2,5-dimethyl imidazolidine-4-thione or in an initially acidic supercritical carbon dioxide-water (scCO₂-H₂O) two-phase environment⁴⁹⁻⁵⁴. In contrast to the nucleoside mixtures containing catalytic imidazolidine-4-thiones as in-situ phosphorylating agent⁵², or those with 20 mol equivalents urea and 3 mol equivalents NaH₂PO₄ or hydroxyapatite heated at 94°C in scCO₂-H₂O, where 5'-nucleotides dominated followed by carbamoyl nucleosides and acyclic 3'- and 2'-monophosphates⁵⁴, our hot neat solid-liquid conditions always produced a high fraction of 2',3'-cyclic phosphates (Supplementary Fig. 131). This fraction was also expectedly higher than that found in the presence of borate⁵⁵. The phosphorylation of neat mixtures of nucleosides and their complementary nucleoside-5'-monophosphates (in the sense of Watson-Crick base pairing) did have an enhancing effect when compared to that of pristine nucleosides, especially in the presence of a purine nucleoside-5'-monophosphate, however, this enhancement was comparable to the efficacy of the phosphorylation and phosphodiester formation of an equimolar mixture of all four canonical ribonucleosides in the initial absence of nucleoside-5'-monophosphates (Supplementary Fig. 125). Dinucleotide (phosphodiester bond) formation represented typically between 2.4 % up to 18.4 % of the Pᵢ consumption (= "conversion", Supplementary Tables 41, 43, 44 and 47). No evidence for the formation of longer than dinucleoside phosphates has been found, neither by NMR spectroscopic (Supplementary Figs. 128, 139, 142) nor mass spectrometric means (Supplementary Figs. 122, 124, 130, 145). Not much irreversible chemical nucleoside modification (denoted 'other' in Fig. 2) occurred when heated neat at 115 °C for 5 days (Supplementary Figs. 122B and 124): hypoxanthine, inosine and inosylate were found by MS indicating adenosine N⁶-hydrolysis, once xanthosine was identified, but not quantified, hinting at the odd guanosine N²-hydrolysis, and about 4-5 % hydrolysis of cytosine-bearing nucleotides to uridylates were perceived from strictly equimolar ribonucleoside mixtures (Fig. 2 and Supplementary Figs. 130-133), see also the Supplementary Discussion (section 7.5.8 in the SI).

Of note, there were no proton-decoupled ³¹P{¹H} NMR spectroscopic signatures of either poly-, cyclic meta- or branched ultraphosphates created in dry conditions⁴⁷, and no condensed nucleoside di- and
triphosphates⁵⁶ could be found in the crude mixture containing all four nucleotides (no δₚ-doublets and -triplets for NDP = ppN or NTP = pppN). Lowering the reaction temperature to 75°C, replacing urea (2a) with cyanamide (1), or Pᵢ with cTMP or SPᵢ brought nucleoside phosphorylation virtually to a halt, although pyrimidine nucleotides formed from 1 and Pᵢ to some extent (Supplementary Fig. 104). As shown by others, the phosphorylation of nucleosides with cTMP is comparingly weak, and requires dry-wet or hot-cold cycling in the presence of water where sufficient magnesium or transition metal ions are mandatory⁵⁷,⁵⁸.

All the above is consistent with previous works on the long-term hydrolytic stability of nucleosides⁵⁹,⁶⁰ and sets a firm prebiotic rostrum for the early production of mono- and dinucleotide-type catalysts and scaffolds that could serve as feedstock for their refinement into the extant mono- and dinucleotidic enzyme co-factors, and also longer nucleic acids at a later stage of molecular evolution⁶¹⁻⁶³. Our findings, that a significant amount of nucleoside phosphates was found carbamoylated when phosphorylated in urea + Pᵢ blends (Supplementary Figs. 111, 122, 124, 130), especially adenosine at its exocyclic amino group, lends support to the thesis that a primeval translation could indeed have started with this kind of tether on N⁶-carbamoyl adenylate-bearing oligoribonucleotides⁶⁴.

Remarkably, the presence of L-valine suppressed neat D-ribonucleoside phosphorylation by about 34 %, whereas the presence of D-valine suppressed it by merely about 11 % (Supplementary Fig. 136 and Table 49), which could be explained by the fact that L-amino acids more tightly interact with D-ribonucleosides than D-amino acids do. This is consistent with the findings that naturally configured oligoribonucleotides direct the synthesis of naturally configured peptides more readily than of their enantiomeric counterparts⁶⁵,⁶⁶ and L-amino acids tend to generate a bias in favor of D-glyceraldehyde from formaldehyde and glycolaldehyde⁶⁷.

Neat hot equimolar mixtures of all ribonucleosides, glycerol and four alkanoic (fatty) acids in one pot produced within 48 hours up to diglyceryl, dinucleoside and glycerylnucleoside phosphates (cf. section 7.6 in the SI, Supplementary Figs. 137-148). Striking in this experiment was the fact that we could not find any sign of carboxyester formation, that is, no sign of glyceryl esters of any of the prebiotically plausible alkanoic acids. This makes us conclude that other prebiotic sources, ammonia-poor reaction conditions or environments, must be searched for the making of mono-alkanoic glyceryl esters (such as MPG) from glycerol, or glyceryl phosphates, and alkanoic acids, also other sources than from the partial deacylation of diacylglyceryl phospholipids such as BTG or DOG phosphates, that gave in only very small amounts 3-tridecanoylglyceryl-1,2-cyclic phosphate (5cTGP) and 3-monooleoylglyceryl-1,2-cyclic phosphate (5cMOGP), see below.

Phosphorylation of monools 7, 8, 9, 10 and pyruvic acid 16 (summary from sections 7.7-7.11 in the SI): We chose the mono-functional model alcohols 7-10, two of which are diacylglycerols (7, 8) and two alk(en)yl alcohols (9, 10) for two reasons, prebiotic and mechanistic. BTG (7) is a plausibly prebiotic precursor because of its relatively short and fully saturated long-carbon-chain residues, with a high membranogenic potential in the context of a phospholipid; but 7 is for the same reason quite an immobile material, which may have had an important bearing on the neat reaction conditions that we studied. Therefore, we chose to test in addition a chemically similar yet much less prebiotic model alcohol, DOG (8), unlikely to have existed in the late Hadean because of its cis-configured unsaturations (trans-alkenes are more stable). Its oily aspect, however, would allow us to conclude on whether the higher physical mobility, or rather the chemical reactivity of a single alcohol group would impact more on the outcome of the tested neat, hot phosphorylation reaction conditions. Alkanols such as dodecanol (9) could have been precursors of long straight-chain alkyl phospholipids without glycerol in between the chain and the polar headgroup, that could assemble to primitive versions of bilayer membranes⁶⁸. An alternative version of primeval membranogenic phospholipids may have been Archaea-like branched isoprenoid phosphates the prebiotic precursors
and membranophilicity of which just have started to be explored¹⁹,⁶⁹. Both kinds of alcohols, unbranched and branched, are primary alcohols. Dodecanol (9) and geraniol (10) are monools that therefore served us as model precursor compounds of phospholipids for bilayer membranes involved in the evolution of cell walls of Bacteria and, respectively, Archaea at some later stage. Pyruvic acid (16) is a recognised crucial protometabolite that could have been generated under prebiotic conditions⁷⁰.

A sobering fact was that the monools **7, 8** and **10** were not phosphorylated in dry (neat) equimolar mixtures of Pᵢ and urea after heating at 115 °C, if not for organo-phosphates in barely detectable trace amounts mostly of deacylated **7** (Supplementary Fig. 152) and **8** (Supplementary Figs. 156-157). Previous work did show that significant amounts of **8** could be phosphorylated under conditions akin to those studied here, but only using a large excess urea (**2a**) with respect to the starting alcohol **8**, combined with an excess of more proton-rich phosphate sources: ammonium dihydrogenphosphate or phosphoryl ethanolamine⁷¹. Heating **9** and aqueous Pᵢ in the presence of up to 10 equivalents **2ac** at 115 °C for 4-5 days generated no organic phosphates whatsoever, only inorganic condensed phosphates PPᵢ and PPPᵢ. By contrast, when neat **9/1/Pᵢ** (1:1:1) mixtures were heated at 115 °C, the Pᵢ consumption was almost 80 %, some organic phosphates (Supplementary Fig. 177) and quite persistent carbamoylated products were found, even more of the latter at 130 °C (Supplementary Fig. 165). In the labelled mixtures using **9/1/[¹⁸O₄]Pᵢ** we have found ¹⁸O-enrichment in the not phosphorylated dodecyl carbamate and carbonate side-products as well (Supplementary Tables 55-56). Evidently, the intermediate inorganic carbamoyl phosphate (CPᵢ) slowly exchanged ¹⁸O with ¹⁶O isotopes prior to dodecanol attacking the carbon or phosphorous atoms of CPᵢ (cf. ochre zone of Fig. 4B). This attack on the carbon atom of CPᵢ did not happen to any of the diols and triols **5, 6** and **11-15**, since no ¹⁸O enrichment in the corresponding not phosphorylated side-products was found whatsoever. This, in turn, means that the alcoholysis of CPᵢ by means of an attack of an OH group of a diol or triol at the phosphorous atom of CPᵢ is just too fast, accelerated by the neighbouring OH group, to allow for a competitive attack on the carbonyl group of CPᵢ. When **1** and Pᵢ were used on neat **10**, it generated what appeared to be 5-membered ring cyclic phosphates indicating the addition of Pᵢ to geraniol's first C=C double-bond, and other less well characterised by-products; probably cyclic monoterpene and geranyl-cyanamide derivatives (Supplementary Fig. 183). Finally, in hot neat mixtures containing **16** the inorganic phosphate sources Pᵢ, cTMP and SPᵢ condensed separately to PPᵢ, PPPᵢ and, respectively, SPPᵢ, SPSPᵢ and SPPPᵢ (Supplementary Figs. 185, 187 and 188) while **16** oligomerised to create, through up to four new C-C bonds, novel polyketides (Supplementary Fig. 196) some of which appeared to be partly phosphorylated (Supplementary Fig. 189).

For the geochemical context: by far the easiest, environmentally most robust and atom-friendly way of phosphorylating polyfunctional alcohols such 1,2,3-triols, 1,2-diols and 1,3-diols like **5, 6, 11-15**, but not monools like **7-10** and **16a**, accompanied by a minimal quantity of wasted atoms under conditions that involve hardly any water, no molar excess of inorganic phosphate over the alcohol, no excess ammonia, imidazole¹⁵ or liquid sulfur dioxide⁵⁰ and no direct intervention of transition metals, borate minerals, silica or clays, or super-critical carbon dioxide⁵¹,⁵³⁻⁵⁵, is the presence of large amounts of hot urea (**2a**). Abiotic urea consists of a thermally stabilised solid form of reactive ammonium cyanate that, by irreversibly evaporating ammonia and leaving cyanate to react, actually drives the phosphorylation reaction at temperatures close to that of boiling water and above (and below). Even at a mere equimolar ratio of alcohol and phosphate, urea can act as an effectual chemical fuel of phosphorylation that will 'burn' anyway, that is, hydrolyse in the presence of water to produce ammonia and carbon dioxide. Therefore, out of the here tested dehydration and phosphorylation agents, urea in combination with Pᵢ (= **2a** + Pᵢ) are geochemically the likeliest environment for prebiotic phosphorylations of simple organic precursor compounds, precisely because they bear the lowest chemical potential of all: solid urea-Pᵢ mixtures could have acted on organic molecules for the longest periods of time before vanishing through hydrolysis. The urea-
assisted phosphorylation of glycerol (5), MPG (6) and the nucleosides (11-15) produced carbamoyl-
and cyclic carbonate derivatives in addition to the phosphate esters; those carbonates generated
from 1,2-diols persisted in 5-15 mol% fractions even 5 days at 115 °C in the dry state.

The tracking of ¹⁸O isotopes showed that there is no organo-catalytic potential in urea (Fig. 4A) when it comes to phosphorylations, the only operative reaction pathway is the thermal dissociation of urea to ammonia and carbamoyl phosphate (Fig. 4B and section 6.3 in the SI). Our *ab initio* calculations have shown that urea's interactions with water and methanol are relatively weak when compared to those with inorganic dihydrogenphosphate (Pᵢ). The dissociative phosphate activation pathway (Fig. 4B) clarifies, how Pᵢ accelerates as a so-called acid-base catalyst the degradation of urea to carbon dioxide and ammonia, viz. through catalysing the direct or water-relayed formation of a zwitterionic tautomer of urea (→ Int-1 in Fig. 4B) predisposed to break one carbon-nitrogen bond and generate ammonium cyanate NH₄NCO (TS_2 and Int-2 in Supplementary Fig. 15). The *computed potential energy surfaces* gives a rationale for how inorganic carbamoyl phosphate (CPᵢ) is generated in neat urea-phosphate 2a + Pᵢ mixtures through transitional NH₄⁺ + NCO⁻ + Pᵢ clusters (Int-3 in Fig. 4B and Supplementary Fig. 15), and how CPᵢ acts as a universal reaction intermediate with alcohols (Int-5). Unlike phosphorylation reactions taking place in water¹¹, those in neat urea as well as in a milieu containing both urea and water (3:2 mol/mol) are favorable and calculated to be exergonic in the beginning formation of organic phosphoric monoesters, even though the decomposition of inorganic carbamic acid (CAᵢ) to NH₃ and CO₂ was calculated to be endergonic by 16-17 kcal/mol (Int-7 → Int-8 → methylphosphate + 2 NH₃ + CO₂). Of course, it happens to completion anyway in an open system like the Earth's surface. Hence, both kinetically and thermodynamically, the pathway in which urea dissociates into ammonium cyanate wins (Fig. 4A versus 4B and Supplementary Figs. 14 vs. 15). However, the alcoholysis of CPᵢ was calculated to be quite slow for methanol (highlighted in cyan in Fig. 4B), which is consistent with our experimental outcome that 1,2,3-triols, 1,2-diols and 1,3-diols like 5, 6, 11-15 are incomparingly more efficiently phosphorylated than the monools 7, 8, 9, 10 and enol 16a.

Cyanamide (1), too, can directly react with inorganic phosphate and alcohols to produce phosphate esters and carbamates (Fig. 2, Supplementary Figs. 57, 60-62, 71, 77, 78, 85, 104, 105, 155, 158, 167, 168), but can also add to alcohols without the inference of phosphate, cf. section 7.11 in the SI (also Supplementary Figs. 174-176, 181-184, 190 and Supplementary Tables 55 and 56). However, monobasic (slightly acidic) phosphate is known for long to efficiently catalyse the hydrolysis of 1 to 2a⁴⁰. Therefore, especially during cool night-time and water-condensing or rain periods, cyanamide (1) should be seen for kinetic reasons to primarily take the geochemical role of being an important precursor of urea refurbishment. The tested formamides (3abc) and acetamides (4abc) are no alternative as urea surrogates, but formamide can enhance, as a substitute for liquid water, during very hot day-time conditions, urea-assisted phosphorylations of the only tested alcohols that remain solid (immobile) at 70-115 °C, viz. the nucleosides. Chemically internally activated inorganic phosphate sources, such as condensed phosphates P<sub>ni</sub> (n = 2, 3, ...) generated by heat (trimetaphosphate, cTMP) or crystallised in minerals (canaphite), and thiophosphate (SP<sub>i</sub>) present in hydrogen sulfide-containing acidic volcanic waters during hours, days or longer¹⁹, can indeed phosphorylate alcohols, but not as long-lasting as urea and inorganic orthophosphate (2a + P<sub>i</sub>). It seems that, over longer episodes including aqueous wet periods, the condensed P<sub>ni</sub> were mainly hydrolytic reservoirs of P<sub>i</sub>. In an alkaline milieu, however, cTMP is a potent and valid phosphate source, particularly, in contrast to the 2a + P<sub>i</sub> couple or SP<sub>i</sub> that actually profit from available protons and slightly acidic conditions. Only cyanamide (1) has a higher chemical potential than cTMP (Fig. 1); cTMP at 115 °C is able to form cyclic carbonates from atmospheric CO₂ and 1,2-diols present in diglyceryl phosphates (Supplementary Figs. 66-69). Carbonylisation stabilises such organic phosphate esters and, as mentioned before in the context of hot urea + P<sub>i</sub> blends, makes them more resistant against degradation over longer periods of time. However, ribonucleosides and monools
are hardly or not at all phosphorylated by cTMP in alkaline neat conditions, at least without
abundant magnesium or transition metal ions⁵⁷,⁵⁸. Both, SPᵢ and cTMP degrade to Pᵢ when the pH
drops, which is a key argument in the context of an atmosphere that bore huge amounts of carbon
dioxide that would importunately acidify the waters by hydrolysing to aqueous carbonic acid⁷².
Therefore, the best immediate prebiotic value of cTMP is its role as a persistent condensing agent
based on its efficiency in creating peptides from amino acids⁴⁶,⁷³,⁷⁴, and that of SPᵢ could be drawn in
particularly alkaline arid environments, where the urea + tribasic Pᵢ blend is an unreactive
phosphorylation agent while tribasic SPᵢ still is (section 7.4.1 and Supplementary Figs. 74-78), and
from its photochemical reducing power¹⁹.

In summary, this work shows that, in conditions that were poor in available (soluble) phosphate
sources and limited in non-volatile reduced-nitrogen compounds — as should be expected to have
been the case on the earliest subaerial landmasses being scarce in the late Hadean era — single-
chain (mono-alkanoyl) glyceryl phosphate derivatives appear to be the most likely chemical starting
line for the first robust bilayer-forming phospholipids. As a preliminary result to be followed more
systematically, we have shown by epifluorescence microscopy that crude extracted, dried and
rehydrated phosphorylation mixtures, where MPG, Pᵢ and urea (1:1:1) have been heated to 115 °C
for 5 days in the dry (neat) state, did form giant vesicles showing diameters of 10-30 µm (Section 8 in
the SI). Hence, when episodically wetted with salty water, mono-alkanoyl glyceryl phosphates could
have let vesicles emerge that enclosed short nucleotides, amino acids and perhaps peptides⁷⁵⁻⁷⁸
inside them. It is less clear at present exactly how alkanoic 'fatty' glyceryl esters and longer than
dinucleotides could have amassed in such arid conditions, perhaps through wet-dry cycling. Most if
not all of the prebiotically available monool starting compounds, and their derivatives that would be
generated, for instance, in neat hot inorganic phosphate-urea blends, could have taken some role as
a co-surfactant in primitive membranes, stabilising or modulating their permeabilities, shapes
(positive versus negative curvatures), membrane-membrane interactions (vesicle fusion versus
cluster formation), growth and division (ease of fission) and robustness (chemical and physical)⁶⁸,⁷¹,
but they are unlikely to have been the major players irrespective of whether they were glyceryl
derivatives or not. Monools must have been outcompeted by prebiotically available mono-alkanoyl
glycerols, glycerol, and probably also carbohydrates that weren't studied here. Taken together, we
have constrained the geochemical space for the earliest possible prebiotic phosphorylation
reactions, setting the stage for their assembly to form protocells in shallow basins once liquid water
arrived more recurrently⁷⁹ and stayed longer in place, while increasingly notable concentrations of
other dissolved small-molecular-weight precursor molecules, such as α-keto acids, α-amino acids,
short peptides and carbohydrates, and N-heterocycles, were able to impact on the protocell's
content¹⁶,⁷⁰,⁸⁰⁻⁸².

Our results should provide valuable data for the theory on the outbreak of life in seafloor
hydrothermal-sedimentary systems⁸³ or deeper hydrothermal-vent systems⁸⁴,⁸⁵ that offer dielectric
barriers as compartments for efficient chemiosmotic work to be casted⁸⁶ albeit, as such, with a
limited evolvability potential. We should think of the possibility that probably neither of these initial
subaerial and seafloor systems could build up sufficient complexity to become alive on their own.
The subaerial locations lacked congregated long-term energy sources to unfailingly push forward the
disequilibrium to a further level of complexity, and the seafloor was not a locum apt to amass
sufficient organic phosphates from the elimination of water out of their precursor molecules. We
need to consent to a common leeway in which the subaerial and the seafloor locations physically
communicated with one another, given the Hadean weather, through strong winds²⁸ bearing
aerosols⁸⁷,⁸⁸ and the intense rhythm of flood and ebb, back and forth to the ocean, of water that
contained sinking phospholipid protocells originating from subaerial milieus, arid conditions and
harbouring dense amounts of nucleotides and peptides. Once sunken to the ocean ground, they
could fuse with seafloor compartments⁸⁹,⁹⁰ and take over their sheltered proto-metabolic reaction
networks²⁰. Only the combined ability to produce true polymers by installing the recurring
translation of a simple genetic code from scarce but well soluble nucleic acids (poly-
phosphodiesters) into functional proteins (poly-carboxamides) more robust and more abundant in
numbers, could provide and sustain more, and more specific catalysis (proteic enzymes), and
hereafter the spreading of the first primary metabolic networks, which made these next-level
entities self-evolvable and gradually more independent of their primitive energy sources and mineral
catalysts, over periods of time much longer than was the average lifetime of their molecular
components.

**Methods:**

**Experimental materials.** Two of three compounds commercially unavailable as racemates, i.e., racemic MPG (6) and racemic BTG (7) have been synthesised and purified for their spectroscopic identification and their quantification in the crude product mixtures (cf. section 1.6 in the SI). Racemic DOG (8) was available from our earlier work. Solid sodium dihydrogenphosphate highly enriched in the ¹⁸O isotope, NaH₂P[¹⁸O₄] (= [¹⁸O₄]Pᵢ), was prepared from PCl₅, H₂[¹⁸O] and Na₂CO₃ according to a newly established protocol (see section 1.6.4 of the SI). All other organic or inorganic starting compounds were commercially available.

**Experimental methods** Several hundred milligrammes to several grammes of the neat components (0.5 to 30 mmol per compound, cf. reaction scales in Supplementary Tables 11 and 19) were premixed usually in equimolar ratios, sometimes dissolved in water, lyophilised or let evaporate to a visibly dry state, yet containing residual amounts of water (often about 15 w%, cf. Supplementary Table 20 and section 5.4 in the SI). These neat mixtures were kept in an open system for up to five days at a constant 60, 75, most often 115 or, when using dodecanol also 130 °C (section 2.1 in the SI). Each reaction condition was carried out in triplicate. In those experiments where the reaction kinetics and stable isotope distribution was followed, the neat reactions were carried out in a closed system under a steady argon flow to carry over volatiles to two traps containing reactive solutions (toluene containing excess benzoyl chloride in trap 1, and tetrahydrofuran containing excess phenylmagnesium bromide in trap 2). After the heating period, the trap liquids for volatiles were periodically sampled, acidified if necessary (trap 2), and diluted in a standard solvent mixture for LC-MS analysis (section 2.2 in the SI). The non-volatiles present in the heated neat reaction mixtures were sampled by extraction with either dimethyl sulfoxide or DMSO-d6, water or D₂O, or in some cases methanol (cf. respective sections 1.2, 1.3, 7.6 and 8 in the SI) for subsequent ¹H, ¹³C and ³¹P NMR, 2D correlation including diffusion-ordered ³¹P{¹H} spectroscopy (DOSY), and LC-MS analyses using reversed phase (RP) and hydrophilic-ion liquid chromatography (HILIC) (ultra)high-performance separation techniques (HPLC and UHPLC) coupled with UV-spectroscopic and evaporative light scattering detectors (ELSD) or an electrospray-ionisation-high-resolution mass spectrometer (ESI-HRMS). The results of essentially all of the spectroscopic, chromatographic and spectrometric data of more than 430 individual experiments are recapitulated in Figs. 2, 3 and 4C, and presented in a detailed and processed form in the sections 4-7 of the SI, accompanied by an appropriate supplementary text and conclusions, and in section 1 of the Appendix. The preparation of lipid films from crude mixtures and lipid vesicles using the natural swelling method, and fluorescence microscopy images thereof, are shown in section 8 of the SI.

**Theoretical methods.** A quantum theoretical study using the Density Functional and Coupled Cluster Theories was carried out on an ab initio-assembly of dihydrogenphosphate (1 molecule), urea (3 molecules), water (0 or 2 molecules) and methanol (1 molecule). We performed a comparative analysis along the calculated Intrinsic Reaction Coordinate (IRC) using the M06-2X and CCSD methods in combination with the 6-31G(d,p) and 6-311++G(2d,2p) basis sets. The results of four distinct reaction pathways, calculated at the M06-2X/6-311++G(2d,2p)//M06-2X/6-31G(d,p) level of theory are presented in a highly condensed form in Fig. 4A and 4B. The whole computational study is detailed in section 1.5 (quantum theoretical method) and section 3 (detailed results) of the SI,
accompanied by an appropriate supplementary text and conclusion. The Gibbs free energies of all computed stationary (transition and intermediate) states are listed in section 2 of the Appendix; the atomic coordinates optimised at the M06-2X/6-31G(d,p) level of theory are made available as a separate Supplementary Dataset.

**Data availability:** All data needed to support the conclusions of this manuscript are included in the main text, the Supplementary Information and in 8 Supplementary Datasets.

**References**

1. Bar-On, Y. M., Phillips, R. & Milo, R. The biomass distribution on Earth. *Proc. Natl. Acad. Sci. USA* **115**, 6506-6511 (2018). https://doi.org/10.1073/pnas.1711842115

2. Khachikyan, A., Milucka, J., Littmann, S., Ahmerkamp, S. Meador, T. Könneke, M. Burg, T. & Kuypers, M. M. Direct cell mass measurements expand the role of small microorganisms in nature. *Appl. Environm. Microbiol.* **85**, e00493-19 (2019). https://doi.org/10.1128/AEM.00493-19

3. Goldford, J. E., Smith, H. B., Longo, L. M., Boswell, A. W. & McGlynn, S. E. Primitive purine biosynthesis connects ancient geochemistry to modern metabolism. *Nature Ecol. Evol.* **8**, 999-1009 (2024). https://doi.org/10.1038/s41559-024-02361-4

4. Walton, C. R., Ewens, S., Coates, J. D., Blake, R. E., Planavsky, N. J., Reinhard, Ju, C., Hao, P. J. & Pasek, M. A. Phosphorus availability on the early Earth and the impacts of life. *Nature Geosci.* **16**, 399-409 (2023). https://doi.org/10.1038/s41561-023-01167-6

5. Russel, M. & Ponce, A. Six ‘must-have’ minerals for life’s emergence: olivine, pyrrhotite, bridgmanite, serpentine, fougerite and mackinawite. *Life* **10**, 291 (2020). https://doi.org/10.3390/life10110291

6. Ritson, D. J., Mojzsis, S. J. & Sutherland, J. D. Supply of phosphate to early Earth by photogeochemistry after meteoritic weathering. *Nature Geosci.* **13**, 344-348 (2020). https://doi.org/10.1038/s41561-020-0556-7

7. Toner, J. D. & Catling, D. C. A carbonate-rich lake solution to the phosphate problem of the origin of life. *Proc. Natl. Acad. Sci. U. S. A.* **117**, 883-888 (2020). https://doi.org/10.1073/pnas.1916109117

8. Walton, C. W., Shorttle, O., Jenner, F. E., Williams, H. M., Golden, J. S., Morrison, M., Downs, R. T. Zerkle, A., Hazen, R. M. & Pasek, M. A. Phosphorus mineral evolution and prebiotic chemistry: From minerals to microbes. *Earth-Sci. Rev.* **221**, 103806 (2021). https://doi.org/10.1016/j.earscirev.2021.103806

9. Postberg, F., Sekine, Y., Klenner, F., Glein, C. R., Zou, Z., Abel, B., Furuya, K., Hillier, J. K., Khawaja, N., Kempf, S., Noelle, L., Saito, T., Schmidt, J., Shibuya, T., Srama, R. & Tan, S. Detection of phosphates originating from Enceladus’s Ocean. *Nature* **618**, 489–493 (2023). https://doi.org/10.1038/s41586-023-05987-9

10. Haas, S., Sinclair, K. P. & Catling, D. C. Biogeochemical explanations for the world's most phosphate-rich lake, an origin-of-life analog. *Commun. Earth Environ.* **5**, 28 (2024). https://doi.org/10.1038/s43247-023-01192-8

11. Pasek. M. A. Thermodynamics of prebiotic phosphorylation. *Chem. Rev.* **120**, 4690-4706 (2020). https://doi.org/10.1021/acs.chemrev.9b00492

12. Zimmermann, J., Mayer, R. J. & Moran, J. A single phosphorylation mechanism in early metabolism — the case of phosphoenolpyruvate. *Chem. Sci.* **14**, 14100-14108 (2023). https://doi.org/10.1039/D3SC04116F
13. Ter-Ovanessian, L. M. P., Rigaud, B., Mezzetti, A., Lambert, J.-F. & Maurel, M.-C. Carbamoyl phosphate and its substitutes for the uracil synthesis in origins of life scenarios. *Sci. Rep.* **11**, 19356 (2021). https://doi.org/10.1038/s41598-021-98747-6

14. Yi, J., Kaur, H., Kazöne, W., Rauscher, S. A., Gravillier, L. A., Muchowska, K. B. & Moran, J. A nonenzymatic analog of pyrimidine nucleobase biosynthesis. *Angew. Chem. Int. Ed.* **61**, e202117211 (2022). https://doi.org/10.1002/anie.202117211

15. Maguire, O. R., Smokers, I. B. A. & Huck, W. T. S. A physicochemical orthophosphate cycle via a kinetically stable thermodynamically activated intermediate enables mild prebiotic phosphorylations. *Nature Commun.* **12**, 5517 (2021). https://doi.org/10.1038/s41467-021-25555-x

16. Patel, B. H., Percivalle, C., Ritson, D., Duffy, C. D. & Sutherland, J. D. Common origins of RNA, protein and lipid precursors in a cyanosulfidic protometabolism. *Nature Chem.* **7**, 301–307 (2015). https://doi.org/10.1038/nchem.2202

17. Becker, S., Feldmann, J., Wiedemann, S., Okamura, H., Schneider, C., Iwan, K., Crisp, A., Rossa, M., Amatov, T. & Carell, T. Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides. *Science* **366**, 76-82 (2019). https://doi.org/10.1126/science.aax2747

18. Bonfio, C., Caumes, C., Duffy, C. D., Patel, B. H., Percivalle, C., Tsanakopoulou, M. & Sutherland, J. D. Length-selective synthesis of acylglycerol-phosphates through energy-dissipative cycling. *J. Am. Chem. Soc.* **141**, 3934-3939 (2019). https://doi.org/10.1021/jacs.8b12331

19. Ritson, D. J. & Sutherland, J. D. Thiophosphate photochemistry enables prebiotic access to sugars and terpenoid precursors. *Nature Chem.* **15**, 1470-1477 (2023). https://doi.org/10.1038/s41557-023-01251-9

20. Camprubí, E. J., de Leeuw, W., House, C. H., Raulin, F., Russel, M. J., Spang, A., Tirumalai, M. R. & Westall, F. The emergence of life. *Space Sci. Rev.* **215**, 56 (2019). https://doi.org/10.1007/s11214-019-0624-8

21. Sasselov, D. D., Grotzinger, J. P. & Sutherland, J. D. The origin of life as a planetary phenomenon. *Sci. Adv.* **6**, eaax3419 (2020). https://doi.org/10.1126/sciadv.aax3419

22. Damer, B. & Deamer, D. The hot spring hypothesis for an origin of life. *Astrobiology* **20**, 429-452 (2020). https://doi.org/10.1089/ast.2019.2045

23. Heller, R., Duda, J. P., Winkler, M., Reitner, J. & Gizon, L. Habitability of the early Earth: liquid water under a faint young Sun facilitated by strong tidal heating due to a closer Moon. *Pal. Z.* **95**, 563–575 (2021). https://doi.org/10.1007/s12542-021-00582-7

24. Farhat, M., Auclair-Desrotour, P., Boué, G. & Laskar, J. The resonant tidal evolution of the Earth-Moon distance. *Astron. Astrophys.* **665**, L1 (2022). https://doi.org/10.1051/0004-6361/20224345

25. Schreiber, U., Locker-Grütjen, O. & Mayer, C. Hypothesis: Origin of life in deep-reaching tectonic faults. *Orig. Life Evol. Biosph.* **42**, 47-54 (2012). https://doi.org/10.1007/s11084-012-9267-4

26. Mayer, C., Schreiber, U. & Dávila, M. J. Periodic vesicle formation in tectonic fault zones—an ideal scenario for molecular evolution. *Orig. Life Evol. Biosph.* **45**, 139-148 (2015). https://doi.org/10.1007/s11084-015-9411-z

27. Shibuya, T. & Takai, K. Liquid and supercritical CO₂ as an organic solvent in Hadean seafloor hydrothermal systems: implications for prebiotic chemical evolution. *Prog. Earth Planet. Sci.* **9**, 60 (2022). https://doi.org/10.1186/s40645-022-00510-6

28. Brady, M. P., Tostevin, R. & Tosca, N. J. Marine phosphate availability and the chemical origins of life on Earth. *Nature Commun.* **13**, 5162 (2022). https://doi.org/10.1038/s41467-022-32815-x

29. Bernstein, M. P., Sandford, S. A., Allamandola, L. J., Chang, S. & Scharberg, M. A. Organic compounds produced by photolysis of realistic interstellar and cometary ice analogs containing methanol. *Astrophys. J.* **454**, 327-344 (1995). https://doi.org/10.1086/176485
30. Britvin, S., Murashko, M., Vapnik, Y., Polekhovsky, Y. S. & Krivovichev, S. V. Earth's phosphides in Levant and insights into the source of Archean prebiotic phosphorus. *Sci. Rep.* **5**, 8355 (2015). https://doi.org/10.1038/srep08355

31. Pirim, C., Pasek, M. A., Sokolov, D. A., Sidorov, A. N., Gann, R. D. & Orlando, T. M. Investigation of schreibersite and intrinsic oxidation products from Sikhote-Alin, Seymchan, and Odessa meteorites and Fe₃P and Fe₂NiP synthetic surrogates. *Geochim. Cosmochim. Acta* **140**, 259-274 (2014). https://doi.org/10.1016/j.gca.2014.05.027

32. Gull, M., Cruz, H. A., Krishnamurthy, R. & Pasek, M. A. Phosphorylation of nucleosides by P-N bond species generated from prebiotic reduced phosphorus sources. *Commun. Chem.* **8**, 187 (2025). https://doi.org/10.1038/s42004-025-01577-0

33. Osumah, A. & Krishnamurthy, R. Diamidophosphate (DAP) : A plausible prebiotic phosphorylating reagent with a Chem to Biochem potential? *ChemBioChem* **22**, 3001-3009 (2021). https://doi.org/10.1002/cbic.202100274

34. Ashe, K. Fernández-García, C., Corpinot, M. K., Coggins, A. J., Bučar, D.-K. & Powner, M. W. Selective prebiotic synthesis of phosphoroaminonitriles and aminothioamides in neutral water. *Commun. Chem.* **2**, 23 (2019). https://doi.org/10.1038/s42004-019-0124-5

35. Becker, S., Schneider, C., Okamura, H., Crisp, A., Amatov, T., Dejmek, M. & Carell, T. Wet-dry cycles enable the parallel origin of canonical and non-canonical nucleosides by continuous synthesis. *Nature Commun.* **9**, 163 (2018). https://doi.org/10.1038/s41467-017-02639-1

36. Slabaugh, M. R., Harvey, A. J. & Nagyvary, J. The possible role of inorganic thiophosphate as a prebiotic phosphorylating agent. *J. Mol. Evol.* **3**, 317-321 (1974). https://doi.org/10.1007/BF01796047

37. Yamagata, Y., Inoue, H. & Inomata, K. Specific effect of magnesium ion on 2', 3'-cyclic AMP synthesis from adenosine and trimeta phosphate in aqueous solution. *Orig. Life Evol. Biosph.* **25**, 47-52 (1995). https://doi.org/10.1007/BF01581572

38. Yamagata, Y. Prebiotic Formation of ADP and ATP from AMP, calcium phosphates and cyanate in aqueous solution. *Orig. Life Evol. Biosph.* **29**, 511-520 (1999). https://doi.org/10.1023/A:1006672232730

39. Schimpl, A., Lemmon, R. M. & Calvin, M. Cyanamide formation under primitive earth conditions. *Science* **147**, 149-50 (1965). https://doi.org/10.1126/science.147.3654.149

40. Lohrmann, R. & Orgel, L. E. Prebiotic synthesis: phosphorylation in aqueous solution. *Science* **161**, 64-66 (1968). https://doi.org/10.1126/science.161.3836.64

41. Mohajer, M. A., Basuri, P., Evdokimov, A., David, G., Zindel, D., Miliordos, E. & Signorell, R. Spontaneous formation of urea from carbon dioxide and ammonia in aqueous droplets. *Science* **388**, 1426-30 (2025). https://doi.org/10.1126/science.adv2362

42. Bada, J. L., Chalmers, J. H. & Cleaves II, H. J. Is formamide a geochemically plausible prebiotic solvent? *Phys. Chem. Chem. Phys.* **18**, 20085-20090 (2016). https://doi.org/10.1039/C6CP03290G

43. Pietrucci, F. & Saitta, A. M. Formamide reaction network in gas phase and solution via a unified theoretical approach: Toward a reconciliation of different prebiotic scenarios. *Proc. Natl. Acad. Sci. USA* **112**, 15030-15035 (2015). https://doi.org/10.1073/pnas.1512486112

44. Ferus, M., Pietrucci, F., Saitta, A. M., Knížek, A., Kubelík, P., Ivanek, O., Shestivska, V. & Civiš, S. Formation of nucleobases in a Miller-Urey reducing atmosphere. *Proc. Natl. Acad. Sci. USA* **114**, 4306-4311 (2017). https://doi.org/10.1073/pnas.1700010114

45. Orgel, L. E. & Lohrmann, R. Prebiotic chemistry and nucleic acid replication. *Acc. Chem. Res.* **7**, 368-377 (1974). https://doi.org/10.1021/ar50083a002

46. Fernández-García, C., Coggins, A. J. & Powner, M. W. A chemist's perspective on the role of phosphorus at the origins of life. *Life* **7**, 31 (2017). https://doi.org/10.3390/life7030031
47. Dürr-Mayer, T., Qiu, D., Eisenbeis, V. B., Steck, N., Häner, M., Hofer, A., Mayer, A., Siegel, J. S., Baldridge, K. K. & Jessen, H. J. The chemistry of branched condensed phosphates. *Nature Commun.* **12**, 5368 (2021). https://doi.org/10.1038/s41467-021-25668-3

48. Burcar, B., Pasek, M., Gull, M., Cafferty, B. J., Velasco, F., Hud, N. V. & Menor-Salván, C. Darwin's little pond : a one-pot reaction for prebiotic phosphorylation and the mobilization of phosphate from minerals in urea-based solvent. *Angew. Chem. Int. Ed.* **55**, 13249-13253 (2016). https://doi.org/10.1002/anie.201606239

49. Gull, M., Feng, T., Smith, B., Calcul, L. & Pasek, M. A. Prebiotic syntheses of organophosphorus compounds from reduced source of phosphorus in non-aqueous solvents. *Life* **13**, 2134 (2023). https://doi.org/10.3390/life13112134

50. Sydow, C., Seiband, C., Siegle, A. F. & Trapp, O. Phosphorylation in liquid sulfur dioxide under prebiotically plausible conditions. *Commun. Chem.* **5**, 143 (2022). https://doi.org/10.1038/s42004-022-00761-w

51. Zhao, Z.-R & Wang, X. A plausible selection of ribose for RNA — formation, dynamic isolation, and nucleotide synthesis based on metal-doped clays. *Chem* **7**, 3292-3308 (2022). https://doi.org/10.1016/j.chempr.2021.09.002

52. Bechtel, M., Hümmer, E. & Trapp, O. Selective Phosphorylation of RNA-and DNA-nucleosides under prebiotically plausible conditions. *ChemSystemsChem* **4**, e202200020 (2022). https://doi.org/10.1002/syst.202200020

53. Gilboa, S., Panz, L., Arbell, N., & Paz, Y. Light-assisted formation of nucleosides and nucleotides from formamide in the presence of cerium phosphate. *Life* **14**, 846 (2024). https://doi.org/10.3390/life14070846

54. Tagawa, S., Hatami, R., Morino, K., Terazawa, S., Akil, C., Johnson-Finn, K., Shibuya, T. & Fujishima, K. Prebiotic nucleoside phosphorylation in a simulated deep-sea supercritical carbon dioxide-water two-phase environment. *Astrobiology* **24**, 1151-1165 (2024). https://doi.org/10.1089/ast.2024.0016

55. Kim, H.-J., Furukawa, Y., Kakegawa, T., Bita, A., Scorei, R. & Benner, S. A. Evaporite borate-containing mineral ensembles make phosphate available and regiospecifically phosphorylate ribonucleosides: Borate as a multifaceted problem solver in prebiotic chemistry. *Angew. Chem. Int. Ed.* **55**, 15816 (2016). https://doi.org/10.1002/anie.201608001

56. Jerome, C. A., Kim, H.-J., Mojzsis, S. J., Benner, S. A. & Biondi, E. Catalytic synthesis of polyribonucleic acid on prebiotic rock glasses. *Astrobiology* **22**, 629-636 (2022). https://doi.org/10.1089/ast.2022.0027

57. Cheng, C., Fan, C., Wan, R., Tong, C., Miao, Z., Chen, J. & Zhao, Y. Phosphorylation of adenosine with trimetaphosphate under simulated prebiotic conditions. *Orig. Life Evol. Biosph.* **32**, 219-224 (2002). https://doi.org/10.1023/A:1016513114799

58. Ozawa, K., Nemoto, A., Imai, E. I., Honda, H., Hatori, K. & Matsuno, K. Phosphorylation of nucleotide molecules in hydrothermal environments. *Orig. Life Evol. Biosph.* **34**, 465-471 (2004). https://doi.org/10.1023/B:ORIG.0000043121.65714.05

59. Levy, M. S. & Miller, L. The stability of the RNA bases: implications for the origin of life. *Proc. Natl. Acad. Sci. USA* **95**, 7933-7938 (1998). https://doi.org/10.1073/pnas.95.14.7933

60. Rios, A. C. & Tor, Y. On the origin of the canonical nucleobases: an assessment of selection pressures across chemical and early biological evolution. *Isr. J. Chem.* **53**, 469-483 (2013). https://doi.org/10.1002/ijch.201300009

61. Szathmáry, E. Coding coenzyme handles: a hypothesis for the origin of the genetic code. *Proc. Natl. Acad. Sci. USA* **90**, 9916-9920 (1993). https://doi.org/10.1073/pnas.90.21.9916
62. Jadhav, V. R. & Yarus, M. Coenzymes as coribozymes. *Biochimie* **84**, 877-888 (2002).
[https://doi.org/10.1016/S0300-9084(02)01404-9](https://doi.org/10.1016/S0300-9084(02)01404-9)

63. Dass, A. V., Wunnava, S., Langlais, J., von der Esch, B., Krusche, M., Ufer, L., Chrisam, N., Dubini, R. C., Gartner, F., Angerpointner, S., Dirscherl, C. F., Rovó, P., Mast, C. B., Šponer, J. E., Ochsenfeld, C., Frei, E. & Braun, D. RNA Oligomerisation without added catalyst from 2',3'-cyclic nucleotides by drying at air-water interfaces. *ChemSystemsChem* **5**, e202200026 (2023).
[https://doi.org/10.1002/syst.202200026](https://doi.org/10.1002/syst.202200026)

64. Müller, F., Escobar, L., Xu, F., Węgrzyn, E., Nainytė, M., Amatov, T., Chan, C.-Y., Pichler, A. & Carell, T. A prebiotically plausible scenario of an RNA-peptide world. *Nature* **605**, 279-284 (2022).
[https://doi.org/10.1038/s41586-022-04676-3](https://doi.org/10.1038/s41586-022-04676-3)

65. Su, M., Schmitt, C., Liu, Z., Roberts, S. J., Liu, K. C., Röder, K., Jäschke, A., Wales, D. J. & Sutherland, J. D. Triplet-encoded prebiotic RNA aminoacylation. *J. Am. Chem. Soc.* **145**, 15971-15980 (2023).
[https://doi.org/10.1021/jacs.3c03931](https://doi.org/10.1021/jacs.3c03931)

66. Węgrzyn, E., Mejdrová, I., Müller, F. M., Nainytė, M., Escobar, L. & Carell, T. RNA-templated peptide bond formation promotes L-homochirality. *Angew. Chem. Intl. Ed.* **63**, e202319235 (2024).
[https://doi.org/10.1002/anie.202319235](https://doi.org/10.1002/anie.202319235)

67. Breslow, R. & Cheng. Z. L. L-amino acids catalyze the formation of an excess of D-glyceraldehyde, and thus of other D sugars, under credible prebiotic conditions. *Proc. Natl. Acad. Sci. USA* **107**, 5723-5725 (2010).
[https://doi.org/10.1073/pnas.1001639107](https://doi.org/10.1073/pnas.1001639107)

68. Fiore, M. & Strazewski, P. Prebiotic lipidic amphiphiles on the early earth. *Life* **6**, 17 (2016).
[https://doi.org/10.3390/life6020017](https://doi.org/10.3390/life6020017)

69. Jordan, S. F., Nee, E. & Lane, N. Isoprenoids enhance the stability of fatty acid membranes at the emergence of life potentially leading to an early lipid divide. *Interface Focus* **9**, 20190067 (2019).
[https://doi.org/10.1098/rsfs.2019.0067](https://doi.org/10.1098/rsfs.2019.0067)

70. Beyazay T., Belthle K. S., Farès, C., Preiner, M., Moran, J., Martin W. F. & Tüysüz, H. Ambient temperature CO₂ fixation to pyruvate and subsequently to citramalate over iron and nickel nanoparticles. *Nature Commun.* **14**, 570 (2023).
[https://doi.org/10.1038/s41467-023-36088-w](https://doi.org/10.1038/s41467-023-36088-w)

71. Fayolle, D., Altamura, E., D'Onofrio, A., Madanamothoo, W., Fenet, B., Mavelli, F., Buchet, R., Stano, P., Fiore, M. & Strazewski, P. Crude phosphorylation mixtures containing racemic lipid amphiphiles self-assemble to give stable primitive compartments. *Sci. Rep.* **7**, 18106 (2017).
[https://doi.org/10.1038/s41598-017-18053-y](https://doi.org/10.1038/s41598-017-18053-y)

72. Catling, D. C. & Zahnle, K. J. The archean atmosphere. *Sci. Adv.* **6**, eaax1420 (2020).
[https://doi.org/10.1126/sciadv.aax1420](https://doi.org/10.1126/sciadv.aax1420)

73. Boigenzahn, H. & Yin, J. Glycine to oligoglycine via sequential trimetaphosphate activation steps in drying environments. *Orig. Life Evol. Biosph.* **52**, 249-261 (2022).
[https://doi.org/10.1007/s11084-022-09634-7](https://doi.org/10.1007/s11084-022-09634-7)

74. Ni, F., Gao, X., Zhao, Z. X., Huang, C. & Zhao, Y. F. On the electrophilicity of cyclic acylphosphoramidates (CAPAs) postulated as intermediates. *Eur. J. Org. Chem.* 2009, 3026-3035 (2009).
[https://doi.org/10.1002/ejoc.200900227](https://doi.org/10.1002/ejoc.200900227)

75. Rabinowitz, J. Recherches sur la formation et la transformation des esters LXXXIII [1]. Réactions de condensation et/ou de phosphorylation, en solution aqueuse, de divers composés organiques à fonctions OH, COOH, NH₂, ou autres, à l'aide de polyphosphates linéaires ou cycliques. *Helv. Chim. Acta* **52**, 2663-2671 (1969).
[https://doi.org/10.1002/hlca.19690520847](https://doi.org/10.1002/hlca.19690520847)

76. Rabinowitz, J. Note on the role of cyanides and polyphosphates in the formation of peptides in aqueous solutions of amino acids, at room temperature, as a possible prebiotic process. *Helv. Chim. Acta* **54**, 1483-1485 (1971).
[https://doi.org/10.1002/hlca.19710540532](https://doi.org/10.1002/hlca.19710540532)
77. Hatanaka, H. & Egami, F. The formation of amino acids and related oligomers from formaldehyde and hydroxylamine in modified sea mediums related to prebiotic conditions. *Bull. Chem. Soc. Jap.* **50**, 1147-1156 (1977). https://doi.org/10.1246/bcsj.50.1147

78. Šponer, J.E., Coulon, R., Otyepka, M., Šponer, J., Siegle, A. F., Trapp, O., Ślepokura, K., Zdráhal, Z. & Šedo, O. Phosphoric acid salts of amino acids as a source of oligopeptides on the early Earth. *Commun. Chem.* **7**, 185 (2024). https://doi.org/10.1038/s42004-024-01264-6

79. Ross, D. & Deamer, D. Prebiotic oligomer assembly: what was the energy source? *Astrobiology* **19**, 4 (2019). https://doi.org/10.1089/ast.2018.1918

80. Muchowska, K. B., Varma, S. J. & Moran, J. Synthesis and breakdown of universal metabolic precursors promoted by iron. *Nature* **569**, 104–107 (2019). https://doi.org/10.1038/s41586-019-1151-1

81. Peters S., Semenov, D. A., Hochleitner, R. & Trapp, O. Synthesis of prebiotic organics from CO₂ by catalysis with meteoritic and volcanic particles. *Sci. Rep.* **13**, 6843 (2023). https://doi.org/10.1038/s41598-023-33741-8

82. Lopez, A., Vauchez, A., Ajram, G., Shvetsova, A., Leveau, G., Fiore, M. & Strazewski, P. From the RNA-peptide world: prebiotic reaction conditions compatible with lipid membranes for the formation of lipophilic random peptides in the presence of short oligonucleotides, and more. *Life* **14**, 108 (2024). https://doi.org/10.3390/life14010108

83. Westall, F., Hickman-Lewis, K., Hinman, N., Gautret, P., Campbell, K. A., Bréhéret, J. G., Foucher, F., Hubert, A., Sorieul, S., Dass, A. V., Kee, T. P., Gorgelin, T. & Brack, A. A hydrothermal-sedimentary context for the origin of life. *Astrobiology* **18**, 259-293 (2018). https://doi.org/10.1089/ast.2017.1680

84. Martin, W. & Russell, M. J. On the origin of biochemistry at an alkaline hydrothermal vent. *Phil. Trans. R. Soc. B: Biol. Sci.* **362**, 1887-1926 (2007). https://doi.org/10.1098/rstb.2006.188

85. Konn, C., Charlou, J. L., Holm, N. G. & Mousis, O. The production of methane, hydrogen, and organic compounds in ultramafic-hosted hydrothermal vents of the Mid-Atlantic Ridge. *Astrobiology* **15**, 381-399 (2015). https://doi.org/10.1089/ast.2014.1198

86. Nitschke, W., Farr, O., Gaudu, N., Truong, C., Guyot, F., Russell, M. J. & Duval, S. The winding road from origin to emergence (of life). *Life* **14**, 607 (2024). https://doi.org/10.3390/life14050607

87. Dobson, C. M., Ellison, G. B., Tuck, A. F. & Vaida, V. Atmospheric aerosols as prebiotic chemical reactors. *Proc. Natl. Acad. Sci. USA* **97**, 11864-11868 (2000). https://doi.org/10.1073/pnas.200366897

88. Tuck, A. The role of atmospheric aerosols in the origin of life. *Surv. Geophys.* **23**, 379-409 (2002). https://doi.org/10.1023/A:1020123922767

89. Jordan, S. F., van Zuilen, M. A., Rouillard, J., Martins, Z. & Lane, N. Prebiotic membrane structures mimic the morphology of alleged early traces of life on Earth. *Commun. Earth Environ.* **5**, 234 (2024). https://doi.org/10.1038/s43247-024-01372-0

90. Geisberger, T., Diederich, P., Kaiser, C. J., Vogele, K., Ruf, A., Seitz, C., Simmel, F., Eisenreich, W., Schmitt-Kopplin, P. & Huber, C. Formation of vesicular structures from fatty acids formed under simulated volcanic hydrothermal conditions. *Sci. Rep.* **13**, 15227 (2023). https://doi.org/10.1038/s41598-023-42552-w

91. Xu, J., Tsanakopoulou, M., Magnani, C., Szabla, R., Šponer, J. E., Šponer, J., Góra, R. W. & Sutherland, J. D. A prebiotically plausible synthesis of pyrimidine β-ribonucleosides and their phosphate derivatives involving photoanomerization. *Nature Chem.* **9**, 303-309 (2017). https://doi.org/10.1038/nchem.2664
**Acknowledgements:**

We thank Robert Pascal (Université Aix-Marseille) for his initiative in the earliest stages of our work on the reaction mechanism and his donation of H₂[¹⁸O], Peter Goekjian (Université Claude Bernard Lyon 1) for insightful NMR interpretations, and Marc Vedrenne (Institut de Chimie de Toulouse, Université Paul Sabatier) for taking NMR spectra using a QCI probe. We are grateful to the Volkswagen-Stiftung (Az 92 850 for P.S.) and the LABEX Institut Lyonnais des Origines for financial support within the program Investissements d'Avenir (LIO PhD program 2020 for A.S.) of the French government operated by the Agence Nationale de la Recherche (ANR-10-LABX-0066).

**Author contributions:** P.S. designed and supervised the research, and wrote the paper. M.F. and I.D. helped to design the study and with supervising A.S. H.C. supervised the ab-initio calculations and helped with writing the quantum theoretical sections. A.S. performed all experiments, analyses, curve fittings, designed Fig. 2 and wrote the Supplementary Information (SI) document, quantum theoretical and conclusion sections exempt. L.M. conceptualised and performed all ab-initio calculations and the reaction pathways depicted in the SI. A.L. analysed lipids for the reaction kinetics. E.F. performed and analysed with A.S. the mass spectra. A.B. performed and analysed with A.S. the NMR spectra. M.F. synthesised with P.S. labelled phosphate, with A.S. and A.L. reference compounds, performed the experiments on dodecanol, the microscopy of vesicles and contributed to writing the SI.

**Competing interests:** The authors declare no competing interests.

**Supplementary Information and Supplementary Datasets:**

Materials and Methods, Supplementary text and conclusions, Supplementary Figs. 1 to 197, Supplementary Tables 1 to 63, Supplementary Literature References 1-47, Appendix Figs. 1 to 25, Appendix Tables 1 to 7. Seven Supplementary Dataset files contain the ³¹P {¹H} NMR spectra that gave rise to the quantifications shown in Supplementary Figures 57, 61, 62, 71, 77, 78 and 85. The spectra in these files were ordered and named as in the corresponding Supplementary Figures. One eighth Supplementary Dataset contains the atomic coordinates (xls) of all stationary states as determined at the M06-2X/6-31G(d,p) level.
**Figure Legends:**

**Fig. 1.** Dehydrating agents (1, 2), liquid additives (3, 4), inorganic phosphate sources, and alcohols (5-16a) used in this work. **2a, 3a, 4a:** natural isotope-abundance; **2b, 3b, 4b:** enriched in ¹⁵N; **2c:** enriched in ¹³C. All alcohols and other compounds, except urea (2abc), the nucleosides (11-15) and the phosphate sources, are liquid at 80 to 130°C. Known standard free enthalpies of hydrolysis (at 25 °C in water) are marked next to arrows (left) or added in brackets (middle), to be compared with ΔG° = -2.2 kcal/mol for the hydrolysis of glyceryl phosphate, -3 to -5 kcal/mol for that of various sugar and nucleoside monophosphates, -7 to -12 kcal/mol for phosphoric anhydrides (di- and triphosphates), up to -14.8 kcal/mol for the hydrolysis of phosphoenolpyruvate¹¹. * Urea⁴⁵,⁴⁶,⁴⁸ and formamide⁹¹ (and, by analogy, other carboxamides) have been claimed in the literature to act as possible organo-catalysts in the phosphorylation of alcohols, cf. Fig. 4A and Supplementary Information (section 3.1).

**Fig. 2.** Maximum percent conversion of $P_i$, cTMP, PP$_i$ and SP$_i$ after 5 days at 115°C in neat conditions. Coloured boxes: Highest % $P_i$ conversion (from ³¹P NMR peak integration) to phospholipid precursors from alcohols 5, 6 (R = palmitoyl) and 7-10 blended with the phosphate source (black circles or ovals) and the dehydrating agent 1 or 2 (red stars), 1 mol equivalent each, all measured reaction scales included, maximum values for each class of compounds (acyclic, cyclic 1,2 and cyclic 1,3). Pie charts (right): Summary of nucleotide distribution (larger) and nucleobase identity (smaller) after heating the neat ribonucleosides (N) at 115°C, mol ratio 11:12:13:14:2:$P_i$ 0.25:0.25:0.25:0.25:4:1 and 1 eq 3a as liquidiser (from signal integration of UV$_{260}$nm using HPLC-HRMS). Other = non-canonical nucleobases (B) and nucleos(t)ides, see Supplementary Figs. 129-130.

**Fig. 3.** Time dependence of urea (filled circles) decay (black) and nitrogen exchange (red), amounts of glyceryl (diamonds)/MPG (squares) carbamate (green), glyceryl/MPG cyclic carbonate (blue), and $P_i$ consumption (cyan-violet) during phosphorylation of glycerol/MPG heated neat at 115°C for 120 hours [insert: 0-8 hours]. Molar ratios either [¹⁵N$_2$]urea/natural urea/NaH$_2$PO$_4$/glycerol 0.5:0.5:1:1, natural urea/NaH$_2$PO$_4$/MPG or [¹³C]urea/NaH$_2$PO$_4$/MPG 1:1:1. Relative values [%] obtained from time-dependent NMR peak area integration (Supplementary Figs. 42-53), the error margins (bars shown) resulted from NMR signal-to-noise ratios (Supplementary Tables 5, 7-8, 13-15, 17). SNR-weighted exponential data fitting gave the associated apparent kinetic rate constants $k$ [per hour] (and half-times, see Supplementary Tables 9, 10 and 18 and section 5.4 in the Supplementary Information).

**Fig. 4.** Chemical reaction mechanism of urea-assisted phosphorylations calculated using DFT (pink and pale green), and identified experimental products (white and pale ochre) using ¹³C (red), ¹⁵N (dark blue) and ¹⁸O isotopes (turquoise). A) and B) Ab initio-calculations on the reactivity of urea + $P_i$ with methanol (MeOH) in the presence of two assisting urea molecules (total urea and water molecules = U3W0, assisting urea not shown) and, additionally, two assisting water molecules (= U3W2, assisting molecules not shown). Elementary steps (magenta): tautomerization (taut), nucleophilic addition (A$_N$) and elimination (E). Most important calculated transition state (TS), intermediate states (Int) and associated free enthalpies of activation ΔG'' and standard free enthalpies of reaction ΔrG° = 0 kcal/mol for urea + $P_i$ (U3W0) and (U3W2); [kcal/mol] values from M06-2X/6-311++G(2d,2p)//M06-2X/6-31G(d,p) level of theory. Yellow text highlight = trajectory-determining transition state, i.e., fastest calculated route of all four. Cyan text highlight = ΔG'' for methanolysis of carbamoyl phosphate C$_P_i$ to methyl phosphoric acid and inorganic carbamate CA$_i$. C) Time-dependent formation of main products observed for the neat phosphorylation of glycerol (5) and MPG (6, R' = palmitoyl C16:0) using urea + $P_i$, isotope-labelled urea (2b, 2c) and [¹⁸O$_4$]P$_i$. All details, including nucleotide products (structures not shown here), see Supplementary Information.