Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions

Article abstract of DOI:10.1039/c7ob00708f

Giant lipid vesicles resemble compartments of biological cells, mimicking them in their dimension, membrane structure and partly in their membrane composition. The spontanenous appearance of closed membranes composed of bilayers of self-assembling amphiphiles was likely a prerequisite for Darwinian competitive behavior to set in at the molecular level. Such compartments should be dynamic in their membrane composition (evolvable), and sufficiently stable to harbor macromolecules (leak-free), yet semi-permeable for reactive small molecules to get across the membrane (stay away from chemical equilibrium). Here we describe bottom-up experiments simulating prebiotic environments that support the formation of simple amphiphilic molecules capable of self-assembling into vesicular objects on the micrometer scale. Long-chain alkyl phosphates, together with related amphiphilic compounds, were formed under simulated prebiotic phosphorylation conditions by using cyanamide, a recognized prebiotic chemical activator and a precursor for several compound classes. Crude dry material of the thus obtained prebiotic mixtures formed multilamellar giant vesicles once rehydrated at the appropriate pH and in the presence of plausibly prebiotic co-surfactants, as observed by optical microscopy. The size and the shape of lipid aggregates tentatively suggest that prebiotic lipid assemblies could encapsulate peptides or nucleic acids that could be formed under similar chemical prebiotic conditions. The formation of prebiotic amphiphiles was monitored by using TLC, IR, NMR and ESI-MS and UPLC-HRMS. In addition we provide a spectroscopic analysis of cyanamide under simulated prebiotic conditions in the presence of phosphate sources and spectroscopic analysis of O-phosphorylethanolamine as a plausible precursor for phosphoethanolamine lipids.

Full text of DOI:10.1039/c7ob00708f

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Giant vesicles from rehydrated crude mixtures containing
unexpected mixtures of amphiphiles formed under plausibly
prebiotic conditions
Received 00th January 20xx,
Accepted 00th January 20xx
Michele Fiore,a,* Warren Madanamoothoo,a Alexandra Berlioz‐Barbier,a Ofelia Maniti,a Agnès
DOI: 10.1039/x0xx00000x
Girard‐Egrot,a René Bucheta and Peter Strazewskia,* †
www.rsc.org/
Giant lipid vesicles resemble compartments of biological cells, mimicking them in their dimension, membrane structure
and partly in their membrane composition. The spontanenous appearance of closed membranes composed of bilayers of
self‐assembling amphiphiles was likely a prerequisite for Darwinian competitive behavior to set in at the molecular level.
Such compartments should be dynamic in their membrane composition (evolvable), sufficiently stable to harbor
macromolecules (leak‐free), yet semi‐permeable for reactive small molecules to get across the membrane (stay away from
chemical equilibrium). Here we describe bottom‐up experiments simulating prebiotic environments that support the
formation of simple amphiphilic molecules capable of self‐assembling into vesicular objects on the micrometer scale. Long‐
chain alkyl phosphates, together with related amphiphilic compounds, were formed under simulated prebiotic
phosphorylation conditions by using cyanamide, a recognized prebiotic chemical activator and precursor for several
compound classes. Crude dry material of thus obtained prebiotic mixtures formed multilamellar giant vesicles once
rehydrated at the appropriate pH and in the presence of plausibly prebiotic co‐surfactants, as observed by optical
microscopy. The size and the shape of lipid aggregates tentatively suggest that prebiotic lipid assemblies could encapsulate
peptides or nucleic acids that could be formed under similar chemical prebiotic conditions. The formation of prebiotic
amphiphiles was montiored by using TLC, IR, NMR and ESI‐MS and UPLC‐HRMS. In addition we provide a spectroscopic
analysis of cyanamide under simulated prebiotic conditions in the presence of phosphate sources and specroscopic
analysis of O‐phosphorylethanolamine as
a
plausible precursor for phosphoethanolamine lipids.
recent years, considerable progresses were made to find plausibly
prebiotic conditions leading to the formation of pyrimidine and
purine nucleotides on the primordial Earth. Different chemical
approaches have been considered. John Sutherland & colleagues
have shown that several important classes of biomolecules (2’,3’‐
cyclic pyrimidine nucleotides, various–amino acids and glyceryl
Introduction
How primitive protocells gradually transformed into the three
domains of life (Archaea, Bacteria and Eukaryota) is a challenging
issue addressed in systems chemistry and synthetic biology. Life as
we know it today has probably evolved from very simple chemical
phosphate) may have hydrogen cyanide and formaldehyde as
systems that assembled spontaneously under favorable conditions.
5,6
common chemical precursors.
Mauro
Raffaele Saladino, Ernesto Di
1
A
crucial step for life’s development has been the
&
colleagues obtained many prebiotically relevant
compartmentalization of the essential components required for
replication, translation, and transcription, in lipidic or otherwise
amphiphilic closed bilayer membranes.2 Over the past 60 years,
research in prebiotic chemistry has given several plausible
explanations for the formation, by condensation, of important
biomolecules such as amino acids and nucleotides. At first, a
significant boost was given by the pioneering experiments of
Stanley Miller,3 later revisited by Jeffrey Bada & colleagues.4 In the
compounds, including polyhydroxy alcohols, aldehydes, carboxylic
acids, amino acids, urea, guanidine, nitriles, N‐heterocycles and,
most notably, pyrimidine nucleosides and purine nucleosides, from
liquid formamide after irradiation with a proton beam (simulating
7
the Solar wind) and catalyzed by various meteorite powders.
Hendrik Zipse, Thomas Carell & colleagues obtained several purine
nucleosides from the double cyclization of formamido pyrimidines
in the presence of ribose or lower sugars under aqueous conditions.
8
Most recently, Mattew Powner & colleagues demonstrated how
pyrimidine nucleotides and 8‐oxopurine nucleotides could be
formed under similar prebiotic conditions.9 Lipid amphiphiles can
be generated under comparable chemical conditions.10 The classic
example is David Deamer’s pioneering work on the simulated
prebiotic synthesis of phosphatidic acids (PA), phosphatidyl glycerol
a. Institut de Chimie et Biochimie Moléculaires et Supramoléculaires,
Université de Lyon, Claude Bernard Lyon 1, 43 bdv du 11 novembre 1918,
69622 Villeurbanne Cedex, France
*E‐mail: michele.fiore@univ‐lyon1.fr, strazewski@univ‐lyon1.fr
†Electronic Supplementary Informaꢀon (ESI) available: NMR, ESI‐MS in positive ion
and negative ion modes, ESI‐HRMS and UPLC‐HRMS. See DOI: 10.1039/x0xx00000x
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(PG), and phosphatidyl glycerophosphate (PGP) by condensation of amine and 1‐decanoyl‐rac‐glycerol, were tested for the formation
glycerol, n‐dodecylic acid (or aldehyde) in the presence of sodium of vesicles. Vesiculation of pure decyl phosphate occurred only at
dicyanamide and disodium hydrogen phosphate.11 Recently, Damer pH 2, more complex mixtures grew vesicles at higher pH. The
and Deamer pointed out those fluctuating hydrothermal pools critical vesicle concentrations (CVC) were in the millimolar range
(FHPs) could be considered as plausibly prebiotic reactors for the (0.7–6.4 mM).
synthesis of several key molecules for the development of life,
Cyanamide as plausibly prebiotic precursor of urea for the
phosphorylation of n‐alkanols. In our rationale we assumed that
including lipids, nucleic acids and peptides.12 FHPs could be the
receptacles of organic, moderately hydrophobic compounds that
various alkanols, with even or odd number of carbons, can be
precipitated, fell into an FHP and accumulated, like a bathtub ring,
phosphorylated under the same conditions as reported for the
around its borders at the fluctuating water‐atmosphere interface.
preparation of decylphosphate.29 We wished to extend the
On the other hand, the role of single chain amphiphiles like mono‐
procedure for the preparation of mono‐alkyl phosphoryl
alkyl phosphates (APs) and fatty acids has enjoyed a growing
ethanolamines (polar head PE), since no example for the
interest ever since the pioneering studies of Hargreaves and
preparation of these compounds was reported in the literature, but
Deamer.13 Only few experiments were reported in the literature
an obvious advantage for the growth of vesicles was to be expected
from this type of zwitterionic polar headgroup. We selected
undecan‐1‐ol (1) that may have existed on early Earth, formed
under hydrothermal conditions by the reduction of the formic acid
in the presence of iron.30 As condensing agent we envisaged to use
cyanamide (2a) and not only urea (2b). Cyanamide was extensively
concerning the abiotic formation of AP precursors like n‐alkanols, n‐
alkanoic acids, n‐alkenes, n‐alkanes and alkanones from simple
organic compounds. UV irradiation of n‐alkanes followed by the
oxidation and reduction through radical intermediates generated n‐
alkanols (n‐ROH).14 Mixtures of cold condensed gases (CH4, NH3 and
H2O) produced aldo‐ and keto‐compounds of medium length.15
used as condensing agent for the formation, under simulated
Fischer‐Tropsch type reactions from the disproportion of formic or
prebiotic conditions, of acylglycerols,31 phosphatidic acids,32
oxalic acid under simulated hydrothermal conditions produced
glycerophosphates33 and phosphatidylcholines.34 Cyanamide also
long‐alkyl chain amphiphilic material that was hydrated to give
promoted short peptide formation under different conditions.4,35‐37
liposome‐like objects.16,17 Here we focus on APs as the driving
Most recently, Richert & coll. tested cyanamide for the concomitant
amphiphiles for the growth of primitive vesicles. We reason that,
condensation of adenosine monophosphate (AMP) and some amino
given favourable geochemical conditions (phosphate source, n‐
acids.38,39 As source of inorganic orthophosphate Pi we used
NH4H2PO4, (3a) a plausible source of inorganic phosphates and used
alkanols, dehydrating agents), APs could have been possible early
products originating from FHPs. In this work we asked the question
for similar purposes.20,29 For the preparation of undecyl
whether crude ‘prebiotic’ phosphorylation mixtures containing APs
phosphoethanolamine (5) we selected 2‐aminoethyl phosphate (3b)
would favour or disfavour vesiculation upon rehydration. In
as plausibly prebiotic organophosphate source.40 Concerning the
particular, in view of preliminary work done by others (vide infra),
geochemical origins and availability of soluble Pi and cyanamide on
we wished to test the growth of giant vesicles from the rehydration
the early Earth, we refer to published reviews on these topics21,41
of crude phosphorylation mixtures that contained naturally
and to the most recent developments in the field.42,43
occurring co‐surfactants, instead of externally added co‐surfactants.
Results
Prebiotic phosphorylation of n‐alkanols to APs. While the
Control experiments in the absence of the undecan‐1‐ol (1) were
phosphorylation of carbohydrates and nucleosides under simulated
carried out to monitor the stability‐reactivity profile of the
prebiotic conditions has been extensively studied,18‐20 only few
cyanamide (2a) and 2‐aminoethyl phosphate (3b) under simulated
examples have been reported on the phosphorylation of long chain
alcohols.21 Two mechanisms, not yet proven, can be considered for
the phosphorylation of long chain alcohols. An associative reaction
mechanism involving organocatalysis by urea,22 in which a mixed
prebiotic conditions. The experiments carried out on 2a had a
double purpose, as negative control and to optimize later
phosphorylation reaction conditions. The experiments on 3b were
carried out to better understand the formation of some unexpected
side products, vide infra. TLC, 1H NMR, 13C NMR and ESI‐MS or
UPLC‐HRMS analyses followed all the experiments. 31P NMR served
to confirm the presence of phosphate species in prebiotic mixtures
and control experiments.
Cyanamide under simulated prebiotic conditions.† We observed
that a 0.1 mM solution of 2a hydrolyses to urea (2b) in 24 hrs at pH
4.5 in the presence of 1 mM ammonium dihydrogen phosphate (3a)
at 65, 80 and 100°C (Table S1). 13C NMR spectra in H2O/D2O 9:1 v/v
showed that the chemical shift corresponding to that of 2a (118.1
ppm) disappeared with the apparition of another signal at 163.2
carbamidic phosphoric anhydride could offer urea as good leaving
23‐24
group20,
for the replacement with the n‐alkanol and/or any
other alcohol. Alternatively, and more in accord with studies on the
degradation of urea in water,25‐28 a dissociative mechanism could
explain the effective phosphorylation of alcohols with urea and
phosphate at elevated temperatures, whereby urea eliminates
ammonia to give isocyanic acid which is trapped by Pi to provide
carbamoyl phosphate as the phosphorylating agent. Monnard &
Sutherland phosphorylated 1‐decanol with urea and Pi and
identified by ESI‐MS decyl phosphate, accompanied by its sodium
salt, and didecyl pyrophosphate.29 Furthermore, rehydrations at pH
2, 7 and 12 of the dry crude phosphorylation mixtures, in the
absence and presence of added co‐surfactants like 1‐decanol, decyl
ppm
characteristic
of
the
carbonyl
of
2b.
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Figure 1. Structure of n‐alkanol 1 and reagents used in the presented experiments. Condensing agents: cyanamide (2a) and urea (2b). Phosphorylating
agents: ammonium dihydrogen phosphate (3a) and 2‐aminoethyl phosphate (3b). Cyanoguanidine (2c) and melamine (2d), both in red, result from
homologation of 2a. Compounds 4–15 obtained using 2a or 2b under simulated prebiotic phosphorylation reactions occurred in the presence of added
ammonium dihydrogen phosphate (3a) or 2‐aminoethyl phosphate (3b). Products 17–20 were obtained by direct transformation of 3b while 20–23 were the
direct products of polymerization and co‐polymerization of 3b and 17. The structures of 4–20 are consistent with the ESI‐MS and/or UPLC‐HRMS analyses of
the crude mixtures and where possible, confirmed by NMR spectroscopy. Standards 5s and 7s were prepared from undecan‐1‐ol (1) via undecanol‐2‐oxo‐
1,3,2‐dioxaphospholane (6). Condensation of 7s in presence of 2b produced both 13 and 14.
31P NMR showed after 6, 12, 24 and 48 hours no other chemical reanalyzed in DMSO‐d6 using as control compounds pure
shift than the signal of 3a at –0.17 ppm, typical of phosphates, commercial samples of 2a‐d (Figure S3). 13C NMR spectra of
irrespective of whether the sample was kept at ambient commercial cyanamide (2a), urea (2b), cyanoguanidine (2c) and
temperature or 80°C. When a sample containing 0.1 mmol 2a was melamine (2d) were recorded in DMSO‐d6 and compared to those
heated to 65, 80 or 100 °C under neat conditions or in neutral water of an analytical sample obtained from heating equimolar amounts
(pH 7) to dryness, the 13C NMR spectrum in H2O/D2O 9:1 v/v of 2a + 3a to dryness. When the temperature was kept at 25 or 40
showed new signals at 120.1 and 166.0 ppm. While the starting °C at neutral or acidic pH for 48 hours, 2a did not react to give
material 2a was still present (117.2 ppm), a signal at 163.2 ppm neither 2b, nor 2c or 2d. In fact, when 2a was hydrolyzed under
would match the chemical shift of 2b (Figures S1 and S2). As a acidic conditions at 65°C for 24 hours, 2c was obtained as the minor
further control, all the samples analyzed in H2O:D2O were product with residual 2a and 2b. No apparent reaction between 2b
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and 3a occurred at pH 4.5 and 80°C (Figure S5). Melamine (2d) was experiments, the starting material undecan‐1‐ol (1, Rf = 0.90)
produced in negligible amount, and was not detected in control disappeared with the concomitant appearance of more polar
experiment sample dissolved in DMSO‐d6 and it was detected by products (Rf = 0.62 and 0.40). The purification by conventional
ESI‐MS (vide infra). In spite of the elevated reaction temperatures chromatography (preparative TLC, column chromatography) of the
(80‐100°C), only the dimer 2c and cyclic trimer 2d but no higher individual components of Mixtures A or B failed due to the
oligomers of cyanamide were observed by direct ESI‐MS analysis of tendency of the mixture’s components to co‐migrate in any of the
the crude mixture. 44 To confirm NMR analysis, sample analyzed by examined eluent systems. The reaction conditions used and the
ESI‐MS showed that two derivatives of cyanamide were formed corresponding results are summarized in Table 1
under the conditions used: cyanoguanidine (2c) and melamine (2d)
Analysis of crude Mixtures A and B. To monitor the formation of
APs, crude reaction mixtures were submitted to infrared analysis.
The anti‐symmetrical phosphate vibration and the symmetrical
with characteristic ionization peaks [M+H]+ at m/z 85 and 127,
respectively (Figure S4).
Table 1.
Entry
phosphate vibrations appear in the region of 1300‐900 cm–1 of IR
spectra. The position of the phosphate bands is sensitive not only to
the chemical nature of phosphate ester‐bonds but also to their ionic
environment such as divalent metal ions and pH. Such positions of
phosphate bands may reveal mixtures of different phosphate
esters, for example, distinguish monoesters from diesters. The IR
spectra of crude Mixtures A (figure 2, upper spectra) are very
similar and show three bands located at 1157, 1078 and 940 cm‐1,
reminiscent of the three peaks of the 3a (figure 2, bottom lane). An
additional weak but distinct band at 989 cm–1 indicates the
presence of another chemical specimen than 3a in the crude
mixture, which could be assigned to 4 on the basis of the ESI‐MS
analysis of the product of the reaction reported below. IR spectra of
crude Mixtures B obtained from the reaction with 2a or 2b in the
presence of 3b, are quite similar. Minor differences, indicates
distinct populations of chemical species (figure 2, middle spectra).
The bands of 3b located at 1187‐83, 1078 and 1029 cm–1 (Figure 2,
bottom spectrum) were also found in both IR spectra of crude
mixture B, with an additional band at 978 cm–1 (figure 2 middle
spectra) indicating another chemical specie than 3b (figure 2,
bottom lane).45,46 The presence of undecyl phosphate (UnPA, 4) in
Mixture A was confirmed by direct ESI‐MS analysis of the crude dry
mixtures. Surprisingly, 4 was accompanied by a plethora of related
amphiphilic compounds 8–10 most of which with higher molecular
weight (figure 1). The structures of the side products 8–10 were
tentatively deduced only from the analysis of ESI‐MS spectra
recorded in positive and negative ion modes. UnPA (4) was
detected essentially in the negative ion mode at m/z 251 [M–H]–.
Bis‐(undecyl) pyrophosphate (8) was observed as m/z 244 [M+2H]2+,
255 [M+Na+H]2+. Ureyl phosphoramidate 9 was detected as m/z
294 [M+H]+, 317 [M+Na]+ and 293 [M–H]–. Undecyl isourea 10,
formally the adduct of 2a on 1, was observed as m/z 215 [M+H]+
also in presence of 2b (Table S2, entries 1‐7 and figures S23S27
and S28).† Mainly 4 was formed when the reaction of 1 was carried
out not only at 65°C but, surprisingly, also at 80°C for 360 hrs.
Unexpectedly, 2‐aminoethyl undecyl phosphate (5) was not
detected by direct ESI‐MS in any of the crude Mixtures B, instead,
MS signals attributed to compounds 10–15 were found. By direct
ESI‐MS at low resolution either compounds 11 or 12 were observed
as m/z 338 and were impossible to distinguish from one another.
Supposed (isomeric) 13 or 14 were detected as m/z 288 [M+2H]2+
and ether 15 was observed only in presence of 2a and was detected
as m/z 348 as [M+Na–H]– (Table S2, entries 8‐14 and figures
Added
Compounds
1 + 2a + 3a
Temp/time
Products
Vesicles
1
65°C/24h
4 & 10

Yes*
2
3
1 + 2a + 3a
1 + 2b + 3a
80°C/24h
100°C/24h
4, 8–9
4, 8–9

4
5
1 + 2b + 3a
1 + 2a + 3b
80°C/360h
100°C/24h
mainly 4
11–15
Yes§


6
7
1 + 2a + 3b
1 + 2b + 3b
80°C/24h
80°C/72h
10 & 11
13 & 14

8‡
9‡
1 + 2b + 3b
7s + 2b
80°C/48h
80°C/48h
5, 7, 12–14
13 & 14
Yes#



10
11
12
1 + 2a
5s + 2a
7s + 2a
80°C/48h
80°C/48h
80°C/48h
10
n.c.
n.c.

Structures of compounds 4 – 15 are shown in figure 4.
‡ from UPLC – HRMS *Vesicles obtained only at pH 2.0 and pH 9.21 (50
mM Bicine); §Vesicles formed in the presence of 10%w/w synthetic co‐
surfactants 5s or 7s (vide infra), the sample had a poor tendency to
vesiculate ; #Vesicles obtained at pH 7.8 (100mM Tris‐HCl) and pH 9.10
(50mM Bicine). n.c = no conversion. Details reported in Supporting
Informaꢀon Table S2.†
Formation of APs under prebiotic conditions.† Alkanol 1 was mixed
with the condensing agents, 2a or 2b and 3a or 3b, respectively. All
reactions were carried out using molar ratios 1:10:10 for 1/2a‐b/3a‐
b. We will refer to “Mixture A” for those analytes obtained from the
reaction with either 2a or 2b in the presence of 3a (PA polar
headgroups) and to “Mixture B” obtained from the reaction with 2a
or 2b in the presence of 3b (PE polar headgroups). The reaction
conditions were optimized by testing different temperatures (T),
reaction times (t) and reaction conditions. Two main conditions
were tested. In the first case, a mixture suspended in water was let
dry for a period of time between 24 and 48 hrs. The second tested
reaction mixtures did not contain any added water, the neat
reaction components were kept partly melted at the indicated
temperatures (65, 80 and 100 °C) for 24 or 48 hrs. Neat reaction
conditions required additional 24 hrs for a virtually complete
consumption of the starting alkanol. In certain cases, the
experiments were prolonged up to 2 weeks to check the stability of
formed compounds. For all conditions, except in control
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S29S35).† The unexpected results obtained for Mixture B made us (yield 90%, figures S12S20)† accordingly to what was previously
investigate more closely in two directions. First, we synthesized an reported by de Jongh and de Kruijfft.50
analytical sample of 2‐aminoethyl undecyl phosphate (5s) as a
reference compound. Second, we analyzed Mixture B by NMR
NMR and HR‐MS analysis of crude Mixture B. The NMR analysis of
crude Mixture
B performed in DMSO‐d6 or CDCl3 yielded
spectroscopy and UPLC‐HRMS (Ultra Performance Liquid
Chromatography coupled with High Resolution Mass Spectrometry)
for a more complete analysis of the compounds formed under
simulated prebiotic conditions. When reactions conditions required
cyanamide (2a) in all ESI‐MS spectra, the peaks of cyanoguanidine
(2c) and melanine (2d) were found.
unsatisfactory results due to the overlapping of the aliphatic signals
of the compounds present in the mixture (figures S37‐44).† Only a
31P NMR spiking experiment carried out in CDCl3 with standard
compounds 5s and 7s showed by comparison with the un‐spiked
original spectra that the major components of the mixture were 5
and 7 (figures S45‐48).† UPLC–HRMS of the same Mixture B showed
that 5 (RT 10.2 minutes, m/z 294.1844 as [M‐H]–) was detected
together with 7 (RT 10.8 minutes, m/z 295.1685 as [M‐H]–) and 12
(RT 10.3 minutes, m/z 337.1890 as [M‐H]–). These compounds were
accompanied by other two compounds (RT 17.4 and 18.0 min) both
having m/z 573.3342 for [M+H]+ and supposed structures 13 and 14
as shown in figure 4 (figure S49).† Part of dried Mixture B (20 mg)
was purified by preparative TLC using the eluent CHCl3/MeOH/H2O
65:45:4 v/v/v. A single TLC band was collected, extracted and
analyzed by UPLC‐HRMS. Spiking with 5s and 7s confirmed by UPLC‐
HRMS the presence of only 5 and 7. No traces of compounds 11‐14
were could be found (figure S50).† To confirm that 13 and 14 were
produced by the condensation of 7 under simulated prebiotic
conditions, synthetic 7s was heated at 80°C for 24 hrs in the
presence of urea (2b) with a molar ratio 1:10 (Table 1, entry 9). A
mixture of more polar compounds, inseparable by TLC, was
obtained. UPLC‐HRMS analysis confirmed the presence of the two
compounds with same molecular formula C26H55O9P2 and for both
m/z 573.3327 as [M‐H]– with retention times 17.4 and 18.0 minutes
and with complete disappearance of the starting material 7s (RT
10.8 minutes). The presence of these two distinct chemical
compounds at RT 17.4 and 18.0 minutes was confirmed by 1D and
2D NMR analysis and revealed a molar ratio o 2:1 (figures S52‐S57).
† In addition, 1 was heated under neat conditions for 48 hours in
the presence of 10 equiv. dry 2a to give 10 together with the
starting material 1 (Table S2, entry 7 and figure S36).† Neither 11
nor 12 were obtained by direct condensation of 5s or 7s in presence
of 2a (or 2b) under the conditions described above (Table 1, entries
11 and 12). At this point it was clear that, in Mixtures B, UnPE 5 was
generated only as a minor component by direct condensation of 1
with 3b in presence of 2a or 2b, and that the other detected
compounds (7, 12, 13 and 14) were a direct product of 1 with 3b to
give ureido derivative 17 followed by 18‐20 (figure 1) and the
products of the direct condensation of 7 under the same simulated
prebiotic conditions. To better understand the reactivity of 3b
under simulated prebiotic conditions, we set up a series of
experiments monitored by NMR and UPLC–HRMS analysis.
Figure 2. Infrared spectra of crude Mixtures A and B, respectively, containing
different APs obtained under simulated prebiotic conditions. Top spectra:
crude mixtures A obtained either with cyanamide (2a) or with urea (2b) in
the presence of Pi (3a). Middle spectra: crude Mixtures B from the reaction
with either 2a or 2b in the presence of 2‐aminoethyl phosphate (3b).
Bottom spectra: IR spectra of phosphates 3a and 3b used as phosphorylating
agents. All the samples were prepared in 100 mM Tris buffer pH=8.1
.
Synthesis of standard compounds for the analysis of Mixture B.†
Synthetic 2‐aminoethyl undecyl phosphate (5s) was prepared by
reacting reasonably anhydrous ammonia with 2‐(undecyloxy)‐1,3,2‐
dioxaphospholane 2‐oxide (6, figure 4, figures S7‐11†)47 as reported
for the synthesis of analogues of phospholipids48 and APs.49 The
synthesis of pure 5s was more difficult than anticipated, owing to
the formation of an another less polar compound that formed
under the same reaction conditions and not easy to separate from
5s. This side product was isolated pure and identified as 2‐
hydroxyethyl undecyl phosphate (7s). The chemical shifts of this
compound were very similar to those of 5s and, even though the
structure was elucidated by mono‐ and bi‐dimensional NMR
spectroscopy, only high resolution mass analysis (HR‐MS) could
confirm that 7s was the product obtained from ring opening of 6 by
a molecule of residual water (figures S21‐22).† To ascertain this
hypothesis, we prepared directly 7s from 6 with an excess of water
Reactivity of 2‐aminoethyl phosphate (3b) under prebiotic
conditions. Compound 3b was fully characterized by NMR
spectroscopy and used as reference compound (figures S58‐S62).†
A 1M solution of 3b (1 mmol) together with 1 mmol of urea (2b)
was heated to dryness for 16 and 48 hrs and the crude mixtures
were analyzed by NMR spectroscopy showing a plethora of signals.
COSY showed the presence of four major compounds together with
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several minor ones in the region between 4.5 and 2.5 ppm (figures and 5s on their own, formed oil droplets when hydrated at, both,
S64 and S69).† 13C and DEPT experiments confirmed the presence
pH 7.81 and 9.21 as shown in figure 3de, whereas 7s formed
of methylenes only (CH2) and several quaternary carbons (urea similar oil droplets at pH 7.81 and oil droplets accompanied by lipid
or ureyl groups at 163.0 and between 162.1 and 161.5 ppm, figures aggregates or giant vesicles (GVs of ≤ 5µm diameter) at pH 9.21
S65 and S70).† The main difference between the spectra registered (figure 5f). When 1 (4 % mol:mol) was added to 5s, the hydrated
after 16 and 48 hrs was the increase of the signals with respect to mixed film produced lipid aggregates and GVs at pH 7.81 (5µm
the residual 3b (figures S63, S68 and S74).† Furthermore, 31P NMR diameter); their size increased with increasing amounts of 1 (up to
spectra showed that two different patterns of signals were present 10 % mol:mol) and higher pH (9.21, figures 3g‐i). Dried thin films of
in the mixtures. Phosphates resonate in the region between 0.24 7s containing 1 (10% mol:mol) gave small assemblies at pH 7.82 (≤
and –0.02 ppm and pyrophosphates, present only in the sample 2µm diameter, figure 5l) but when hydrated at pH 9.21 produced
obtained after 48 hrs reactions, showed very small signals between interesting huge (> 40 µm diameter) lipid aggregate looking like
–10.40 and –11.30 ppm. (figures S67, S73 and S75).† The presence
of residual 3b was confirmed by 13C spiking experiment carried out
on the mixture obtained after 48 of heating (figure 76).† Direct HR‐
MS analysis and UPLC‐HRMS analysis confirmed that, together with
3b, six major compounds were present in the mixtures (figures
multilamellar and multivesicular GVs (figure 3mn). Surprised by
this result, we decided to investigate the capacity of 5s and its
analogue 7s to act as co‐surfactant in Mixture A that did contain
UnPA (4) and side products 8‐10 but had a poor tendency to
vesiculate or to even form hydrated aggregates in unbuffered
S7783).† Undecyl(2‐ureidoethyl) hydrogen phosphate (17) showed water.
at m/z 185.0321 as [M+H]+ (figure S84). † This compound was
Role of 1, 5s and 7s in the vesiculation of Mixture A. As a first
reported as being of high industrial interest and produced on a
large scale under similar condensation conditions.51 1‐(2‐
Hydroxyethyl)urea (18) was found at m/z 105.0659 as [M+H]+
(figure S85)† together with monophosphate 19 at m/z 98.9841 as
[M+H]+ (figure S84).† 2‐Hydroxyethyl dihydrogen phosphate (20)
was not found by HR‐MS but its presence is plausible in other
mixtures that allowed forming products 7 by direct condensation of
20 with 1 in presence of 2a or 2b as hypothesized in figure 1, middle
scheme. Three distinct polymers 21‐23 were also found in the
mixture as products of the direct polymerization and co‐
polymerization of 2b and 17 (figures S83 and S84).
experiment, we added to a ‘prebiotic’ Mixture A (10 mg mL–1) small
amounts of 1, 5s and 7s in ratio 1:1:1 w/w/w. GVs (>10m)
appeared when the amount of co‐surfactants reached 10% w/w.
We used unbuffered Milli‐Q water with a measured pH 7 for the
hydration (figures 6ac). In a second experiment, 1 (5% w/w) was
added together with 5s (5% w/w). Hydration at pH 7.0 (unbuffered
Milli‐Q water) gave neither GVs nor oil droplets (data not shown)
but when the sample was hydrated at pH 7.8 (Tris‐HCl) GVs (figure
6d) appeared together with worm‐like objects and other lipid
assemblies (not shown). A third experiment was carried out by
adding to mixture A 10% w/w of 7s. GVs and lipid aggregates very
similar to those observed for added 5s+1 were obtained, but no
Formation of vesicles and lipid aggregates upon hydration. The
crude (dry or wet) reaction mixtures A or B were hydrated using the
gentle hydration method.52 These suspensions, containing 1 to 5 mg
of membranogenic compounds and co‐surfactants, were hydrated
with an adequate volume of hydration buffer (or unbuffered Milli‐Q
water) to a final concentration of 10 mg mL–1. First trials were made
on Mixtures A (Table 1, entry 2). These mixtures formed, in
accordance with the results on crude decyl phosphate reported by
addition of
1 was needed this time (figures 6ef). These
experiments demonstrate that an important role is played by 5s
and 7s in the vesiculation of mixtures that require acidic or basic pH
values, in order to vesiculate or to aggregate from those mixtures
(Mixtures A and B) that had a poor tendency to form GVs or lipid
aggregates at neutral pH (being in our experiments unbuffered
Milli‐Q water).
29
Monnard & Sutherland small supramolecular structures at pH 2
Discussion
and surprisingly, aggregate lipids looking like vesicles (with 5µm
diameter) at pH 9.21 (figures 3ab). Hydration of Mixture B
produced small vesicle‐like structures when hydrated at pH 7.81
(not shown), while larger vesicle‐like structures were formed at pH
9.21 (figures 3c). Mixture B had the tendency to form oil droplets
that disappeared upon increasing the pH value. Decreasing the pH
(upon hydration with unbuffered Milli‐Q water) did not allow to
observe objects larger than 0.5 µm diameter accompanied by an
increasing number of oil droplets (data not shown). We also tried to
vesiculate mixtures that did not contain 5 or 7 (Table 1, entries 5‐7).
These mixtures did not yield any vesicles or oil droplets and the film
stayed as if not hydrated at any examined pH (data not shown). At
this point, we decided to compare the microscopic images of re‐
A non negligible, if not crucial, aspect of the described results is that
long‐chain monoalkyl phosphatidic acids (n‐ROPAs) and
phosphatidylethanolamines (n‐ROPEs) could both have been
formed under the same prebiotic conditions. Our experimental
findings showed that long‐chain monoalkyl phosphates (UnPA, 4)
and monoalkyl phosphoethanolamines (UnPE, 5) were formed
under simulated prebiotic conditions by condensing undecan‐1‐ol
(1) and ammonium phosphate (3a) or phosphorylethanolamine (3b)
in the presence of cyanamide (2a) or urea (2b). The reaction
conditions led to the formation of both products accompained by a
plethora of amphiphiles shown to act as co‐surfactants and
possessing chemical structures close or related to those of 4 and 5.
The use of 2a produced more amphiphilic side products than the
same reactions promoted by 2b. For Mixture B, the formation of
UnPE (5) was accompanied by chemically related compounds that
hydrated crude prebiotic Mixtures
A and B with those of
“reconstituted” mixtures obtained from hydrating thin films of
known amounts of synthetic products 1, 5s and 7s. Compounds 1
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All samples were prepared at 10 mg mL–1. All the samples prepared as
10mM in 3 mL of buffer and were stained with NileRed (1.2. mM,
results unexpected respect to those obtained for Mixture A. In fact,
under prebiotic conditions, 2‐aminoethyl phosphate (3b) reacts to
give at least six compounds that, once formed, can condense with
alkanol (1) yielding unexpected mixtures of compounds that can act
as co‐surfactants. Some of those, like 7, 11 and 12 seem to be the
direct product of the transformation of UnPE (5) under the prebiotic
condensation reaction conditions carried out in presence of 2a or
2b. The latest results on the phosphorylation of glycerol with
geochemically more plausible soluble phosphate sources in a urea‐
ammonium formate‐formamide‐water eutectic do suggest that 7
might have prebiotic roots as well.41

[excit]=561 nm). Scale bar 20µm for all micrographs in this figure.
Concerning the formation of the other co‐surfactants, their
synthesis under prebiotic conditions was possible only for
compound 10 (by condensation of 1 and 2a) and for the products
13 and 14 (formed by condensation of 7s in presence of 2b). In
particular, even if present only in close to negligible amounts, UnPE
(5) was formed under simulated prebiotic conditions with a certain
analogy to the biochemical route evolved for the biosynthesis of
1,2‐diacyl‐sn‐glycero‐3‐phosphoethanolamines (PEs). In modern
cells PEs are biosynthesized, among other known pathways, via the
cytidine diphosphate‐ethanolamine pathway (CDP‐ethanolamine or
“Kennedy” Pathway) using ethanolamine as the substrate.52 Since
the major function of PEs in modern biological membranes is to
fusion and promote membrane curvature,53 it is plausible that small
amounts of UnPE (5) generated under prebiotic conditions might
have played a role in promoting and forming membranes in first
protocells, especially if the available membranogenic compounds
were n‐ROPAs that required narrow pH ranges for vesiculation.54,55
Unfortunately, no paleontological evidence exists to confirm this
hypothesis, since the oldest micro‐fossils date back to 3.7 Gya.56,57
Certain is that in our experiments the role of 5 and its unexpected
analogue 7 (UnPOH, the major of the two compounds formed)
acting as co‐surfactants was demonstrated by adding small
amounts of those compounds, together with starting material 1, to
crude Mixtures A, that on their own had a poor tendency to
vesiculate. The GVs observed upon hydration of thin films showed
that not only mixtures of 5 and 1 promoted vesiculation but that 7
played and unexpected role in GV formation in the absence of 1.
The size and the shape of generated GVs or lipid aggregates having
not necessarily bilayer structures tentatively suggest that prebiotic
APs assemblies could be able to encapsulate polymers such as
peptides or nucleic acids that could be formed under similar
prebiotic reaction conditions (time, temperature, pH, presence of
condensing agents like urea‐phosphate mixtures), which remains to
be shown by experiment. It is plausible that one single chemical
environment, such as a fluctuating hydrothermal pools (FHP), might
have contained all the prebiotic reagents to form APs: mono‐alkyl
alkanols were in contact with sufficient amounts of chemicals acting
as condensing agents (cyanamide, urea) and phosphorylating
agents (organophosphates or phosphites).58 To date, the first proto‐
cellular membranes are mostly assumed as being formed from
hydrating either single‐chain “fatty” (carboxylic) acids or double‐
chain phospholipids (glyceryl phosphate esters) rather than APs.
APs do, however, unify two favourable properties that are not
combined in either of the other amphiphiles. Thus, joining small pH‐
robust polar headgroups with comparingly short and easy‐to‐make
hydrophobic single‐chains can give rise to just the right truncated‐
cone geometry to produce curved bilayers needed for vesiculation
and compartmentation. The facts that the found amphiphilic
species 414 were formed at mild temperatures, i.e. conditions that
are compatible with the prebiotic condensation of amino acids to
peptides, indicate that these molecules might also be formed
Figure. 3. Micrographs of giant vesicles and lipid aggregates obtained
from the gentle hydration of crude reaction Mixtures A and B and GVs
obtained from gently hydrating films of crude Mixture A enriched with of
1, 5s and 7s alone or in combination. a) Mixture A at pH 2; b) Mixture A
at pH 9.21; c) Mixture B at pH 9.21; d) 1 at pH 9.21 (Bicine 50mM); e) 5s
pH 9.21 (Bicine 50mM); f) 7s at pH 9.21; g) 5s+1 (96:4%mol:mol) at pH
7.81 (Tris‐HCl 100mM); h‐i) 5s+1 (90:10%mol:mol) at pH 9.21 (Bicine 50
mM); l) 7s+1 (90:10%mol:mol) at pH 7.81 (Tris‐Bicine). m‐n) 7s+1
(90:10%mol:mol) at pH 9.21 (Bicine 50mM). Micrograph h was taken
under bright field illumination. o‐q) GVs or lipid aggregates obtained
from gently hydrating Mixture A that contained mainly 4 enriched with
added 10 %mol:mol:mol synthetic 5s, 7s and 1 (ratio 1:1:1) as co‐
sufactants. r) Mixture A+(5s+1) (90:10% w/w) pH 7 (unbuffered Milli‐Q
water); s‐t) Mixture A+7s (90:10% w/w) pH 7 (unbuffered Milli‐Q water)
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59
together with nucleotides and nucleic acids.
Furthermore our General procedure for hydrolysis of cyanamide in prebiotic
findings confirm that cyanamide, being a plausible reservoir of
urea,21 is the precursor of related molecules, such as the dimer
cyanoguanidine (2c) and its cyclic trimer melamine (2d) that could
have played a crucial role in the prebiotic synthesis of biomolecules
in the early stage of life. In particular 2c is reported as efficient
condensing agent for peptides4 and as phosphorylation promotor of
nucleosides and sugars at low pH through the presumed
conditions. In a typical experiment, an Eppendorf tube or a
glass test tube (volume 2 ml), was filled with 1 mmol (42 mg)
of cyanamide (2a) in the presence of 1 equivalent of 3a or 3b
(115 or 141 mg, respectively). Samples were left heated (where
required) without any cap until dryness of the water/ethanol
mixture in a thermo‐shaker apparatus set to 80°C for 48 hrs.
Dried samples were dissolved in DMSO‐d6 or D2O and then
analyzed mixtures were compared to the commercial or synthetic
intermediacy
Melamine instead can undergo the equivalent of Watson‐Crick base
pairing with barbituric acid once ribosylated and
of
N‐[O‐(phosphatidyl)carbamoyl]guanidine.23
standard solutions. Cyanamide (2a) 13C NMR (DMSO‐d6): δC
=
117.2; 13C NMR (D2O): δC = 118.1; Urea (2b) 13C NMR (DMSO‐d6): δC
= 159.3; 13C NMR (D2O): δC = 163.2; Cyanoguanidine (2c) 13C NMR
(DMSO‐d6): δC = 119.1, 163.4; 13C NMR (D2O): δC = 120.0, 163.3;
Melamine (2d) 13C NMR (DMSO‐d6): δC = 167.4; 13C NMR (D2O): δC =
166.0.
ribophosphorylated under simulated prebiotic conditions to give
nucleoside‐ and nucleotide‐like compounds that spontaneously
assemble in non‐covalent hexad‐stacked supramolecular fibrous
polymers.60 Again, orthogonal reactions could occur in similar
heterogeneous media allowing the formation of covalent fibrous
assemblies needed to kick off the start for the emergence of the
first replicating proto‐cells and primitive self‐replicating organisms.
General procedure for transformation of 2‐aminoethyl phosphate
in prebiotic conditions. In a typical experiment, an Eppendorf
tube or a glass test tube (volume 2 ml), was filled with 1 mmol
(141 mg) of 2‐aminoethyl phosphate (3b) in the presence of 1
equivalent of 2b (60 mg). Samples were left heated (where
required) without any cap until dryness of the water/ethanol
mixture in a thermo‐shaker apparatus set to 80°C for 48 hrs.
Dried samples were dissolved in D2O and the analyzed mixtures
were compared to the commercial or synthetic standard solutions.
Mixture of compounds (selected signals) 1H NMR (D2O), mixtures of
aliphatic compounds: δH = 2.95, 3.12 (3b), 3.18, 3.25, 3.56, 3.77,
3.81, 3.95 (3b), 4.42; 13C NMR (D2O): δC = 40.2 (3b, from spiking),
40.3, 40.7, 40.8, 41.4, 42.2, 57.8, 60.8, 61.4 (3b, from spiking), 61.6,
64.5, 64.6, 161.8, 161.9, 162.0, 163.2 (2b). 31P NMR (D2O): δP = 0.24
(3b, from spiking) ‐0.02, ‐10.40, ‐10.60, ‐11.20, ‐11.30 ppm.
Experimental
General procedure for simulated prebiotic formation of
amphiphiles. In a typical experiment, an Eppendorf tube or a
glass test tube (volume 2 ml), was filled with 0.1 mmol of
1
(17.2 mg) and mixed with 10 equivalents (1mmol) of 2a or 2b
(42 or 60 mg, respectively) in the presence of 10 equivalents
(1mmol) of 3a or 3b (115 or 141 mg, respectively). The
heterogeneous mixture was suspended in 0.9 mL of ultrapure
water and 0.1 mL of ethanol were added in order to dissolve 1
in water and avoid the formation of giant oil droplets. The
obtained suspension was vortexed (1 minute), sonicated (5
minutes) and the obtained clear solution was left heated
without any cap until dryness of the water/ethanol mixture in
a thermo‐shaker apparatus set up at 80°C between 24 and 48
hrs. Reactions mixtures were monitored by analytical TLC using
eluent A and eluent B. A variant of this procedure was to mix
all the reactants and to let them melt at 80°C (neat
conditions). For all reaction conditions, a solid yellowish
residue was obtained. An aliquot was directly analysed by ESI‐
MS, UPLC‐HRMS, IR and NMR (where possible).
Amphiphile films: In a 5 ml round bottom flask, 1 ml of CHCl3
containing the mixture of amphiphiles (10 mg mL–1 or 1 mM for 1
mL of buffer solution or unbuffered milli‐Q water) was slowly
evaporated in a rotary evaporator, in order to obtain a film as thin
as possible. The film was further dried for 2 hours under vacuum
(membrane pump) in order to avoid any presence of residual
organic solvents. The solutions were hydrated by using the
methodologies as reported below.
Mixture B (selected signals) 1H NMR (DMSO‐d6), mixtures of
aliphatic compounds: δH = 0.83 (brt, ‐CH3), 1.18‐1.24 (brs, ‐CH2‐ )
1.37 (t, ‐CH2‐ ), 3.34 (brt, ‐CH2‐); then 3.62 (brt, ‐CH2‐) correlates
with 3.11 (brt, ‐CH2‐) as a ‐CH2‐CH2‐ system; 13C NMR (DMSO‐d6): δC
= 14.3, 22.6, 26.1, 29.1, 29.6, 31.7, 32.9, 40.9 61.3, 64.1 (signals at
3.62 and 3.11 correlates with those at 64.1 and 40.9 accordingly
with HSQC spectra). 31PNMR (CDCl3): δP = ‐0.07 and ‐0.05 ppm.
HRMS (ESI): M+ calcd. for C13H29NO4P [M‐H]‐ 294.1844; found
294.1844; M+ calcd. for C13H28O5P [M‐H]‐ 294.1685; found
294.1685; M+ calcd. for C14H30N2O5P [M‐H]‐ 337.1892; found
337.1890; M+ calcd. for C26H55O9P2 C26H57O9P2 [M+H]+ 574.3437;
found 575.3474.
Gentle hydration: For the preparation of GVs a thin layer of crude
amphiphile mixture obtained under simulated prebiotic conditions
(10 mg mL–1) was hydrated with unbuffered milli‐Q water (pH 7.0)
freshly prepared Tris‐HCl (100 mM, pH 7.8), or Bicine 50mM, at pH
9.2 for 8‐12 hours at 20°C. Once the hydration solution was added,
the samples were vortexed for 5 seconds, then sonicated for further
10 seconds. To be able to observe GVs under the epifluorescence
microscope, and clearly distinguish them from oil droplets or other
assemblies, each suspension (20 to 40 L) was stained with 2.0 μL
of Nile red (NR) prepared as 1.2mM ethanol solution.
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Chemical Systems” (COST Action CM1304) is gratefully
acknowledged.
Summary, conclusion and future perspectives
Undecyl phosphate (4), 2‐aminoethyl undecyl phosphate (5) and 2‐
hydroxyethyl undecyl phosphate (7) were formed from undecan‐1‐
ol (1) under simulated prebiotic conditions described as being
possible in fluctuating hydrothermal pools on an early Earth. The
reaction conditions included the use of either cyanamide or urea
and two different, plausibly prebiotic phosphate sources, soluble
inorganic orthophosphate (Pi) and 2‐aminoethyl phosphate. When
cyanamide was used as condensing agent, the obtained mixtures
were enriched with amphiphilic side products. Most of these side
products turned out to act as co‐surfactants in later experiments.
Urea as the phosphorylation catalyst/mediator was very effective
but gave less varied side products. A study using a combination of
mass spectrometry and 13C NMR spectroscopy showed that
cyanamide partially hydrolyzed to urea under the applied reaction
conditions, but the direct addition of cyanamide to hydroxyl or
amino groups could occur as well. Dried samples of most of the
simulated ‘prebiotic’ crude mixtures produced giant vesicles (GVs)
or lipid aggregates upon rehydration at neutral and weakly basic pH
values. The GVs were always accompanied by (non vesicular) oil
droplets and other supramolecular assemblies. In particular,
mixtures containing 5, or where 5 was present as co‐surfactant, GVs
or lipid aggregates formed at close to neutral pH. A fully synthetic
amphiphile found by serendipity and structural analogue of 5, that
might be a prebiotic product as well, was identified as 2‐
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Acknowledgements
We thank Yoan Dougnon, Éléa Wallisky and Valentin Travers‐
Lesage for their valuable help in the preparation of GVs from
prebiotic mixtures, and the members of the CO2‐Glyco
research group (ICBMS) for technical and scientific support.
The support from the European Coordination of Science and
Technology Action on “Emergence and Evolution of Complex
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