Phosphorylation, oligomerization and self-assembly in water under potential prebiotic conditions

Article abstract of DOI:10.1038/nchem.2878

Prebiotic phosphorylation of (pre)biological substrates under aqueous conditions is a critical step in the origins of life. Previous investigations have had limited success and/or require unique environments that are incompatible with subsequent generation of the corresponding oligomers or higher-order structures. Here, we demonstrate that diamidophosphate (DAP) - a plausible prebiotic agent produced from trimetaphosphate - efficiently (amido)phosphorylates a wide variety of (pre)biological building blocks (nucleosides/tides, amino acids and lipid precursors) under aqueous (solution/paste) conditions, without the need for a condensing agent. Significantly, higher-order structures (oligonucleotides, peptides and liposomes) are formed under the same phosphorylation reaction conditions. This plausible prebiotic phosphorylation process under similar reaction conditions could enable the systems chemistry of the three classes of (pre)biologically relevant molecules and their oligomers, in a single-pot aqueous environment.

Full text of DOI:10.1038/nchem.2878

ARTICLES
PUBLISHED ONLINE: 6 NOVEMBER 2017 | DOI: 10.1038/NCHEM.2878
Phosphorylation, oligomerization and
self-assembly in water under potential
prebiotic conditions
Clémentine Gibard†, Subhendu Bhowmik†, Megha Karki†, Eun-Kyong Kim
and Ramanarayanan Krishnamurthy
*
Prebiotic phosphorylation of (pre)biological substrates under aqueous conditions is a critical step in the origins of life.
Previous investigations have had limited success and/or require unique environments that are incompatible with
subsequent generation of the corresponding oligomers or higher-order structures. Here, we demonstrate that
diamidophosphate (DAP)—a plausible prebiotic agent produced from trimetaphosphate—efficiently (amido)phosphorylates
a wide variety of (pre)biological building blocks (nucleosides/tides, amino acids and lipid precursors) under aqueous
(solution/paste) conditions, without the need for
a condensing agent. Significantly, higher-order structures
(oligonucleotides, peptides and liposomes) are formed under the same phosphorylation reaction conditions. This plausible
prebiotic phosphorylation process under similar reaction conditions could enable the systems chemistry of the three
classes of (pre)biologically relevant molecules and their oligomers, in a single-pot aqueous environment.
hosphorylation of biologically relevant molecules under poten- Results
tial prebiotic conditions is an important step in the origins of Phosphorylation of nucleosides/tides and oligonucleotides by
P
life and has been investigated (especially for nucleosides) over DAP. We began by investigating the phosphorylation of
a wide variety of conditions1–4 with more recent variations5,6. All nucleosides with DAP. Reacting 0.1 M uridine 1 with DAP in
of these approaches use differing phosphorylation sources and are aqueous solution, under a range of conditions (1–25 equiv. DAP,
limited in substrate scope due to their reaction conditions, or pH 5.5–10, r.t.–50 °C, with and without Zn2+ or Mg2+ or
require unique non-aqueous environments to enable the phos- imidazole and slowly over days to weeks, Supplementary Table 1),
phorylation process7,8. Many of these approaches are therefore we observed the formation of uridine-2′,3′-cyclophosphate (5,
incompatible with the next step of generating the corresponding oli- c-UMP, Fig. 1a) and traces of the 5′-amidophosphate of 5. While
gomers and higher-order structures (towards RNA or pre-RNA Zn2+ or Mg2+ accelerated the reaction, Zn2+ also lowered the pH,
worlds) from these phosphorylated substrates, necessitating leading to the hydrolysis13/condensation of DAP, necessitating the
spatially separated processes and other discrete mechanisms and continuous addition of DAP. The addition of imidazole
chemistries. A universal and efficient phosphorylating agent that (conjugate acid, pKa = 7.05) circumvented this problem at pH 7,
would phosphorylate a wide class of (pre)biological molecules in affording similar conversion while decelerating the hydrolysis/
water under similar reaction conditions and enable the formation condensation of DAP. Cytidine 2 was converted to 2′,3′-c-CMP
of higher-order structures would be of significance in the context (6, 27%) under similar conditions, while purines 3 and 4 reacted
of prebiotic systems chemistry9.
equally well, forming 2′,3′-c-AMP (7, 31%) and 2′,3′-c-GMP (8,
We have previously shown that diamidophosphate (DAP, Fig. 1) 27%), respectively. Further addition of DAP, over days/weeks,
efficiently phosphorylates a wide variety of prebiotically relevant facilitated ongoing conversions to the respective c-NMPs.
sugar molecules and their building blocks10. A prebiotic synthesis
Dry/solid-state and low water-activity prebiotic phosphoryl-
of phosphoenol pyruvate has been demonstrated recently using ation reactions have been widely investigated4,6,14–16. Mixing
DAP as the prebiotic phosphorylating reagent11. In all cases, the DAP, imidazole and 1 in the solid state at room temperature
mechanism involves a nucleophilic attack of the NH2-group of with drops of water led to a paste-like material (‘paste reaction’
DAP on the free aldehyde moiety to facilitate an intramolecular conditions) and efficiently produced
5
(∼80%, 30 days,
phosphorylation of the α-hydroxy-aldehydes (Supplementary Supplementary Table 1). A preparative experiment on a 1 g
Fig. 1). Such a mechanism precludes the phosphorylation of scale afforded 5 in 65% isolated yield (Supplementary Figs 18–21).
hydroxyl groups, which are not next to a carbonyl moiety (for To our delight, oligouridylate (up to the tetramer, Fig. 1b) with
example, nucleosides and glycerol). However, when we observed terminal 2′,3′-cyclophosphate was also observed, indicating
that DAP alone in water at pH 6, or in the presence of ortho- or that oligomerization was taking place under the same mild phos-
pyro-phosphates, produced trimetaphosphate (Supplementary phorylation conditions. Cytidine 2 was cyclo-phosphorylated
Figs 2–5), this suggested that direct phosphorylation of other under similar conditions (42%, 10 days). However, phosphoryl-
nucleophilic groups in aqueous media should be possible by a ation of purine-nucleosides 3 (<5%) and 4 (17%) was less efficient.
direct attack on the phosphorous centre, with the protonated Further investigations are under way to optimize the efficacy of
NH2-group of the DAP acting as the leaving group12.
the paste reactions and the oligomerization process and to
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. †These authors contributed
equally to this work. e-mail: rkrishna@scripps.edu
*
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2878
ARTICLES
a
b
HO
(DAP)
O
5 +
U-p-Ucp
U-p-U-p-Ucp
U-p-U-p-U-p-Ucp +
corresponding open forms
O
U
DAP
HO
HO
P
O
O
P
B
NH2
B
H2N
Method C
(paste reaction)
HO
OH
ONa
R1O
OR2
Methods
A, B or C
O
O
O
ONa
100
90
80
70
60
50
40
30
20
10
0
R1 = R2 = H
1 B = U
M
1 = U-p-U-p-U-p-U-cp
M2 = U-p-U-p-U-p-U-p
Na2M1–H
5 B = U
6 B = C
7 B = A
8 B = G
27–89%
conversion
2 B = C
Na3M1–H
3 B = A
4 B = G
NaM1–H
Na2M2–H
R
1 = PO3Na2
R2 = H
9 B = U
48–87%
conversion
NaM2–H
10 B = C
11 B = A
12 B = G
M1–H
R1 = H R2 = PO3Na2
13 B = C
47–67%
conversion
1,200
1,220
1,240
1,260
1,280
1,300
Mass (m/z)
14 B = A
c
O
P
O
P
O
P
H2N
O
O
NaO
O
NaO
O
O
NaO NaO
n
B
B
DAP
Methods A or B
73–91%
conversion
OH
HO
HO
n = 1,2
OH
n = 1,2
2
2
a = 5'-NMP
b = 5'-NDP
15b
a = 5'-NDPNH
b = 5'-NTPNH
19b
20b
21b
22b
15a
16a
17a
18a
19a
15a
16a
17a
18a
B = U
Zn2+, pH 5.5
16b
17b
18b
20a
21a
22a
B = C
B = A
B = G
d
O
O
O
P
(2')
Oligo
(3')
O
O
O
P
DAP
DAP
P
ONa
ONa
Oligo(3')
O
Oligo(3')
O
O
P
ONa
NH2
ONa
ONa
O
P
O
O
P
Oligo-(3')p: (A4U3A), (G2UGC2AGUC), d(T8)
P
NaO
O
O
ONa
(5')Oligo
NaO
O
(5')Oligo
(5')p-oligo: (U12), (A12), UAU2AU2A, G2U2CUC, d(T10),
d(T12), d(GCTACG)
NH2
ONa
23–66% conversion
Figure 1 | DAP-mediated phosphorylation of nucleosides/tides demonstrating the potential to generate and progress through the successive levels of
nucleotides and oligonucleotides under similar conditions. a–d, Phosphorylation of nucleosides and 2′- and 3′-mononucleotides to give cyclophosphates (a),
uridine under paste-reaction conditions, also forming oligonucleotides (b), 5′-nucleotides (c) and 5′- and 3′-phosphorylated oligonucleotides (d) by
diamidophosphate (DAP). The reactions were carried out by method A (aqueous, pH 5.5), method B (aqueous, pH ≈ 7) with or without Mg2+ or Zn2+ or
imidazole, or by method C (paste reaction). For details see Supplementary Information and Supplementary Tables 1–11.
analyse the nature of the phosphodiester linkages (2′,5′ vs 3′,5′) is of importance in the prebiotic formation of oligonucleotides18.
of oligomers.
The reaction of 3′-nucleoside monophosphates (3′-NMPs, 9–12)
The phosphorylation proceeds through nucleophilic attack of the and 2′-monophosphates (2′-CMP and 2′-AMP, 13–14) with DAP
2′,3′-cis-hydroxyl groups on the protonated DAP, leading to a puta- in water formed the corresponding 2′,3′-cyclophosphates efficiently
tive monophosphoramidate (2′-NMPNH or 3′- NMP ) intermedi- under various conditions (Fig. 1a). The corresponding 3′- and
NH2
2
ate, which then cyclizes to give the 2′,3′-cyclophosphate derivatives 2′-amidodiphosphate derivatives are observed as intermediates, on
5–8. When imidazole is present, the protonated DAP is converted to the pathway to c-NMPs formation.
the amidophosphoimidazolide (Supplementary Fig. 7), which reacts
The 5′-nucleoside monophosphates (5′-NMPs, 15a–18a) were
further. Support for these intermediates was obtained from amidophosphorylated in water at pH 7 by DAP (Fig. 1c and
31P-NMR using 15N-labelled reactants (Supplementary Figs 7–13). Supplementary Tables 7 and 8), with 77–90% conversions to the
This phosphorylation chemistry, in principle, can be extended to corresponding 5′-nucleoside-amidodiphosphates (5′-NDPNH
2
,
other alternative (prebiotically plausible) nucleosides that have the 19a–22a) within 5 days. The 5′-nucleoside diphosphates
adjacent cis-hydroxyl configuration17 (a work that is ongoing).
(5′-NDPs, 15b–18b) were similarly efficiently converted to the
The hydrolysis of nucleoside-2′,3′-cyclophosphates to the 2′/3′- nucleoside-amidotriphosphates (5′-NTPNH , 19b–22b; Fig. 1c and
phosphates, and reactivation to regenerate 2′,3′-cyclophosphates, Supplementary Table 9), with up to 10% of the corresponding
2
2
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ARTICLES
O
a
P
d
H2N
NH2
ONa
HO
O
HO
OH
Imidazole
(paste reaction)
O
P
OH
ONa
23
O
24
Approx. 60%
O
b
O
OH
DAP
7
O
26
O
25
+
2 μm
7
Imidazole
(paste reaction)
O
P
ONa
HO
OH
O
(Reaction mixture)
OH
e
23
c
O
Synthesis
O
O
O
7
O
OH
P
7
ONa
26
O
25
(Authentic sample)
0.5 μm
g
f
h
2 μm
2 μm
2 μm
Figure 2 | DAP-induced phosphorylation and esterification of glycerol with short-chain fatty acids give rise to simple mimics of phospholipids, leading to
formation of protocell-like structures under the same reaction conditions, signifying the single-pot transition of simple building blocks to higher-order
self-assemblies. a,b, Phosphorylation reaction of DAP with glycerol (a) and with nonanoic acid and glycerol (b) under paste-reaction conditions. c, Synthesis
of authentic cyclophospholipid 26. d, TEM image of a sample (15 mg in 1 ml water) from the crude reaction in b, showing the formation of vesicle-like
structures with a diameter of ∼9.2 µm. e, TEM image of a sample (1 mg in 1 ml water) of authentic phospholipid 26 from Fig. 2c showing the formation of
vesicle-like structures with a diameter of ∼3.5 µm. f–h, Confocal laser scanning microscopy fluorescence images of vesicles prepared with authentic
phospholipid 26 (1 mg in 0.1 ml water) with dye encapsulation. In f, green fluorescence indicates hydrophilic pyranine dye encapsulated within the cavity of
the liposome. In g, red fluorescence indicates rhodamine B dye labelling the bilayer phospholipid membrane of the liposome. In h, a fluorescence merged
image is shown of a phospholipid vesicle prepared with both rhodamine B dye and pyranine dye. For details see Supplementary Information.
2′,3′-cyclophosphates. At pH 5.5 with Zn2+, the 5′-NDPNH
(19a–22a) hydrolyse back to the starting 5′-NMP (15a–18a).
Phosphorylation of glycerol and fatty acids by DAP leading to
(self-assembly) vesicles. Because cis-disposed 2′,3′-hydroxyl
2
This ‘phosphorylation followed by hydrolysis’ raises the possi- groups of nucleosides are converted to cyclophosphates, glycerol
bility of using DAP as a (re)activating agent for 5′-, 2′- and 3′- 23 was reacted with DAP with the purpose of accessing the
nucleoside phosphates in water for ligation/oligomerization reac- building blocks of phospholipids (Fig. 2a). Mixing 23 with DAP
tions, providing an alternative to the current methods of activation and imidazole in water at r.t. led to the formation of glycerol-1,2-
in prebiotic oligomerization and replication studies19. cyclophosphate 24 (∼12%), with the paste reaction demonstrating
Phosphorylations of selected oligonucleotides with 5′-phosphate efficient conversion (∼60%) via the amidophosphorylated intermediate
and 3′-phosphate terminal ends were briefly investigated and (24i; Supplementary Table 12). Heating the paste reaction (50 °C)
found to produce the respective oligo-5′-DPNH and the corre- accelerated the reaction, but also led to the formation of the
2
sponding oligo-2′,3′-cyclophosphate (via the oligo-3′-DPNH ), par- diglycerol- phosphodiester, indicating that the cyclophosphate is
2
alleling observations made for mononucleotides (Fig. 1d and attacked by another molecule of glycerol. Based on studies by
Supplementary Tables 10 and 11). Investigations probing the Deamer20, we probed the ability for DAP to mediate ester bond
scope of DAP for template-mediated ligation/oligomerization formation by reacting nonanol with ammonium acetate under
are ongoing.
paste-reaction conditions and found that DAP increased the
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2878
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O
P
a
O
R
O
NH2
H2N
+
H
ONa
+
N
H3N
O–
O–
H3N
H2O, imidazole
R
O
R
n
R = H (27)
Oligopeptides
R = CH2CO2H (28)
R = CH2CH2CO2H (29)
n = 1–8
b
1H NMR
O
+
H3N
O–
O
O
P
27
Reference
H2N
c
α-proton region
13C NMR
Reaction mixture
d
O
ONa
NH2
13C NMR
27i
Reaction mixture (40 days)
Control reaction; no DAP (40 days)
4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9
{H-coupled} 31P NMR
47
46
45
44
43
42
41
178.5
177.5
176.5
175.5
174.5
e
O
P
Reaction mixture
f
O
P
{H-coupled} 31P NMR
g
NaO
N
H
CO2Na
NaO
H2N
N
H
CO2Na
HO
{H-coupled} 31P NMR
O
O
H2N
P
O
ONa
NH
27i
14.0
13.8
13.6
13.4
13.2
9.2
9.0
8.8
8.6
–0.2
–0.4
–0.6
Figure 3 | DAP phosphorylates amino acids in water while activating them towards the formation of oligopeptides in the same reaction setting.
a, Reaction of DAP with glycine, aspartate or glutamate leading to oligopeptides. b–g, Reaction of glycine with DAP leading to (quantitatively) phosphorylated
species that are intermediates on the way to oligopeptide formation, as seen by 1H-NMR (b), 13C-NMR (c,d) and 1H-coupled-31P-NMR (e–g) of the reaction
mixture. For conditions and details see Supplementary Information.
efficiency of ester bond formation when compared to the control 132–135). The opaque solution generated from the synthetic cyclo-
(Supplementary Fig. 117 and Supplementary Table 13). Encouraged phospholipid 26 in water was analysed by TEM, which showed
by these observations, we performed a one-pot paste reaction with ‘vesicle-like’ structures (Fig. 2e) comparable to those obtained
nonanoic acid 25 (ref. 20) and glycerol in the presence of DAP and from the crude paste-reaction mixture. This demonstrates that
imidazole (Fig. 2b). Analysis of the crude reaction mixture cyclophospholipid 26, generated in the crude paste-reaction
suggested the formation of a cyclophospholipid 26, the structure of mixture, is indeed capable of forming these vesicle-like structures,
which was confirmed by comparison with synthesized authentic 26 perhaps even better in heterogeneous mixtures with glycerol20. To
(Fig. 2c and Supplementary Figs 118–123). Such cyclophospholipid prove that the structures are indeed liposomes, we performed dye-
structures (for example, cyclophosphatidic acid) are known in inclusion experiments20, and confocal microscopy images showed
extant biology, but in a diagnostic and pharmacological context21.
that the red rhodamine dye was retained within the bilayer, while
Mixing a portion of this crude paste reaction containing 26 in the green pyranine dye was encapsulated within the cavity of the
water (pH of ∼8.5) resulted in an opaque solution, which became liposome (Fig. 2g,f). Preparing the liposomes with both dyes afforded
clear upon sonication. This was filtered (0.2 µm) and analysed by a merged-image suggesting an ‘uneven-enclosed’ structure (Fig. 2h),
dynamic light scattering (DLS) and transmission electron probably arising from changes in internal osmotic pressure22 and
microscopy (TEM) techniques, revealing the formation of micelles interlayer distances23 when both dyes are present. Preliminary reac-
and vesicle-like bilayer and multilamellar structures in the range tions of octanoic acid with DAP under the paste-reaction conditions
of 30–110 nm (Supplementary Figs 124 and 137). TEM analysis gave rise to smaller vesicle-like structures (Supplementary Fig. 131).
of the opaque solution itself (without sonication and filtration) dis- That a library of prebiotically plausible fatty acids, available through
played liposome-like structures with diameters around 280 nm meteoritic delivery24 or other prebiotic means20, could form stable
(Supplementary Fig. 125). In addition, we were surprised to also phospholipid liposomes such as giant vesicles by phosphorylation
find TEM images implying the formation of giant bilayer vesicle- in the presence of glycerol, while unprecedented, does align with
like structures with diameters of 9.2 µm on average (Fig. 2d), with the advantage provided by the physical and chemical heterogeneity
some structures nearing 20 µm (Supplementary Fig. 126). The in vesicles in the context of protocells25.
control reaction lacking DAP, but containing only nonanoic acid
and glycerol (Supplementary Fig. 133), or each of the components Phosphorylation of amino acids by DAP leading to oligopeptides.
alone (for example, nonanoic acid alone), did not show the Motivated by these observations we briefly explored—as a proof of
formation of these ‘vesicle-like’ structures (Supplementary Figs principle—the phosphorylation of carboxylic acids of three
4
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2878
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representative prebiotic amino acids 27–29 (Fig. 3a) under aqueous and alternative pathways at milder pH, such as corrosion of phos-
,
conditions3. When glycine 27 was reacted with DAP in water, phide minerals (schreibersite)39 40, towards the formation of DAP
quantitative phosphorylation of the α-amino and α-carboxyl would be desirable. In that context, the detection of PN-containing
,
groups was observed (Fig. 3b–g and Supplementary Figs 138–140). compounds in interstellar media41 42 and star-forming regions43
Concomitant formation of peptides up to an octamer was suggests that nitrogenous versions (or forerunners) of phosphate
observed by mass spectral analysis (Supplementary Figs 141–143). may not be unreasonable to contemplate32,44. The phosphorylation
Controls lacking DAP did not show any oligo-glycine formation of small molecules by DAP and its analogues may also prove to be
(Fig. 3b). The acyl-phosphoramidate of glycine 27i observed by useful in synthetic chemistry32.
NMR (Fig. 3d,g) is proposed to be the active intermediate26 that
leads to amide bond formation. There may be other possible
Methods
mechanistic pathways27,28
,
the establishment of which need
Full details are provided in the Supplementary Information.
further detailed investigations. Diketopiperazine (DKP) was also
detected. With Gly-Gly 30, the formation of oligomers up to an
octamer was observed by mass spectrometry (Supplementary
Fig. 144). The reaction of DAP with aspartic acid 28 and
glutamic acid 29 (Fig. 3a) also showed the formation of
oligopeptides up to tetramers (Supplementary Figs 145 and 147).
General phosphorylation protocols
DAP in water. DAP, with or without imidazole (and/or zinc chloride and/or
magnesium chloride), was added to the substrates (nucleosides, nucleotides,
oligonucleotides, glycerol, nonanoic acid or amino acids) in water. The pH was
adjusted and maintained between 5.5 and 8 by the addition of 4 M HCl (aq) and
agitated at room temperature. Additional DAP was added based on the progress of
the reaction and its consumption, as monitored by NMR analyses.
1
In the case of 28, H-NMR shows ∼23% conversion to higher-
order products with minimal DKP (Supplementary Fig. 146),
DAP in the ‘paste reaction’. The substrates (nucleosides, nucleotides,
while for 29, it was ∼15% conversion to higher-order products
oligonucleotides and glycerol) and DAP, with or without imidazole, were ground
together with a few drops of water. Additional DAP was added based on the progress
of the reaction and its consumption as monitored by NMR analyses.
(DKP + peptides,
Supplementary
Fig.
148).
Accurate
determination of oligopeptide yields (currently complicated by
interference from the various phosphorylated species),
optimization of reaction conditions for increasing oligopeptides
yields, the nature of mechanistic pathways and elucidation of the
nature of connectivity (α- versus β-) are some of the issues that
need to be addressed and are underway. The promising
preliminary results of oligopeptide formation in water warrant in-
depth and systematic investigations to determine the scope and
selectivity of the oligopeptide-forming process and to address the
issues associated with random peptide synthesis such as the rapid
increase in diversity of even short peptide sequences29.
Phosphorylation reaction analyses. The progress of the reactions was monitored by
the following techniques: 1H, 13C and 31P NMR spectroscopy (nucleosides,
nucleotides, glycerol, fatty acids and amino acids), fast protein liquid
chromatography (FPLC; oligonucleotides), liquid chromatography–mass
spectrometry (LC–MS; glycerol and amino acids) and matrix-assisted laser
desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF-MS;
oligouridylate, oligonucleotides and amino acids).
Vesicle preparation. Fatty acid (1 equiv.), glycerol (1 equiv.), DAP (5 equiv.) and
imidazole (5 equiv.) were mixed together with a few drops of water and left at room
temperature. An aliquot of this crude reaction mixture was mixed with water,
vortexed (with and without sonication and filtration) to allow for the formation of
micelles/vesicles.
Discussion
The similar phosphorylation conditions for all three classes of pre-
biological molecules (nucleosides, fatty acids and glycerol and
amino acids) suggest that they could be combined and conducted
in a single pot. Moreover, the commonality of conditions for the oli-
gomerization of different building blocks suggests that productive
and mixed chemistries might be possible30,31, such as cross-catalysing
the oligomerization and self-assembly process, and the resulting
higher-order structures could, in turn, increase the efficiency of
phosphorylation. This approach could create opportunities that go
beyond the generation of phosphorylated building blocks, leading
to the co-formation and coexistence of building blocks and their oli-
gomers in the same locality, which would be conducive for the
emergence of primordial synergistic systems within confined
(aqueous) environments towards an RNA or pre-RNA world(s).
Although there are similarities with extant biochemical pathways
using such P–N activation chemistries32 (such as N-phosphoryl
transfers27,28), any comparison must be viewed with caution given
the pitfalls of extrapolating extant biochemical pathways backwards
all the way to prebiotic chemistry and vice versa33.
Vesicle characterization. The structures obtained from the vesicle preparation
described above (and also from synthetic phospholipid 26) were characterized by
dynamic light scattering and visualized by TEM with negative staining and by
confocal laser scanning microscopy with dye incorporation.
Data availability. The data that support the findings of this study are available from
the corresponding author upon reasonable request.
Received 14 April 2017; accepted 22 September 2017;
published online 6 November 2017
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The results from this work and previous DAP-phosphorylation
investigations10,11,35 demonstrate that such an expansion of the
phosphorylation scenario to include the prebiotically plausible
nitrogenous version of phosphate (such as DAP) provides an effi-
cient and alternative solution to the prebiotic-phosphorylation
problem. Although DAP has been used as a prebiotically plausible
reagent11,35, it has been produced from the prebiotically available tri-
,
metaphosphate36 by reaction with ammonia37 38, at relatively high
pH. Because the results demonstrated in this work proceed at
lower pH, an investigation of additional, prebiotically plausible
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2878
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Acknowledgements
The work was supported by a grant from the Simons Foundation to R.K. (327124) and
NASA (NNX14AP59G). This is manuscript #29523 from The Scripps Research Institute.
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measurements, L. Leman for help with analysis of peptides and the S.F. Dowdy laboratory
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Author contributions
R.K. conceived the project. R.K., C.G., S.B., M.K. and E.-K.K. designed the experiments.
C.G. and S.B. performed the nucleoside/nucleotide/oligonucleotide phosphorylation
experiments. M.K., S.B. and C.G. performed the amino acid phosphorylation experiments.
M.K. performed the liposome studies. E.-K.K. and S.B. made the initial observations of
DAP-mediated phosphorylation. R.K. wrote the paper with input from C.G., S.B., M.K. and
E.-K.K. All authors discussed the results and commented on the manuscript. C.G., S.B. and
M.K. contributed equally to this work.
Additional information
Supplementary information is available in the online version of the paper. Reprints and
permissions information is available online at www.nature.com/reprints. Publisher’s note:
Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Correspondence and requests for materials should be addressed to R.K.
33. Lazcano, A. Complexity, self-organization and the origin of life: the happy
liaison? Origins of Life 2009, 13–22 (2009).
Competing financial interests
The authors declare no competing financial interests.
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