Selective Prebiotic Synthesis of α-Threofuranosyl Cytidine by Photochemical Anomerization

Article abstract of DOI:10.1002/anie.202101376

The structure of life's first genetic polymer is a question of intense ongoing debate. The “RNA world theory” suggests RNA was life's first nucleic acid. However, ribonucleotides are complex chemical structures, and simpler nucleic acids, such as threose nucleic acid (TNA), can carry genetic information. In principle, nucleic acids like TNA could have played a vital role in the origins of life. The advent of any genetic polymer in life requires synthesis of its monomers. Here we demonstrate a high-yielding, stereo-, regio- and furanosyl-selective prebiotic synthesis of threo-cytidine 3, an essential component of TNA. Our synthesis uses key intermediates and reactions previously exploited in the prebiotic synthesis of the canonical pyrimidine ribonucleoside cytidine 1. Furthermore, we demonstrate that erythro-specific 2′,3′-cyclic phosphate synthesis provides a mechanism to photochemically select TNA cytidine. These results suggest that TNA may have coexisted with RNA during the emergence of life.

Full text of DOI:10.1002/anie.202101376

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International Edition:
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doi.org/10.1002/anie.202101376
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Prebiotic Chemistry
Hot Paper
Selective Prebiotic Synthesis of a-Threofuranosyl Cytidine by
Photochemical Anomerization
Ben W. F. Colville and Matthew W. Powner*
Abstract: The structure of lifeꢀs first genetic polymer is
a question of intense ongoing debate. The “RNA world
theory” suggests RNA was lifeꢀs first nucleic acid. However,
ribonucleotides are complex chemical structures, and simpler
nucleic acids, such as threose nucleic acid (TNA), can carry
genetic information. In principle, nucleic acids like TNA could
have played a vital role in the origins of life. The advent of any
genetic polymer in life requires synthesis of its monomers. Here
we demonstrate a high-yielding, stereo-, regio- and furanosyl-
selective prebiotic synthesis of threo-cytidine 3, an essential
component of TNA. Our synthesis uses key intermediates and
reactions previously exploited in the prebiotic synthesis of the
canonical pyrimidine ribonucleoside cytidine 1. Furthermore,
we demonstrate that erythro-specific 2’,3’-cyclic phosphate
Figure 1. Nucleic acid backbones of TNA, RNA (R=OH, R1 =H), ANA
synthesis provides a mechanism to photochemically select
(R=H, R1 =OH), and DNA (R=H, R1 =H). Truncated 3’-2’-phospho-
diester of TNA (blue) compared to natural 5’-3’-phosphodiesters (red).
In TNA the a-anomer of l-threose nucleic acid is observed to Watson–
Crick base pair with RNA/DNA. B=nucleobase.
TNA cytidine. These results suggest that TNA may have
coexisted with RNA during the emergence of life.
N
ucleic acids are essential to modern biology, and several
theories for the origin of life rely on the chemical (non-
enzymatic) emergence of nucleic acids.[1,2] The “RNA world
theory”,[3] for example, which postulates that RNA emerged
as lifeꢀs first genetic polymer, is supported by the universal
dual genetic and phenotypic functions of RNA in biology.[4,5]
It has been suggested that selective chemical synthesis of
RNAꢀs monomers may have foreshadowed its selective
inclusion into life. However, alternative scenarios have been
proposed,[6] and it remains unclear whether RNA was the first
genetic polymer to emerge during the evolution of life,[7]
therefore alternatives must be evaluated. The structure and
composition of a nucleic acidꢀs backbone is a key property
that dictates its ability to form a Watson–Crick duplex, and is
therefore crucial to heritable information transfer. Threose
nucleic acid (TNA), built from a 4-carbon tetrose sugar, was
identified as a potential progenitor to RNA (Figure 1).[8] TNA
can form stable duplexes with not only itself but also with
RNA and DNA.[8–10] Although TNAꢀs backbone connectivity
(whereas RNA and DNA are linked by 5’-3’-phosphodies-
ters), TNAꢀs trans-vicinal phosphodiesters can stretch into
a conformation that can accommodate Watson–Crick base
pairing (Figure 1). Homo-duplexes of TNA are more hydro-
lytically stable than RNA, instead comparable to DNA.[11–14]
Mixed pools of TNA and RNA monomers could form
chimeric oligomers, which have been shown to promote the
formation of homogeneous oligomers during template-
directed ligation.[15] TNAs can also fold into tertiary struc-
tures that bind targets with high affinity and specificity.[16]
These properties of TNA have enabled the in vitro evolution
of TNAs, which can be synthesized by DNA poly-
merases,[10,17–19] RNA-dependent RNA polymerases,[20] and
even TNA polymerases.[21]
Despite TNAꢀs remarkable pairing properties and recent
progress toward the chemical synthesis of RNA mono-
mers,[2,22–30] the prebiotic synthesis of TNAꢀs monomers has
yet to be thoroughly evaluated. TNA is a simpler structure
than RNA, with one fewer chiral carbon atom in its sugar
moiety, and conceptually, TNAꢀs sugar moiety can be
assembled from two achiral C2 building blocks (whereas
RNAꢀs sugar moiety requires one C2 and one chiral C3
building block).[8] TNA nucleosides (adenosine, inosine,
uridine) have been observed in glycosylation reactions,[25,26]
but these glycosylation reactions require preformed nucleo-
bases and invoke formose-type reactions to deliver separately
formed tetrose sugars. We, and others, have recently eluci-
dated prebiotically plausible syntheses of RNA, ANA, and
DNA nucleosides which bypass these challenging glycosyla-
tion reactions,[22,27–30] and now we extend this work to evaluate
this mode of prebiotic synthesis for TNA. A key element of
results from
a truncated 3’-2’-phosphodiester linkage
[*] B. W. F. Colville, Prof. Dr. M. W. Powner
Department of Chemistry, UCL
20 Gordon Street, London, WC1H 0AJ (UK)
E-mail: matthew.powner@ucl.ac.uk
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202101376.
ꢀ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
10526
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these (RNA, ANA, DNA) syntheses has been common
aminooxazoline intermediates, and it is of note that tetrose
aminooxazolines (i.e., threose aminooxazoline, TAO and
erythrose aminooxazoline, EAO) can also be efficiently
synthesized by the same reactions that yield pentose amino-
oxazolines (e.g., ribose aminooxazoline, RAO) (Figure 2).[31]
Moreover, we have previously demonstrated that equimolar
reaction of 2AO with C2 and C3 sugars (glycolaldehyde 5,
glyceraldehyde, and dihydroxyacetone) significantly favours
synthesis of TAO over RAO (7:1, see ref. [27] and Supple-
mentary Figure 19 & 20 therein). Therefore, we envisaged
a prebiotic synthesis that yields TAO would in turn provide
access to TNA.[22,32]
Irradiation with UV-light provides a powerful mechanism
to bring about molecular change in prebiotic chemis-
try,[2,22,24,28,30,33,34] and UV-light would have been an important
energy source on the prebiotic Earth.[35] The prebiotic Earthꢀs
atmosphere is predicted to have been significantly more
transparent to UV-light, due to lower O2/O3 concentration
than the modern atmosphere, leading to more UV-light (l >
204 nm) irradiating the surface of Earth.[36–39] Pioneering work
by Orgel and co-workers demonstrated that irradiation of the
unnatural a-anomer of cytidine gave a 4% yield of the natural
b-anomer 1.[40] While low yielding, this demonstrated the
plausibility of photochemical anomerization for prebiotic
cytidine syntheses. Further studies have revealed that the
competing cyclization to oxazolidinones, via attack of the C2’-
hydroxy on the C2-position of the base, was problematic for
pyrimidine ribonucleoside anomerization.[33] Sutherland and
co-workers have recently demonstrated that irradiation
(254 nm) of a-ribo-thiocytidine in neutral water with hydro-
gen sulfide gave a much higher photoanomerization yield of
thiocytidine 2 than previously observed for anomerizations of
cytidines,[24] even without blocking the 2’-hydroxy moiety to
prevent oxazolidinone formation.[41,42] Proposed TNA pre-
cursor aminooxazolines (e.g., TAO) have been shown to form
alongside those aminooxazolines that can yield RNA,[31] but
the transformation of TAO to TNA has not been reported.
Accordingly, we set out to investigate the protecting-group-
free, prebiotic synthesis of threo-cytidine 3 by UV-light driven
photoanomerization (Figure 2).
Previous work has established that cyanamide 4 and
glycolaldehyde 5 react efficiently to form 2-aminooxazole
2AO (> 80%)[22] and that tetrose aminooxazolines, TAO and
EAO, can be formed in high yields (> 90%) from the reaction
of 2AO with glycolaldehyde 5 (Figure 2).[27,31,32] Here, we also
observed the reaction of TAO and EAO, with cyanoacetylene
(HC3N),[43,44] to give near-quantitative conversion to their
respective anhydronucleosides, 6 and 7 (Figure 3 and Supple-
mentary Figure 1). We next turned our attention to the
synthesis of tetrose nucleoside 3. We envisaged H2S addition
Figure 2. Divergent synthesis of RNA and TNA cytidines through the
reaction of cyanamide with prebiotically plausible aldehydes. The five-
carbon RNA backbone requires sequential reaction of glycolaldehyde 5
(blue) and glyceraldehyde (red) generating four furanosyl pentose
aminooxazolines (RAO, AAO, XAO, and LAO) and one pyranosyl
pentose aminooxazolines (p-LAO). The four-carbon TNA backbone
requires only glycolaldehyde 5 (blue) and only generates two furanosyl
isomers (EAO and TAO).
Figure 3. Synthesis of tetrose nucleosides by photochemical anomeri-
zation. Tetrose aminooxazolines (EAO and TAO) react with cyanoace-
tylene (HC3N) to yield tetrose 2,2’-anhydrocytidines 6 and 7. Reversible
thiolysis of 6 and 7, followed by selective photoanomerization of the
1’,2’-cis-nucleosides 9 and 10 yields 1’,2’-trans-nucleosides 8 and 11.
Oxidative hydrolysis of trans-1’,2’-thiocytidine 8 and 11 furnishes
cytidines 3 and 15, whereas oxidation of cis-1’,2’-thiocytidines 9 and 10
furnishes anhydrocytidines 6 and 7.
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to tetrose anhydronucleosides 6 and 7 would regioselectively
position a sulfur atom at C2 on the nucleobase and enable
photochemical anomerization to yield the TNA monomer 3
(via thiocytidine 8).
As anticipated, we observed smooth thiolysis of 6 and 7 to
their corresponding b-thiocytidines 9 and 10 in formamide
(78%, Figure 3 and Supplementary Figure 2). Therefore, we
sought to investigate the stereochemical inversion of the
anomeric position for the tetrose series to understand if this
would enable delivery of TNA monomers. We then envisaged
the C2-sulfur would be removed by a facile oxidation,[28] or by
hydrolysis,[24] to yield the canonical nucleobase.
After 1 day of irradiation with UV-light (254 nm) in water
at pH 7 with H2S (26 mm) we observed conversion of b-threo-
thiocytidine 9 (25 mm) to a-threo-thiocytidine 8 (46%). After
1.5 days irradiation a-threo-thiocytidine
8 (51%) was
observed to be the major product (Supplementary Figure 6).
Spiking with an authentic standard of 8 (See Supplementary
Information for synthesis of authentic 8) confirmed the
effective photochemical anomerization.
Concomitant photoanomerization of threo- and erythro-
thiocytidine 9 and 10 was observed to occur at a similar rate
(Figure 4), to furnish 8 (40%) and 11 (42%) after 1 day.
Maximum conversion was observed after 2 days (8 52%, 11
51%). Conversely, when 8 and 11 were irradiated (254 nm) in
neutral water for 3 days with hydrogen sulfide we observed
significant formation of 2-thiocytosine 12 from both a-
isomers, but interestingly little reversion to the b-anomers
(Supplementary Figure 14).
As anticipated the sulfur atom at C2 can be facilely
removed, to generate the canonical nucleobase. For example,
reaction with hydrogen peroxide[28] rapidly converted 8 to the
TNA nucleoside, a-threo-cytidine 3, in quantitative yield
(Supplementary Figure 4). Interestingly, however, cis-1’,2’-
isomers 9 and 10 were observed to rapidly cyclize to reform
their corresponding anhydronucleoside 6 and 7 (Figure 3 &
Supplementary Figure 3) upon exposure to hydrogen perox-
ide, rather than undergo conversion to their respective
cytidine hydrolysis products.
Encouraged by the anomerization of tetrose thiocytidines,
we next considered the role and timing of phosphorylation in
the sequence towards TNA. Phosphorylation of prebiotic
nucleosides has been demonstrated in the dry state with
urea[45] and we sought to utilize this simple phosphorylation in
our work. In the ribo-series, photoanomerization and con-
version of thiocytidine 2 to cytidine 1 must occur prior to
phosphorylation.[24] These constraints are imposed by the
formation of 2’,3’-cyclic phosphates which, when in a trans-
configuration with respect to the 2-thiocytidine nucleobase,
undergo (destructive) cyclization to 2,2’-thioanhydronucleo-
tides.[29] We envisage that this process, in the tetrose cytidines,
could furnish the selectivity required to synthesize the threo-
isomer (i.e. TNA cytidine) by selective destruction of the
erythro-isomer of thiocytidine.
Figure 4. 1H NMR (700 MHz, noesygppr1d, H2O/D2O (9:1), 6.1–
8.3 ppm) spectra showing the irradiation (254 nm) of 9 (18 mm) and
10 (16 mm) at pH 7 with H2S (113 mm): a) before the irradiation; and
after b) 1 day; c) 2 days; d) 3 days irradiation. Spectrum e) following
addition of authentic 8. Spectrum f) following addition of authentic 11.
À
À
!
~
*
(C6) H and (C1’) H resonances are labelled: 9 ( ), 10 ( ), 8 ( ),
and 11 ( ).
J
after undergoing photoanomerization from b-anomer to a-
anomer, attack the C2’-position of the sugar moiety, leading
to nucleotide degradation. Pleasingly, upon irradiation we
observed rapid destruction of cyclic phosphate 13; 2-thiocy-
tosine 12 was the sole observed cytosine product after
irradiating 13 for 2 days (Supplementary Figure 18). We
next irradiated a mixture of erythro-cyclic phosphate 13 and
threo-9; after 3 days 8 was the major nucleoside (47%), whilst
13 was observed to degrade in only 1 day (Figure 6 and
Supplementary Figures 16 and 17), demonstrating that irra-
b-erythro-thiocytidine 10 underwent highly selective urea-
mediated phosphorylation,[23,45] with only one equivalent of
ammonium dihydrogen phosphate, to yield 2’,3’-cyclic phos-
phate 13 as the major product (Figure 5A and Supplementary
Figure 15). We anticipated that the C2-sulfur of 13 would,
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Figure 5. Photochemical selection of threo-thiocytidine induced by
erythro-specific 2’,3’-cyclic phosphate synthesis. A) Urea-mediated
phosphorylation of erythro-thiocytidine 10 yields cyclic phosphate 13 in
near quantitative yield. 13 is observed to undergo rapid and complete
photodegradation to free base 12. B) Urea-mediated phosphorylation
of b-threo-thiocytidine 9 yields a mixture of b-threo-thiocytidine phos-
phates that photoanomerize to their a-anomers. Annulation of 9 is
also observed during urea-mediated phosphorylation to yield anhydro-
nucleoside 6, which phosphorylates and rearranges to yield photo-
stable erythro-cytidine 14. R=H or (PO3À)n.
diation, when coupled with erythro-specific 2’,3’-cyclic phos-
phate synthesis, allows the photochemical selection of threo-
thiocytidine 8 from a mixture of erythro- and threo-thiocyti-
dines. The 2’,3’-trans-configuration of threose means that
TNA nucleosides cannot form 2’,3’-cyclic phosphates, and
therefore TNA thiocytidine is inherently protected from
these destructive reactions. Phosphorylation of b-threo-thio-
cytidine 9 cannot furnish b-threo-2’,3’-cyclic phosphates, but
reversibly yields a mixture of nucleoside phosphates (Fig-
ure 5B and Supplementary Figure 20). Irradiation of this
mixture of acyclic phosphates leads to anomerization of b-
threo to a-threo nucleotides, which was unambiguously
confirmed (following treatment with bovine intestinal phos-
phatase) by sample spiking with a-threo-thiocytidine 8
(Supplementary Figure 20). However, the high temperature
required for urea-mediated phosphorylation resulted in some
conversion of 9 back to anhydrocytidine 6 and concomitant
phosphorylation and thermal rearrangement to yield photo-
stable a-erythro-cytidine-2’,3’-cyclic phosphate 14 (Supple-
mentary Figure 20).
Figure 6. 1H NMR (700 MHz, noesygppr1d, H2O/D2O (9:1), 5.85–
8.3 ppm) spectra showing the irradiation (254 nm) of 9 (12 mm) and
13 (12 mm) at pH 7 with H2S (109 mm): a) before the irradiation; and
after b) after 1 day; c) 2 days; d) 3 days; e) 4 days irradiation. Spec-
trum f) following addition of authentic 8. Spectrum g) following
À
À
addition of authentic 12. (C6) H and (C1’) H resonances are labelled
!
~
À
À
*
for 9 ( ), 13 ( ), and 8 ( ). (C6) H and (C5) H resonances are
labelled for 12 ( ).
J
are photostable. A milder phosphorylation strategy would be
required to further enhance the observed selection in the
intermediate thiocytidines and avoid conversion of 9 to 14.[46]
However, the trans-disposition of hydroxy groups in cytidine
3 means that phosphorylation cannot yield a 2’,3’-phosphate,
whereas the cis-disposition of hydroxy groups in 11 (and its
cytidine congener 15) mean that 2’,3’-phosphate formation is
inevitable in erythro-nucleotides. Therefore, the sluggish
chemical reactivity of 2’,3’-cyclic phosphates[47–51] may provide
the final selection required, during polymer synthesis, to
selectively incorporate TNA into nucleic acids. Whilst 3 can
be chemically activated to polymerisation (for example, as an
imidazolide[52,53]), activation in the erythro-isomer 15 will be
Our results demonstrate the TNA monomers can be
accessed by the same photochemical strategy as RNA
monomers. We also observed that, following phosphorylation
and irradiation, threose thiocytidine 8 can be photochemically
selected. However, this selection is only feasible in the
thiocytidine intermediates, as b-erythro-cytidines, such as 14,
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