Prebiotic phosphorylation of 2-thiouridine provides either nucleotides or DNA building blocks via photoreduction

Article abstract of DOI:10.1038/s41557-019-0225-x

Breakthroughs in the study of the origin of life have demonstrated how some of the building blocks essential to biology could have been formed under various primordial scenarios, and could therefore have contributed to the chemical evolution of life. Missing building blocks are then sometimes inferred to be products of primitive biosynthesis, which can stretch the limits of plausibility. Here, we demonstrate the synthesis of 2′-deoxy-2-thiouridine, and subsequently 2′-deoxyadenosine and 2-deoxyribose, under prebiotic conditions. 2′-Deoxy-2-thiouridine is produced by photoreduction of 2,2′-anhydro-2-thiouridine, which is in turn formed by phosphorylation of 2-thiouridine—an intermediate of prebiotic RNA synthesis. 2′-Deoxy-2-thiouridine is an effective deoxyribosylating agent and may have functioned as such in either abiotic or proto-enzyme-catalysed pathways to DNA, as demonstrated by its conversion to 2′-deoxyadenosine by reaction with adenine, and 2-deoxyribose by hydrolysis. An alternative prebiotic phosphorylation of 2-thiouridine leads to the formation of its 5′-phosphate, showing that hypotheses in which 2-thiouridine was a key component of early RNA sequences are within the bounds of synthetic credibility.

Full text of DOI:10.1038/s41557-019-0225-x

Articles
https://doi.org/10.1038/s41557-019-0225-x
Prebiotic phosphorylation of 2-thiouridine
provides either nucleotides or DNA building
blocks via photoreduction
JianfengXuꢀ ꢀ1, NicholasJ.Greenꢀ ꢀ1, ClémentineGibard2, RamanarayananKrishnamurthyꢀ ꢀ2* and
JohnD.Sutherlandꢀ ꢀ1*
Breakthroughs in the study of the origin of life have demonstrated how some of the building blocks essential to biology could
have been formed under various primordial scenarios, and could therefore have contributed to the chemical evolution of life.
Missing building blocks are then sometimes inferred to be products of primitive biosynthesis, which can stretch the limits of
plausibility. Here, we demonstrate the synthesis of 2′-deoxy-2-thiouridine, and subsequently 2′-deoxyadenosine and 2-deoxy-
ribose, under prebiotic conditions. 2′-Deoxy-2-thiouridine is produced by photoreduction of 2,2′-anhydro-2-thiouridine, which
is in turn formed by phosphorylation of 2-thiouridine—an intermediate of prebiotic RNA synthesis. 2′-Deoxy-2-thiouridine is
an effective deoxyribosylating agent and may have functioned as such in either abiotic or proto-enzyme-catalysed pathways to
DNA, as demonstrated by its conversion to 2′-deoxyadenosine by reaction with adenine, and 2-deoxyribose by hydrolysis. An
alternative prebiotic phosphorylation of 2-thiouridine leads to the formation of its 5′-phosphate, showing that hypotheses in
which 2-thiouridine was a key component of early RNA sequences are within the bounds of synthetic credibility.
on-canonical, sulfur-containing nucleobases are utilized Results and discussion
by extant biology1,2, and exert interesting effects on the 5′ Phosphorylation. First, we attempted the phosphorylation of
N
stability, base-pairing properties and replication fidelity of 2-thiouridine 8 in hot formamide7, but increased the concentrations
nucleic acid polymers3. As such—and given the key role of sulfur of phosphate and nucleoside from 100mM to 200mM, compared
in established prebiotic synthetic scenarios4,5—they are poten- with our previous phosphorylation of cytidine (full experimental
tial components of a genetic system that either complemented details are included in the Supplementary Information). After heat-
or perhaps predated, and ultimately evolved into, enzymatically ing at 100°C for 20h, we observed the initially formed phosphory-
replicated canonical RNA and DNA. We recently demonstrated lated product, thiouridine-5′-phosphate 14, in 24% yield alongside
a viable pathway to pyrimidine ribonucleosides and ribonucleo- the starting material 8 in 42% yield and some minor products
tides via thioribonucleosides (Fig. 1)5. Highly crystalline ribo- (Fig. 2 and Supplementary Fig. 39). In hot urea, ribonucleosides
aminooxazoline 1 was shown to react with cyanoacetylene 2 generally undergo initial (kinetic) 5′ phosphorylation8; over time,
to give anhydronucleoside 3, which on thiolysis furnished α-2- the 2′,3′-cyclic phosphate dominates over the 5′-phosphate (ther-
thioribocytidine 4. 4 could be efficiently photoanomerized to modynamic control). The formation of phosphate monoesters in
β-2-thioribocytidine 5, which serves as a precursor to the canoni- hot urea (and formamide) is reversible but slow, such that clear-cut
cal pyrimidine ribonucleosides cytidine 6 and uridine 7, β-2- kinetic or thermodynamic control is not shown by such systems.
thiouridine 8, and oligomerizable6 cyclic phosphates of cytidine Szostak and coworkers3,9 have already shown that non-enzymatic
and uridine (9 and 10, respectively).
RNA replication using the 2-methyl- and 2-amino-imidazole-
Since we had demonstrated that thioribonucleosides 5 and 8 activated derivatives of 14 proceeds with a higher rate and fidelity
are probably formed in primordial pyrimidine synthesis, we set than the corresponding activated uridine nucleotide, but until now
out to further explore prebiotic phosphorylation conditions that a prebiotic provenance of 14 remained unestablished. These results
might yield oligomerizable thioribonucleotides. Herein, we dem- show that the kinetic product of phosphorylation—the 5′ nucleo-
onstrate that phosphorylation of 2-thiouridine 8 can either lead tide 14—is indeed accessible, and would become increasingly plau-
to an oligomerizable nucleotide or initiate a prebiotic synthesis sible if progressively milder prebiotically plausible phosphorylation
of a deoxythionucleoside, 2′-deoxy-2-thiouridine 11, via hydro- procedures are uncovered. Our synthesis of 14 shows that it could
gen sulfide-mediated photoreduction as the key step. We show have been available for non-enzymatic incorporation into primor-
that 11 is a productive transglycosylating reagent in an abiotic or dial RNA sequences, and provides further impetus for the investiga-
proto-enzymatic context; for example, in the supply of 2-deoxy- tion of the potential role of 2-thiouridine in RNA.
ribose 12 and the synthesis of a canonical 2′-deoxynucleoside,
2′-deoxyadenosine 13. These results provide a chemical connec- Thioanhydride formation. Semi-molten urea is an alternative
tion between RNA and DNA in a prebiotic context (that is, in the prebiotic phosphorylation medium to hot formamide8, so we next
absence of biosynthesis).
used it to attempt phosphorylation of 8 (Fig. 3a). Surprisingly, no
1MRC Laboratory of Molecular Biology, Cambridge, UK. 2Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.
*e-mail: rkrishna@scripps.edu; johns@mrc-lmb.cam.ac.uk
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NH2
HO
O
HO
O
HO
HO
O
P
N
N
N
NH
N
O
O
hν
2
N
N
O
O
NH2
O
O
O
O
N
NH2
O
O
P
HO
HO
1
3
O
O
O
O
9
10
HS
Pi1 / formamide
P
i1 / formamide
NH2
NH2
NH2
O
HO
HO
HO
HO
O
O
O
O
N
N
hν
N
N
N
N
N
NH
H2O
S
S
O
O
HO
OH
HO
OH
HO
OH
HO
OH
4
5
6
7
+
O
NH2
N
HN
N
N
OH
O
S
HO
This work: Prebiological
O
N
HO
synthetic link between RNA
and DNA
O
N
NH
N
O
O
S
OH
HO
OH
HO
OH
8
HO
OH
11
12
DNA building blocks
13
Fig. 1 | Summary of previous and present work. Previous work demonstrated the synthesis of the canonical pyrimidine RNA nucleotides 9 and 10
alongside other RNA building blocks, such as 2-thiouridine 8. This work (boxed) demonstrates a link between 8 and a range of DNA building blocks, via
prebiotic phosphorylation and photoreduction. hv, UV irradiation; Pi1, NH4H2PO4/urea.
O
O
OH
O
Activation and
oligomerisation
P
O
O
HO
Pi1
RNA
O
O
N
NH
N
NH
oligomers
Formamide
S
(concentrated)
S
HO
OH
HO
OH
14 (24%)
(Kinetic phosphorylation product)
8
Fig. 2 | 5′ phosphorylation of 2-thiouridine 8 in concentrated formamide solution. Prebiotically plausible conversion of 8 into 2-thiouridine-5′-phosphate
14 in hot formamide potentially enables incorporation of 8 into primordial RNA sequences, by previously developed prebiotic activation of nucleoside
phosphates and oligomerization chemistry3,9
.
2′,3′-cyclic nucleotide derivatives were observed. A major prod- confirm that the major product of phosphorylation of 8 (even before
uct had been formed, but a total of four monophosphate 31P NMR enzymatic dephosphorylation) was indeed 15a. After the crude reac-
resonances suggested that the hydroxyl groups of this product were tion products from the phosphorylation were subjected to alkaline
phosphorylated to varying degrees, complexifying the NMR spectra. phosphatase (Fig. 3a), addition of an NMR integration standard
The crude mixture from the phosphorylation reaction was therefore (sodium formate) revealed that 2′,2-anhydro-2-thiouridine 15a had
treated with alkaline phosphatase, generating a single major nucleo- been formed in 50% yield, accompanied by some glycosidic bond
side product. Extensive analysis of both one- and two-dimensional cleavage (2-thiouracil 19 (15%) and isocytosine 20 (7%)). To obtain
NMR spectra of the dephosphorylated product suggested the forma- the ratio of the anhydronucleoside to its phosphorylated derivatives
tion of 2′,2-thioanhydronucleoside 15a10. Previously, Hampton and before treatment with alkaline phosphatase, the crude mixture from
Nichol11 synthesized a related derivative, 2′,2-anhydro-uridine 16, the phosphorylation of 8 was subjected to analytical high perfor-
when uridine 7 was treated with diphenylcarbonate in hot dimethyl- mance liquid chromatography, which showed that the ratio of 15a:
formamide (Fig. 3b). Under those conditions, intermediate uridine 15b: 15c was 3.7:1:1 (Supplementary Figs. 57 and 58). The same
2′,3′-cyclic carbonate11 17a is a good substrate for ring opening at phosphorylation conditions were also applied to 2-thiocytidine 5
C2′ by 2-O to give cyclonucleoside 16. Thus, under phosphorylation (Fig. 3a), which afforded 2′,2-anhydro-2-thiocytidine 2112 (43%
conditions, we propose cyclic phosphate 18 as the intermediate in the yield), cytidine 6 (8%), diaminopyrimidine 22 (16%), 2-thiocyto-
formation of 15 (R1 =R2 =H or PO3H−), with rapid ring opening by sine 23 (8%) and cytosine 24 (8%). All structures were confirmed
the nucleophilic sulfur atom ensuing to convert 18 to 15. To confirm by spiking experiments, with synthetic standards made in house or
the structure of the product and support our proposed mechanism, purchased (Supplementary Figs. 32 and 35).
2′,2-anhydro-2-thiouridine 15a was unambiguously synthesized
To further support a cyclic phosphate intermediate, we sought
from 2-thiouridine 8 and diphenylcarbonate (Fig. 3b). The fully milder conditions for phosphorylation of 8. We chose diami-
characterized synthetic standard was used for spiking experiments to dophosphate (DAP)—a reagent known to generate not only the
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a
O
O
O
O
O
NH
NH
N
N
N
N
Pi2
N
NH
S
+
+
O
O
S
S
O
Urea
N
H
N
HO
NH2
S
O
OH
H
P
OR1
RO
R2O
O
19 (15%)
20 (7%)
O
OH
O
15 (50% total yield)
15a, R1 = R2 = H
15b, R1 = HOPO2 , R2 = H
15c, R1 = H, R2 = HOPO2
15d, R1 = R2 = HOPO2
(15a:15b:15c = 3.7:1:1)
8
Proposed
Alkaline
intermediate 18
18a, R = H
18b, R = HOPO2
phosphatase
NH
NH2
N
NH2
N
NH2
NH2
N
NH2
N
Pi2
N
N
N
N
N
+
+
+
+
S
S
O
O
O
O
Urea
S
N
O
N
H
HO
N
H2N
OH
OH
H
OR1
HO
R2O
OH
OH
5
21 (43% total yield)
21a, R1 = R2 = H
21b, R1 = HOPO2 , R2 = H
21c, R1 = H, R2 = HOPO2
21d, R1 = R2 = HOPO2
6 (8%)
22 (16%)
23 (8%)
24 (8%)
Alkaline
phosphatase
O
b
O
O
O
NH
NH
N
PhO
OPh
N
N
N
X
X
O
X
DMF,
O
O
HO
NaHCO3,
150 °C
OH
O
HO
HO
OH
O
OH
O
7, X = O
8, X = S
17a, X = O
17b, X = S
16, X = O (59%)
15a, X = S (70%)
Fig. 3 | Prebiotic and synthetic routes to (thio)anhydronucleosides. a, Synthesis of the thioanyhydronucleosides of uridine 15 and cytidine 21 under
prebiotic phosphorylation conditions. 2-Thiouridine 8 and 2-thiocytidine 5 both undergo phosphorylation to form the cyclophosphate intermediate
18 shown for the uridine variant. These intermediates undergo rearrangement to give structures of type 15 and 21, respectively. 15d was not directly
observed, but is inferred from the mechanism. In addition to rearrangement, the nucleosides undergo some cleavage of the glycosidic C–N bond to
yield free nucleobases 19 and 23, as well as their hydrolysis/ammonolysis products 20, 22 and 24, respectively. b, Hampton and Nichol’s synthesis of
2′,2-anhydrouridine 16 and our use of the same conditions to synthesize the sulfur analogue 15a as a standard. 17a and 17b are the proposed intermediates
leading to the formation of 16 and 15a, respectively. Pi2, Na2HPO4/NH4Cl/NH4HCO3.
corresponding 2′,3′-cyclophosphates from nucleosides, but also
We also investigated the reaction of DAP (moist paste) with
short oligonucleotides after prolonged reaction13. Indeed, reaction 2-thiocytidine 5, which barely proceeded under the conditions
of 2-thionucleoside 8 with DAP and imidazole under moist-paste employed for 8. By lowering the pH of the reaction paste from 7 to 4
conditions at room temperature resulted in formation of the cyclic and raising the reaction temperature to 60°C, we were able to detect
phosphate 18a after one week (Fig. 4a). Monitoring the phosphor- cyclic phosphate 28, and later we were able to identify the corre-
1
ylation reaction by 31P, H and 13C NMR, we observed the trans- sponding 3′-phosphate and 3′-amidopyrophosphate of thioanhy-
formation of cyclophosphate 18a to a new species that showed drocytidine (21b and 29) by 31P NMR (Fig. 4b and Supplementary
characteristic NMR, electrospray ionization high-resolution mass Figs. 64 and 65). An accurate conversion was difficult to deter-
spectrometry and ultraviolet spectral signatures of anhydronucleo- mine by 1H NMR, but the combined yield for 28, 21b and 29 was
tide 15b (Supplementary Figs. 60–63). Furthermore, the DAP reac- estimated at slightly greater than 10%. The greater reactivity of
tion of 8 does not stop at 15b; the 3′ phosphate of 15b reacts again 2-thiouridine 8 towards DAP compared with 2-thiocytidine 5 prob-
with DAP, leading to the corresponding 3′-amidodiphosphate of the ably stems from its greater solubility and pKa near neutrality (8.0,
2′,2-thioanhydronucleoside 25. The total conversion (measured by N3–H)2.
1H NMR) of 8 to 15b/25 was 78% after one month at room tempera-
ture. When 4-thiouridine 26 was subjected to the same DAP phos- C2′ reduction. Our syntheses of thioanhydronucleosides 15 and
phorylation reaction as a control, the expected cyclophosphate 27 21 from 2-thionucleosides 8 and 5 are the first prebiotically plau-
was formed after a few days at room temperature, but no further sible routes to nucleoside derivatives containing a sulfur atom con-
transformation to the corresponding 2′,2-anhydride was observed nected to C2′. Such compounds are of significant interest in the
(Fig. 4a and Supplementary Figs. 66 and 67). In this case, cyclization origins-of-life field because the potential conversion of a C–S to
was presumably prevented due to the lower nucleophilicity of the a C–H bond (a chemically facile process14,15) would result in the
oxygen on C2 compared with sulfur.
synthesis of 2′-deoxynucleosides—components of DNA. In mod-
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O
O
a
O
O
NH
N
O
N
N
N
DAP
NH
O
O
N
O
imidazole
HO
HO
N
S
S
S
O
S
O
‘Paste’,
O
O
HO
Room temperature
O
P
O
OH
P
P
OH
O
O
O
O
HO
OH
O
O
P
O
15b
8
18a
NH2
S
25
S
(15b + 25 = 78%)
NH
DAP
NH
O
N
imidazole
N
O
O
x
‘Paste’,
O
Room temperature
O
OH
HO
O
P
O
HO
OH
O
26
27 (27%)
NH
NH2
b
NH2
N
NH
N
N
N
DAP
N
P
N
N
O
imidazole
O
N
S
O
S
S
S
O
‘Paste’,
O
pH 4, 60 °C
O
O
OH
O
HO
HO
P
O
O
O
HO
O
P
O
P
O
NH2
O
OH
HO
OH
O
O
5
28
29
21b
Conversion of 5 to 28 + 21b + 29 > 10%
Fig. 4 | Phosphorylation of thionucleosides under mild conditions with DAP. a, Phosphorylation of 2-thiouridine 8 with DAP leads to the formation of
thioanhydrouridine-3′-phosphate 15b and corresponding 3′-amidodiphosphate 25 via the intermediate cyclophosphate 18a. The same phosphorylation
with 4-thiouridine 26 results in the cyclophosphate 27, which can undergo no further transformation. b, Phosphorylation of 2-thiocytidine 5 with
DAP under slightly more forcing conditions affords thioanhydrocytidine-3′-phosphate 21b and corresponding 3′-amidodiphosphate 29, again via an
intermediate, cyclophosphate 28.
ern biology, ribonucleotide reductases convert ribonucleotides to afforded two new nucleoside derivatives, as well as thiouracil 19 as
deoxyribonucleotides16. This enzymatic reduction proceeds via a major products. Promisingly, one nucleoside showed two 2′-H sig-
1
radical mechanism with a dithiol as the stoichiometric reductant. nals shifted upfield (2.64 and 2.32ppm) in the H NMR spectrum
We wondered if a mechanism involving sulfur-based reductants (Supplementary Fig. 38), strongly suggesting that a deoxyribosyl
might have led to the formation of 2′-deoxyribonucleosides/2′- moiety was present. Comparison of NMR spectra with literature
deoxyribonucleotides on early Earth, in the absence of complex values for deoxyribosyl pyrimidines suggested the formation of
enzymes, from 2′-thio-functionalized ribonucleosides. Nagyvary 2′-deoxy-2-thiouridine 11. We subsequently verified this assign-
and coworkers17–19 first experimentally investigated this idea by syn- ment by synthesizing 11 via a literature route25 and using this mate-
thesizing 2′-thiocytidine (using an enzyme) and studying a variety rial to spike our samples (Supplementary Fig. 40). We speculate
of reduction procedures, including photoreduction. However, the that the second nucleoside product is 2′-α-thio-2-thiouridine 30,
limited published characterization data for the supposed 2′-thio- but attempts to isolate and characterize this compound have so far
cytidine substrate have since been pointed out as apparently incon- been thwarted by its facile decomposition to 2-thiouracil 19 as the
sistent with the structure20. Recently, Powner and coworkers21 only identifiable product. The instability of the glycosidic bonds of
revisited the idea with a proposal to modify the prebiotically plau- 2′-thioribonucleosides has previously been documented26. Addition
sible pyrimidine synthesis5,22 by including a sulfur atom attached of the NMR integration standard, pentaerythritol, to the crude mix-
to C2′ from the outset. This route proved unsuccessful because the ture indicated yields of 33, 25 and 28% for 11, 19 and presumed 30,
sulfur-containing building blocks exhibited different reactivity to respectively. The 5′- and 3′-phosphates of 2′,2-anhydro-2-thiouri-
analogues from the pyrimidine synthesis. Indeed, while a route dine (15b and 15c, respectively) undergo the same photoreduction,
from pure ribose or arabinose to deoxyribose has recently been in similar yields to 15a (Supplementary Figs. 41 and 42).
established23, a plausible abiotic synthesis of 2′-deoxynucleosides
A possible mechanism for this remarkable photoreduction is
themselves—whether by desulfurization or other means—has not proposed in Fig. 5b. Separate control reactions in the dark with H2S
yet been reported. 2′,2-thioanhydronucleosides 15a and 21a were and irradiation without H2S gave no reduction products nor 30, sug-
therefore subjected to ultraviolet irradiation at 254nm in the pres- gesting that initiation is by an electron released from hydrosulfide
ence of aqueous hydrogen sulfide as a reductant (Fig. 5a). Flux under ultraviolet irradiation4,27,28, rather than homolysis of the C–S
measurements from the same experimental setup have been car- bond of 15a. Transfer of this electron to thioanhydrouridine 15a to
ried out within our group previously, from which we determined a form the radical anion 31, and subsequent homolysis of the C–S
flux rate of 2.5×1016 cm−2 s−1 (19mWcm−2) for the 254-nm-centred bond would give intermediate 32. The newly formed C2′ radical can
emission24. Inspection of 1H NMR spectra showed that, under these abstract a hydrogen atom from H2S (pictured) or react with a hydro-
conditions, thioanhydrocytidine 21a gave 2-thiocytosine 23 in 29% gen atom (not pictured), to give 11. Alternatively, reaction with an
yield after 3h of irradiation. However, thioanhydrouridine 15a HS• radical equivalent (HS− with loss of an electron, or H2S2 or a
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of the hydrolysis of 2′-deoxy-2-thiouridine 11 at 60°C revealed half-
lives of 32 and 31h for 2′-deoxy-2-thiouridine 11 in 0.27M phos-
phate (pH7.0) and acetate (pH4.1) buffers, respectively, and 28h
in unbuffered solution (Fig. 6a and Supplementary Figs. 43–50).
The complete and clean conversion of 2′-deoxy-2-thiouridine 11 to
2-thiouracil 19 and deoxyribose 12 suggests that 11 may have been
a prebiotic source of deoxyribose, both in abiotic reactions and/or
for early enzymes, akin to the use of activated ribose in modern
phosphoribosyltransferases30–32. This reactivity of 11 presented an
opportunity for a plausible synthesis of purine deoxynucleosides via
transglycosylation33–35. Indeed, heating of 11 with excess adenine 33
in the dry state36 at 100°C for 31h led to the formation of both
alpha and beta isomers of 2′-deoxyadenosine 13, in 6 and 4% yield
respectively, accompanied by nucleobase loss to give 2-thiouracil 19
in 72% yield (Fig. 6b and Supplementary Figs. 54 and 55) Notably,
replacement of 11 with 2-deoxyribose 12 led only to a complex
mixture that did not contain β-2′-deoxyadenosine (Supplementary
Fig. 56). Such a demonstration of a plausible abiotic pathway stem-
ming from the ribo-pyrimidine synthesis to DNA is significant, and
further efforts are underway in our laboratory to discover more
favourable transglycosylation conditions. Additionally, if promis-
cuous proto-enzymes with ribosylase activity existed, 11 may have
been an efficient source of deoxyribose in an early biosynthesis of
deoxyribonucleotides via transglycosylation. Thus, there are vari-
ous plausible pathways by which the C2′-reduced nucleoside 11
could have facilitated the chemical progression of RNA into DNA.
O
O
a
O
HN
HN
O
hν, 254 nm
N
S
N
S
NaHS, pH 7
N
N
HN
O
+
+
S
H2O
O
O
SH
S
N
H
20 °C–35 °C
OH
HO
OH
11 (33%)
OH
30 (28%)
HO
HO
19 (25%)
15a
(21a: No reduction)
(Proposed structure)
hν
b
O
O
N
HS
e
HS
O
N
N
N
(aq)
N
N
O
S
O
S
O
S
OH
OH
31
HO
OH
HO
HO
15a
32
H2S
SH equivalent
H
H
SH
O
O
HN
HN
O
S
S
N
N
HN
Conclusions
O
O
Our investigation into the phosphorylation of prebiotic RNA pyrim-
idine intermediate 8 has resulted in several significant discoveries.
We have shown that 5′-phosphoryl-2-thiouridine 14 can be obtained
from kinetically selective phosphorylation of 8, demonstrating its
viability as a potentially important primordial non-canonical com-
ponent of nucleic acids. Other phosphorylation conditions lead to
the until-now elusive prebiotic functionalization of the C2′ position
of a nucleoside with sulfur. Moreover, we successfully reduced such a
sulfur-containing derivative to a non-canonical 2′-deoxyribonucle-
S
SH
N
H
OH
19 (25%)
OH
11 (33%)
HO
HO
30 (28%)
(Proposed structure)
Fig. 5 | Photoinduced reduction of thioanhydrouridine with hydrosulfide
(hS–) and its proposed mechanism. a, Photoreduction of 15a gives
the major product 2′-deoxy-2-thiouridine 11, as well as 2-thiouracil 19,
which is possibly formed via facile nucleobase loss from proposed
intermediate 30 (see ref. 26). Nucleotides 15b and 15c—minor products
of the phosphorylation of 8 in Fig. 3—undergo photoreduction under the
same conditions, in similar yields (Supplementary Figs. 41 and 42). b, Our
proposed mechanism is initiated by photodetachment of an electron from
aqueous hydrosulfide to generate a solvated electron. Reduction of 15a
by this electron generates radical anion 31, which undergoes C–S bond
homolysis to generate stabilized radical anion 32. 32 can react with a
hydrogen atom donor or combine with a HS• radical to give major isolated
product 11 or proposed intermediate 30, respectively, which we observed in
crude spectra but were unable to isolate.
a
O
HN
O
HO
S
O
H2O, 60 °C
OH
N
HN
+
pH
Half-life (h)
28
O
S
N
H
HO
Unbuffered
7.0 (phosphate) 32
OH
4.1 (acetate)
31
19 (87–97%)
HO
12 (84–91%)
11
(Furanose and
pyranose isomers)
NH2
b
N
O
NH2
N
HS• radical, generated in previous steps) forms 2′-thionucleoside
30. We conjecture that 21 probably does not undergo this reduction
because the first step, were it to occur, would lead to a radical anion
intermediate with less resonance stabilization than 31.
HN
N
N
H
33
N
N
O
S
(5 equiv.)
N
N
N
HN
+
1’
Dry state,
O
O
S
N
100 °C, 31 h
H
Syntheses of deoxyribose and β-2′-deoxyadenosine. Since our
synthesis of 2′-deoxyribonucleoside 11 takes place under the
same conditions under which amino acid4 and sugar29 precursors
and RNA pyrimidines5,22 are formed, we immediately questioned
whether DNA building blocks such as 11, and related (canonical)
derivatives, may also have been present at the advent of life. In an
attempt to form the canonical 2′-deoxyribonucleoside, deoxyuri-
dine, we investigated the hydrolysis of 11 under the same condi-
tions under which β-2-thioribocytidine 5 is converted to cytidine
(Fig. 1)5. However, in the presence of phosphate buffer, 11 is cleanly
hydrolysed to 2-deoxyribose 12 and thiouracil 19. A detailed study
OH
OH
HO
HO
11
19 (72%)
α-13 (6%)
β-13 (4%)
Fig. 6 | hydrolysis and transglycosylation reactions of 2′-deoxy-
2-thiouridine 11. a, Hydrolysis of 11 at different pH values gives
deoxyribose 12 and the corresponding nucleobase, 2-thiouracil 19.
b, Transglycosylation in the dry state with adenine 33 gives a mixture of
the alpha- and beta- stereoisomers of the canonical deoxynucleoside,
deoxyadenoisine 13, as well as nucleobase loss to yield 19.
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