Synthesis of 2-Thioorotidine and Comparison of Its Unusual Instability with Its Canonical Pyrimidine Counterparts

Article abstract of DOI:10.1021/acs.joc.9b01546

2-Thiopyrimidine nucleosides exhibit properties that are interesting from both a biological/medicinal and origins of life chemistry point of view. We report here the first synthesis of the nucleoside 2-thioorotidine and our observations on its unexpected lability with respect to its N-glycosidic bond when compared with its corresponding canonical pyrimidine counterparts. We hypothesize that the cause of the lability of the N-glycosidic bond is due to the combined steric and electronic effects from the 2-thio- and the 6-carboxyl groups, a lability that could, in turn, be useful for further chemical transformations.

Full text of DOI:10.1021/acs.joc.9b01546

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Article
Synthesis of 2-thioorotidine and comparison of its unusual
instability with its canonical pyrimidine counterparts
Ravi Kumar, Greg Springsteen, and Ramanarayanan Krishnamurthy
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01546 • Publication Date (Web): 14 Oct 2019
Downloaded from pubs.acs.org on October 14, 2019
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Synthesis of 2-Thioorotidine and Comparison of its Unusual Instability with
its Canonical Pyrimidine Counterparts
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Ravi Kumar¶†, Greg Springsteen†‡ and Ramanarayanan Krishnamurthy¶†*
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¶
The Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA
92037
‡ Department of Chemistry, Furman University, Greenville, SC 29613, USA
† NSF/NASA Center for Chemical Evolution, Atlanta, GA 30332, USA
* Corresponding author: rkrishna@scripps.edu
Abstract: 2-Thiopyrimidine-nucleosides exhibit properties that are interesting both from a
biological/medicinal and origins of life chemistry point of view. We report here the first
synthesis of the nucleoside 2-thioorotidine and our observations on its unexpected lability with
respect to its N-glycosidic bond when compared with its corresponding canonical pyrimidine
counterparts. We hypothesize that the cause of the lability of the N-glycosidic bond is due to the
combined steric and electronic effects from the 2-thio- and the 6-carboxyl groups, a lability
which could in turn be useful for further chemical transformations.
H
S
N
O
O
NH
O
O
O
NH
S
O
O
H4NO
O
O
N
O
O
NH
OR
N
HO
S
O
O
O
OH
O
HO
Stereo- and
regio specific
Thioorotidine
-isomer only

Introduction
2-Thiopyrimidines are a class of nucleosides that are naturally occurring and have been
investigated primarily for their properties and use in biological and medicinal chemistry.1 In
addition, there has been a long-standing interest in the use of 2-thiopyrimidines (particularly 2-
thiouridine and 2-thiocytidine) in nucleic acid systems based on the fact that they are
components of transfer RNA.2-4 Subsequently, it has been shown that inclusion of 2-thiouridine
increases the stability of nucleic acid duplexes2, which has led to investigations of the role of 2-
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thiopyrimidines in the context of origins of life. For example, work from the Szostak lab has
shown the beneficial effects of 2-thiouridine with respect to increased fidelity and rates in non-
enzymatic replication of RNA.5-7 The Sutherland6,8 and Powner9 labs have shown that thio
derivatives of pyrimidines and purines could have played a role in producing RNA nucleosides
or its precursors under plausibly prebiotic conditions. Thus, there is an increasing awareness and
interest in the chemical transformation potential of the 2-thio-derivatives of the canonical
nucleobases and nucleosides.10
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In this context, we became interested in the 2-thio-derivatives of orotic acid and orotidine
based on our recent work with orotic acid and orotidine containing RNA.11-13 While 2-thioorotic
acid is well precedented in the literature14,15, 2-thioorotidine is only known as the 2-
thioorotidine-5’-monophosphate which has been prepared enzymatically.16,17 Here we describe
our efforts in synthesizing the 2-thioorotidine, report on its chemical properties and compare its
stability behavior with those of the corresponding canonical pyrimidine nucleosides. We find
that the N-glycosidic bond of 2-thioorotidine is remarkably labile in water and cleanly
regenerates ribose and the nucleobase, 2-thioorotic acid. This lability seems to be unusual when
compared with its natural counterparts and seems to be a consequence of a combination of the
carboxylic group at the 6-position and the sulfur at the 2-position.
Results and Discussions
Synthesis of 2-thioorotidine: We began our synthesis of 2-thioorotidine by employing the
strategy and conditions developed in our lab previously for the successful synthesis of
orotidine.11 The approach was based on an intramolecular nucleosidation strategy to access 2-
thioorotidine in a highly regio- and stereo-specific manner resting on three key steps: a)
esterification of the C(6)-COOH moiety of 2-thioorotic acid with 1-O-acyl-2,3-O-isopropylidine-
D-ribofuranoside (at the 5-O-position), b) intramolecular nucleosidation to facilitate the correct
regio- and stereo-chemistry of the nucleoside and c) hydrolytic ring opening followed by acetal
deprotection to furnish the 2-thioorotidine. It is important to note that in order for the
intramolecular nucleosidation to work (as noted in our earlier work)11, a cyclic protecting group
at the 2',3'-O-positions rigidifying the furanose sugar frame work was necessary. The
intramolecular nucleosidation was unsuccessful when using the acetate or benzoyl protecting
groups at the 2'- and 3'-positions,.11
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We therefore prepared two derivatives, 1-O-acetyl/benzoyl-2,3-O-isopropylidine-D-
ribofuranoside 1a and 1b according to the reported procedure11 in 4 steps starting from D-ribose.
Initially we tested the esterification of this protected sugars (1a/1b) with 2-thioorotic acid (2)
under the previously developed condition using carbonyldiimidazole (CDI), but failed to obtain
the desired esterified products. We then turned to the standard N,N’-dicyclohexyl carbodiimide
(DCC) with catalytic DMAP conditions to carry out the acid-alcohol coupling, which furnished
the desired esterified products 3a and 3b in 67 and 62% yields respectively (Scheme 1). The
formation of the thioorotate ester bond at the desired 5'-O-position of the sugar was further
confirmed by HMBC-NMR spectroscopy by correlating the C(7) carbonyl carbon of 2-
thioorotate with C-H(5') proton of ribofuranose sugar (Supporting Figures S3 and S7).
H
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O
N
S
NH
O
O
C(7)
OH
O
O
O
O
NH
S
O
ref 11
a
b
D-Ribose
OPG
N
O
H(5')
O
OPG
O
O
O
O
O
1a
1b
3a
3b
67%
: PG = Ac,
62%
: PG = Bz,
4:
3a
3b
: PG = Ac
: PG = Bz
52% yield from
51% yield from
Scheme 1: Synthesis of 2-thioorotidine lactone: (a) thioorotic acid (2, 1.1 eq,.), DCC, DMAP, DMF, 0 oC to rt, 2 h,
(b) BSA (2.2 eq.), MeCN (0.010 M), rt 1 h followed by SnCl4 (0.5 eq.), 60 oC, overnight.
Next, the ester derivatives 3a and 3b were subjected to Vorbrüggen-Hilbert-Johnson (VHJ)
nucleosidation18 condition to obtain 5'-O-lactone 4. Again, following the previously reported
condition11 for the orotidine derivative using TMSOTf was not successful in providing the
desired product for the thio-derivative. Changing the Lewis acid activator to tin(IV)chloride from
TMSOTf seemed to do the trick, delivering the title product 5'-O-lactone (4) in 52% yield.
With the desired 2-thioorotidine 5'-O-lactone (4) in hand and having successfully
addressed two of the three key steps we were confident about the remaining pathway, the
hydrolytic opening of lactone ring followed by acetonide deprotection. We adopted one of the
previous protocols developed for orotidine synthesis,11 which involves acetonide deprotection
with In(OTf)3 followed by methanolysis with NaOMe/MeOH to generate the methyl ester of 2-
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thioorotidine, which upon subsequent hydrolysis with aq. NaOH would provide the desired 2-
thioorotidine. However, in the case of the thio-derivative 4, the three-step protocol proved
problematic as we observed only the formation of 2-thioorotic acid (2) after the final step
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(confirmed by H-NMR and ESI-mass analysis), indicating that de-pyrimidation of 2-thioorotic
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acid derivative 4 had taken place (Supporting Figures S40-S41). With this unexpected result, we
decided to move step-wise through the synthesis to understand the issues at hand. We first
investigated the option of deprotecting the acetonide ring of compound 4 to see if we could form
the corresponding 4-diol under acidic conditions. However, we again observed only glycosidic
bond cleavage or decomposition of the starting material 4 (Table 1, For 1H NMR see Supporting
Figure S43).
Table 1: Optimization for the deprotection of the acetonide of 2-thioorotidine 5'-lactone.
O
O
O
NH
O
O
NH
S
O
N
Conditionsa
O
N
O
S
O
O
HO
4-diol
4
OH
Entry
1.
Condition
Time (h)
Temperature
Observation
N-glycosidic bond cleavage
1 M HCl (aq.)
60% TFA (aq.)
10% TFA (aq.)
1
1
1
1
rt
2.
rt
rt
Decomposition of starting material
Decomposition of starting material
Decomposition of starting material
2.
4.
In(OTf)3 in
MeCN:H2O
pTSA (0.5 eq.) in
MeOH
rt to 40 oC
5.
24 h
50 oC
N-glycosidic bond cleavageb
a10 mg of 2-thioorotidine 5’-lactone (4) was dissolved in 0.3 mL of the indicated solvent in a small reaction vial.
The reaction mixtures were analyzed by TLC (direct spotting) and NMR (0.2 mL reaction aliquots in 0.5 mL of
D2O). bThe product of N-glycosidic bond cleavage (from entry 5) was isolated (Scheme 2a).
When we tested organic acids such as pTSA in methanolic conditions (entry 5, Table 1),
the reaction (by TLC) showed complete consumption of starting material forming a new product
without the usual decomposition patterns observed in the previous reactions. But, when the
isolated product was analyzed by 1H- and 13C-NMR, it was found to be 1-methoxy-2,3-OH-5'-O-
thioorotyl-D-ribofuranose 5 (Table 1 entry 5; Scheme 2a). This once again indicated that the N-
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glycosidic bond in 4 was undergoing cleavage readily under the acidic conditions. For
comparison, the acetonide of the 5'-O lactone of orotidine (the 2-oxo version of 4) was
deprotected under 60% aq. TFA to afford the corresponding 2',3'-diol in 80% yield,11 indicating
that there was something atypical with the 2-thio derivative 4 in terms of the N-glycosidic bond
lability.
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H
O
O
N
S
O
NH
O
NH
S
N
a
O
..(a)
O
O
O
O
OMe
O
HO
4
OH
5
O
O
O
O
OH
MeO
MeO
O
O
NH
b
..(b)
N
+
HN
S
O
N
O
O
S
NH
O
NH
O
S
O
O
6
7
4
Scheme 2: Acetonide deprotection and methanolysis of 5'-O-lactone 4 leading to cleavage of the N-glycosidic bond.
Conditions (a) pTSA (0.5 eq.), MeOH, 50 oC, 24 h. (b) 1 M NaOMe (0.2 eq.), MeOH, rt, 24 h.
Therefore, we modified the strategy to first open the lactone ring followed by acetonide
deprotection to get the desired 2-thioorotidine, with the hope that relieving the strain (induced by
the lactone ring) might increase the stability of the N-glycosidic bond. Accordingly, we
attempted to generate the methyl ester of 2-thioorotidine (6) by treating 2-thioorotidine 5’-O-
lactone (4) with 1.0 M NaOMe in methanol; However, depyrimidination to (7) was still the
dominant pathway (based upon TLC and NMR analysis, Supporting Figure S42), The
depyrimidination was further increased by silica gel chromatography (even after the silica gel
was treated with 0.1% triethylamine), and we isolated the acetonide protected 2-thioorotidine
methyl ester (6) in only 27% yield along with 41% of 2-thioorotic acid methyl ester (7) (Scheme
2b). Unfortunately, acetonide deprotection of the methyl ester of 2-thioorotidine (6) using
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pTSA/methanolic condition afforded only 2-thioorotic acid methyl ester (7). Exploring other
mineral acids such TFA, HCl, AcOH in place of pTSA again led to either decomposition of
starting material 6 or depyridimination to give 2-thioorotic acid methyl ester.
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The failure to successfully deprotect the acetonide moiety of 2-thioorotidine 5'-O-lactone
(4), led us to consider that increasing the electron density on the nucleobase (by forming the
anionic carboxylate group by increasing the pH) may decrease the chance of depyrimidination.
Also, since such processes are thought to be catalyzed by protonation of the nucleobase,19,20 we
reasoned that increasing the pH may slow down the depyrimidination process. Therefore, we
proposed to make sodium salt of the acetonide protected methyl ester of 2-thioorotidine 6
(Scheme 3). Accordingly, we treated the methyl ester of 2-thioorotidine (6) with sodium
hydroxide and were gratified to find that the reaction afforded the desired sodium salt of 2,3-O-
isopropylidine 2-thioorotidine (8) in 82% yield. Compound 8 was also accessible, under the
same condition, in one step from 2-thioorotidine 5’-O-lactone (4) in 88% yield. In both cases we
did not observe the loss of the pyrimidine nucleobase which seemed to give credence to our
reasoning above, further reinforced by the instability of the N-glycosidic bond in the methyl ester
derivative 6 during isolation (Scheme 2).
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O
OH
O
O
MeO
O
OH
NaO
O
H4NO
O
a
HO
O
N
b
O
N
N
O
O
NH
NH
O
6
O
NH
S
S
O
S
HO
OH
9
8
O
O
82%
88%
6
4
Yield from
Yield from
a
O
NH
S
N
O
O
O
4
Scheme 3: Hydrolysis of methyl ester of 2-thioorotidine. Condition (a) Crushed NaOH (2 eq.), MeCN:H2O (1:1,
v/v), rt, overnight. (b) 1M HCl in MeOH, rt, 6 h followed by 30% NH4OH to pH ~9-10.
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The sodium salt of 2-thioorotidine derivative 8 was then subjected to different acetonide
1
deprotection conditions (Table 2, entries 1-6, For H NMR see Supporting Figure S44) and the
best condition was found to be the use of methanolic HCl (Table 2, entry 6). After complete
conversion of starting material, the reaction mixture was basified with NH4OH to produce the
ammonium salt of 9, which was stable to column chromatographic conditions to furnish the
desired target molecule in 91% yield. The regio- and stereo- chemistry of the product was
confirmed by HMBC-NMR spectroscopy (Supporting Figure S28).
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8
9
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Table 2: Optimization of the deprotection of acetonide 8.
O
O
OH
OH
-O
O
NaO
O
Conditionsa
N
N
S
O
O
NH
O
NH
HO
O
S
OH
8
Entry
1.
Condition
Time (h)
Temperature
Observationb
1 M HCl (aq.)
60% TFA (aq.)
10% TFA (aq.)
1
1
1
1
rt
N-glycosidic bond cleavageb
N-glycosidic bond cleavageb
N-glycosidic bond cleavageb
2.
rt
rt
2.
4.
In(OTf)3 in
MeCN:H2O
pTSA (0.5 eq.) in
MeOH
rt to 40 oC
Decomposition of starting material
Decomposition of starting material
5.
6.
2 h
6 h
40 oC
rt
1 M HCl in MeOH
Trace amount of N-glycosidic bond
cleavage observed with complete
conversion of starting materialb
a10 mg of 2’,3’-O-isopropylidine--2-thioorotidine sodium salt (8) was dissolved in 0.3 mL of the indicated solvent
in a small reaction vial. The reaction mixtures were analyzed by TLC (direct spotting) and NMR (0.2 mL reaction
aliquots in 0.5 mL of D2O). bObservations based upon TLC and NMR analysis.
Based on our previous work11 we wanted to know if the direct VHJ-nucleosidation with
2-thioorotic acid would furnish the N-1 or the N-3 regioisomer of the product, since the 2-thio
group may interfere as it has done with the N-glycosidic bond stability. It is known in case of
orotidine that such direct nucleosidation approaches provide the N-3-regioisomer as the major
product.21-23 We had also noted in our previous work11 that even protecting the N-3-position (as
N(3)-benzoyl) of the orotic acid methyl ester still afforded the N-3 isomer (isoorotidine), and
none of the N(1)-orotidine was formed by direct nucleosidation.
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7
O
H4NOOC
MeOOC
O
O
8
9
O
HN
BzO
b
a
HN
OAc
HN
N
N
O
OMe
O
+
S
N
H
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BzO
S
S
BzO
O
OH
OBz
OBz
HO
OH
OBz
7
10
11
12
Scheme 4: Direct intermolecular nucleosidation of 2-thioorotidine methyl ester 7. Conditions: (a) BSA (2.0 eq.),
MeCN, 50 oC, 30 minutes followed by TMSOTf (1.0 eq.), 50 oC, 3 h. (b) Crushed NaOH (10.0 eq.), MeCN:H2O
(1:1, v/v), rt, overnight.
For the thio-derivative, we tested the same reaction for the direct nucleosidation of the methyl
ester of 2-thioorotic acid (7) with the protected ribose sugar (1-O-acetyl-3,4,5-O-benzoyl β-D-
ribofuranoside, 10) under VHJ conditions. Under these conditions, we observed only the
formation of compound 11, which upon deprotection of benzoyl and ester groups afforded
product 12 (Scheme 4). This was shown to have N(3)-regioselectivity as confirmed by NMR data
comparison of 2-thioorotidine (9); iso-2-thioorotidine (12) showed complete dissimilarity in the
chemical shift of anomeric and olefinic protons (Figure 1). Further confirmation was obtained
by comparison of UV-data and HMBC-correlation studies by correlating the H-1’ proton with
thiocarbonyl (C-2) and carbonyl carbon (C-4) of 2-thioorotic acid (Supporting Figures S37-S39).
Thus, the intramolecular nucleosidation strategy provides the desired N(1)-2-thioorotidine with a
β-linkage, which may be useful in the context of our ongoing prebiotic and origins of life studies.
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-O2C
O
O
HO
N
NH
8
9
S
OH
5
HO
2
5'
10
11
12
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Thioorotidine
1'
3'
2'
4'
-
O
CO2
NH
HO
N
O
S
OH
HO
1
Isothioorotidine
5
1'
2'
3'
5'
5'
4'
7.4
7.2
7.0
6.8
6.6
6.4
6.2
6.0
5.8
5.6
5.4
f1 (ppm)
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
Figure 1: 1H-NMR comparison of 2-thioorotidine (9) with iso-2-thioorotidine (12) in D2O at 600 MHz
Studies on the instability of 2-thioorotidine: Even following the successful protocol reported
for orotidine,11 the unexpected lability of the N-glycosidic bond of 2-thioorotidine was
problematic. The lability of the N-glycosidic bond is unusual when compared to not only
orotidine but also to other thionucleosides. For example, 2-thiouridine is a stable compound, and
has been studied extensively in RNA oligonucleotides.2-7 It is also known that 2'-deoxy-2'-
mercaptouridine is fairly stable in neutral or slightly acidic aqueous solution at room
temperature.24 Therefore, the relative instability of the 2-thioorotidine (9), which has the
combination of the 6-carboxylic acid (of orotidine), 2-thio and the 2'-hydroxyl groups (of
thiouridine) was surprising. Therefore, we undertook an investigation to learn how unstable the
2-thioorotidine derivative is compared to its pyrimidine counterparts, orotidine, 2-thiouridine and
uridine. We subjected each of the nucleosides to a variety of aqueous pH conditions and
followed their stability on a reverse phase HPLC using UV-detection. Accordingly, we prepared
the aqueous solution of 2-thioorotidine 9 in water (unbuffered, pH~6.2) and in 10 mM sodium
phosphate buffer at pH 3.0, 6.0, 7.0, 8.4 and 12.5. In order to compare the unusual behavior of 2-
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thioorotidine, we also prepared the same aqueous solutions of orotidine, 2-thiouridine, uridine
and iso-2-thioorotidine (12) and analyzed the samples at different intervals of time via reverse
phase HPLC. As evident from the HPLC traces (Figure 2), the N-glycosidic bond in 2-
thioorotidine is labile in acidic or neutral solution forming 2-thioorotic acid, but relatively stable
in highly alkaline media (Figure 2b). The N-glycosidic bond is cleaved under acidic conditions
(pH 3) with about 30% of 2-thioorotidine 9 remaining, while at an alkaline pH (12.5) 80% of 2-
thioorotidine is still present after 2 weeks. (Figure 3a) The other three nucleosides, orotidine,
thiouridine and uridine were found to be stable at all pH ranges with no discernible
decomposition under similar conditions, as evidenced by the HPLC traces (Supporting Figures
S51-S68). From the HPLC chromatograms, it is clear that the stability of 2-thioorotidine
increases with increasing pH suggesting that the depyrimidination is probably taking place via a
protonation on the nucleobase (perhaps at N-1).
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pH 12
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a
Thioorotic acid 2
2-Thioorotidine 9
pH 8
pH 7
pH 6
pH 3
2-Thioorotidine 9
pH 12
pH 8
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pH 6
pH 3
time (min.)
time (min.)
Figure 2: HPLC traces of 2-thioorotidine (~ 30 µM) at different pH values in 10 mM sodium phosphate. (a) HPLC
traces of 2-thioorotidine at t = 0 h at unbuffered (pH ~ 6.2), pH 3.0, 6.0, 7.0, 8.4 and 12.5 in sodium phosphate
buffer. (b) HPLC traces of 2-thioorotidine after 14 days (336 h) at unbuffered (pH ~ 6.2), pH 3.0, 6.0, 7.0, 8.4 and
12.5 in sodium phosphate buffer. pH adjustment was carried out by addition of 0.1 N HCl or 0.1 N NaOH.
The fact that under even basic conditions, 2-thioorotidine is still decomposing is perhaps
suggestive of the extent to which even minimal proton concentration is enough for catalyzing
this reaction. For example, when compound 4 was treated with sodium methoxide in methanol,
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pH 3.0
unbuffered
pH 6.0
pH 7.0
pH 8.4
pH 12.5
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24 h
48 h
96 h
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Figure 3: (a) Percentage of 2-thioorotidine remaining over different intervals of time (0 to 336 h) at different pH
values (3.0 - 12.5) in sodium phosphate buffer (For HPLC analysis conditions see supporting information).
Unbuffered pH was ~ 6.2. (b) The rate of change in 2-thioorotidine concentration over time (pseudo-zeroth order)
and the pH-rate constant correlation (insert in 3b) obtained from HPLC data in figure 3a, showing the decrease in
the deglycosylation rate of 2-thioorotidine with increasing in pH with a dramatic drop of the rate at pH 12.5.
N-glycosidic bond cleavage was minimized compared to acidic conditions, but still observed; it
appears that a proton transfer that results in the transient protonated species of 2-thioorotidine
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would cause this deglycosylation. Moreover, when the pH is controlled at 12.5, well above the
pKa of N3(H) of 2-thioorotdine, there is a prominent stabilization when compared to acidic pH;
but there is still a slow(er) and perceptible cleavage of the glycosidic bond (figure 3a and 3b
insert). The linear correspondence in the change in 2-thioorotidine concentration with time over
the range of pH values studied is indicative of a pseudo zeroth order rate (Figure 3b). This
suggests that the proton accelerated decomposition is magnifying other internal structural factors
that are causing the inherent instability of this molecule.
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As to why the N-glycosidic bond of 2-thioorotidine (versus the other nucleosides) is highly
labile, we hypothesize that it must be a combination of effects of steric and electronics from both
the C(6)-CO2H and the C(2)=S group (Figure 4). With regards to the electronic effects, the
replacement of the C(2)=O with a C(2)=S is known to change the preferred protonation sites
with a strong preference for C(2)=S site when compared to uridine (where there is equal
preference for 2,4H tautomer and C(4)=O protonation), and that a protonation at the C=S site
leads to a strong preference for the anti-conformation around the nucleosidic bond.25,26 This
phenomenon alone cannot be the cause of instability, since 2-thiouridine is found to be quite
stable under comparable conditions. Therefore, the C(6)-COOH group must also play a role
(keeping in mind orotidine is quite stable under these conditions as well). Thus, it is reasonable
to conclude that it is not one or the other, but a combination of the two (the C=S and the COOH
group) that is responsible for the bond instability: while the C=S increases the anti-
conformation,2,27 it has to contend with the steric hindrance caused by the C(6)-COOH group.12
This combination of the increased size of sulfur atom (versus the oxygen atom) plus the steric
clash induced by the COOH group, weakens the N-glycosidic bond (Figure 4). Thus, the
protonation (of the C=S bond) at lower pH values enhances the lability of the glycosidic bond in
2-thioorotidine (Figure 4). This behavior is similar to the instability experienced by many of the
pyrimidine nucleosides that are attached at N(3)-position of the pyrimidine nucleobases, where
there is steric hindrance from the carbonyl groups at the C-2 and C-4 positions. Support for such
an assertion comes from the observation that iso-2-thioorotidine (12) was significantly more
labile than 2-thioorotidine (9) under mildly acidic conditions (pH 6.0) and glycosidic bond
cleavage proceeded relatively rapidly (1.6x, after 24 h) (Supporting Figures S47 versus S71).
Further work on iso-2-thioorotidine was limited as it begins to decompose immediately once
purified and isolated.
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Figure 4: Postulated steric and electronic factors that may be contributing to the weakening of the N-glycosidic
bond in 2-thiouridine. Protonation of the C=S bond typically favors the anti-conformation to relieve steric
interactions between the large sulfur atom and the 5’ OH.25,26 However in 2-thioorotidine, this anti conformation is
also strained by a steric clash between the C(6)carboxylate and the 5’ hydroxyl.12
In conclusion, we have developed a successful intramolecular nucleosidation strategy and
a synthetic protocol that affords us regioselectively the N(1)-2-thioorotidine in good yield.
However, this thionucleoside was found to have unusual glycosidic bond lability, which we
attribute to the combined effect of the C(6)-COOH group and the C(2)=S moiety. Further
investigations (theoretical as well as experimental) are ongoing to understand the exact nature of
the contributions leading to this unusual instability. We propose that such lability could be of use
in a prebiotic context as recently demonstrated by the work of Sutherland and co-workers8 in a
trans-nucleosidation approach affording access to other nucleosides, an endeavor that is ongoing
in our laboratory.
Experimental Section
Thioorotic acid was purchased from Sigma Aldrich (CAS # 6953-78-2). 4-Dimethylamino
pyridine (DMAP), N,N'-Dicyclohexylcarbodiimide (DCC) and Bis(trimethylsilyl)acetamide
(BSA) were purchased from Acros Organics. Tin(IV)Chloride was purchased from Sigma
Aldrich. Anhydrous solvents were purchased from EMD Chemicals. Unless otherwise noted, all
experiments were performed under a nitrogen atmosphere. Thin layer chromatography (TLC)
was performed on silica gel 60 Å F254 from Angela Technologies, and it was visualized by UV
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lamp and/or a stain solution of phosphomolybdic acid (PMA) in ethanol. Flash column
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chromatography was performed on silica gel 60 Å with particle size 35-70 μm purchased from
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Acros Organics. NMR spectra were recorded on Bruker DRX-600 or AV-600 (600 MHz for H
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and 150 MHz for C). NMR Chemical shifts δ are given in ppm relative to the residual signals
1
of tetramethylsilane in CDCl3 or deuterated solvent DMSO-d6 for H and 13C NMR.
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Multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublets (dd), doublet of
triplets (dt), triplet (t), quartet (q), multiplet (m), broad singlet (bs). Mass spectra were measured
with an Agilent ESI-TOF or ThermoElectron Finnigan LTQ ion trap mass spectrometer. The
purity and characterization of these compounds were further established using HRMS (ESI-TOF)
mass analysis. Mass spectra of all compounds were recorded in positive ion mode on a mass
spectrometer in the Scripps Mass Spectrometry Center. HPLC was performed by ion-pair reverse
phase HPLC (Phenomenex Aqua analytical column [250 mm x 4.6 mm (i.d.), 5 µm particle size]
protected by a Phenomenex Security Guard [4 mm x 3.0 mm (i.d.)] C18 ODS column) on a
Vanquish UHPLC purification system.
1ʹ-O-Acetyl-2ʹ,3ʹ-O-isopropylidene-5ʹ-O-thioorotyl-D-ribofuranoside (3a): To a solution of
thioorotic acid (1.22 g, 7.10 mmol) in dry DMF (6.5 mL) was added 1-O-acetyl-2.3-O-
isopropylidine-D-ribofuranoside (1.5 g, 6.45 mmol) and DMAP (79 mg, 0.64 mmol) at room
temperature. The reaction was then cooled to 0 oC and a solution of DCC (1.46 g, 7.10 mmol) in
6.5 mL of dry DMF was added dropwise. After complete addition of DCC, the reaction was
warmed to room temperature and stirred for additional 2 h. After complete consumption of
starting material (ribofuranose sugar) as indicated by TLC, the reaction mixture was passed
through a short bed of celite (to remove dicyclohexyl urea) and washed with ethyl acetate (2 x 20
mL). Filtrate was then collected and concentrated in a rotary evaporator to produce a residue,
which was purified by silica gel column chromatography (40% ethyl acetate:hexane to 60% ethyl
acetate:hexane) to afford 3a as pale yellow solid. Rf = 0.42 (hexane:ethyl acetate, 1:1), Yield =
1.64 g (67%). 1H NMR (600 MHz, CDCl3) δ 6.67 (s, 1H, H-5), 6.29 (s, 1H, H-1’), 4.79-4.77 (m,
2H, H-2’, H-3’), 4.61-4.56 (m, 2H, H-4’, H-5’), 4.39 (m, 1H, J = 10.6 Hz, 4.8 Hz, H-5’), 2.11 (s,
13
3H, Ac), 1.54 (s, 3H, Me), 1.38 (s, 3H, Me) ppm. C{H} NMR (151 MHz, CDCl3) δ 175.1
(C=S, thioorot.), 169.2 (Ac; C=O), 160.3 (thioorot.), 159.3 (thioorot.), 139.6 (thioorot.), 113.8
(quat. C), 108.4 (C-5), 102.0 (C-1’), 85.0 (C-2’), 84.9 (C-4’), 81.0 (C-3’), 67.2 (C-5’), 26.5 (C-
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Me), 25.1 (C-Me), 21.2 (C-Ac) ppm, HRMS (ESI-TOF) calcd for C15H18N2O8SNa [M+Na]+
409.0682 found 409.0692.
1ʹ-O-Benzoyl-2ʹ,3ʹ-O-isopropylidene-5ʹ-O-thioorotyl-D-ribofuranoside (3b): To a solution of
thioorotic acid (965 mg, 5.61 mmol) in dry DMF (5.0 mL) was added 1-O-benzoyl-2.3-O-
isopropylidine-D-ribofuranoside (1.5 g, 5.09 mmol) and DMAP (62 mg, 0.51 mmol) at room
temperature. The reaction was then cooled to 0 oC and a solution of DCC (1.15 g, 5.61 mmol) in
5.0 mL of dry DMF was added dropwise. After complete addition of DCC, the reaction was
warmed to room temperature and stirred for additional 2 h. After complete consumption of
starting material (ribofuranose sugar) as indicated by TLC, the mixture was then passed through
a short bed of celite (to remove dicyclohexyl urea) and washed with ethyl acetate. The filtrate
was then collected and concentrated in a rotary evaporator to produce a residue, which was
purified by silica gel column chromatography (eluted with 40% ethyl acetate:hexane to 60%
ethyl acetate:hexane) to afford 3b as pale yellow solid. Rf = 0.30 (hexane:ethyl acetate, 1:1),
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1
Yield = 1.41 g (62%). H NMR (600 MHz, DMSO-d6) δ 12.71 (bs, 1H, NH), 12.31 (bs, 1H,
NH), 7.92 (dd, J = 8.4 Hz, 1.4 Hz, 2H, Bz), 7.68 (tt, J = 7.3 Hz, 1.3 Hz, 1H, Bz), 7.52 (dd, J =
8.3 Hz, 7.4 Hz, 2H, Bz), 6.31 (s, 1H, H-5), 6.27 (s, 1H, H-1’), 5.08 (dd, J = 5.9 Hz, 1.1 Hz, 1H,
H-2’), 5.01 (d, J = 5.9, 1H, H-3’), 4.68 (td, J = 6.0 Hz, 1.1 Hz, 1H, H-4’), 4.47 (dd, J = 11.6, 5.9
Hz, 1H, H-5’), 4.35 (dd, J = 11.6 Hz, 6.3 Hz, 1H, H-5’), 1.47 (s, 3H, Me), 1.34 (s, 3H, Me) ppm.
13C{H} NMR (151 MHz, DMSO-d6) δ 176.6 (thioorot. C=S), 164.8 (Bz, C=O), 160.9 (thioorot.),
159.7 (thioorot.), 141.7 (thioorot.), 134.3 (Bz), 129.7 (Bz), 129.4 (Bz), 129.2 (Bz), 112.6 (quat.
C), 108.7 (thioorot. C-5), 103.4 (C-1’), 85.2 (C-4’), 84.9 (C-2’), 81.2 (C-3’), 66.6 (C-5’), 26.7
(C-Me), 25.2 (C-Me) ppm, HRMS (ESI-TOF) calcd for C20H21N2O8S [M+H]+ 449.1019 found
449.1012.
2ʹ,3ʹ-O-Isopropylidene-2-thioorotidine 5ʹ-lactone (4): To a solution of 1ʹ-O-acetyl-2ʹ,3ʹ-O-
isopropylidene-5ʹ-O-thioorotyl-D-ribofuranoside 3a (1.28 g, 3.33 mmol) in anhydrous MeCN
(333 mL, 0.01M) under a nitrogen environment, was added BSA (1.8 mL, 7.33 mmol) at room
temperature and the mixture was stirred for an additional 1 h. Then, SnCl4 (195 µL, 1.66 mmol)
was added at room temperature and the reaction mixture was transferred to preheated oil bath at
o
60 C. The reaction was then stirred overnight until completion of the reaction as indicated by
TLC. Solid NaHCO3 was added to the reaction mixture and stirred for 30 minutes at room
temperature. Reaction was then filtered through sintered funnel and solvent was concentrated in
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rotary evaporator to produce a residue, which was further purified by silica gel column
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chromatography using CH2Cl2:acetone as eluent (95:5 to 90:10) to afford 4 as white solid. Rf =
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0.28 (Hexane:Ethyl Accetate, 1:2), Yield = 564 mg (52%). H NMR (600 MHz, DMSO-d6) δ
8
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6.21 (s, 1H, H-5), 6.17 (s, 1H, H-1’), 5.28 (d, J = 6.0 Hz, 1H, H-2’), 4.74 (dd, J = 10.7 Hz, 5.3
Hz, 1H, H-4’), 4.62-4.58 (m, 2H, H-3’, H-5’), 4.20 (t, J = 12.0 Hz, 1H, H-5’), 1.45 (s, 3H, Me),
1.29 (s, 3H, Me) ppm. 13C NMR (151 MHz, DMSO-d6) δ 174.9 (thioorot. C=S), 164.7 (thioorot.
C-7), 159.3 (thioorot.), 144.0 (thioorot.), 111.6 (quat. C), 107.2 (C-5), 97.8 (C-1’), 87.2 (C-2’),
85.9 (C-4’), 79.2 (C-3’), 65.6 (C-5’), 26.1 (C-Me), 24.6 (C-Me) ppm, HRMS (ESI-TOF) calcd
for C13H15N2O6S [M+H]+ 327.0651 found 327.0653.
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1ʹ-O-methyl-5ʹ-O-thioorotyl-D-ribofuranoside (5): 2-Thioorotidine 5’-lactone 4 (250 mg, 0.77
mmol was dissolved in anhydrous MeOH (10 mL) under nitrogen environment and was added
para-toluene sulfonic acid (66 mg, 0.38 mmol) at room temperature and reaction mixture was
o
then heated at 50 C and stirred for overnight. Next day, after completion of the reaction as
indicated by TLC, solvent was removed in rotary evaporator and the residue thus obtained was
purified by silica gel column chromatography using CH2Cl2:acetone (95:5 to 80:20) and afforded
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5 as white solid. Rf = 0.28 (CH2Cl2:acetone, 8:2), Yield = 78 mg (32%). H NMR (600 MHz,
DMSO-d6) δ 12.76 (bs, 1H, NH), 12.26 (bs, 1H, NH), 6.36 (s, 1H, H-5), 5.19 (d, J = 6.0 Hz, 1H,
OH, D2O exchangeable), 5.07 (d, J = 6.0 Hz, 1H, OH, D2O exchangeable), (4.66 (s, 1H, H-1’),
4.48 (dd, J = 11.7 Hz, 3.0 Hz, 1H, H-5’), 4.25 (dd, J = 11.7 Hz, 4.9 Hz, 1H, H-5’), 4.10-4.03 (m,
13
2H, H-4’, H-3’), 3.78-3.77 (m, 1H, H-2’), 3.20 (s, 3H, OMe) ppm. C{H} NMR (151 MHz,
DMSO-d6) δ 176.7 (thiooro. C=S), 161.2 (thioorot.), 159.9 (thioorot.), 142.0 (thioorot.), 108.8
(C-5), 108.4 (C-1’), 79.8 (C-2’), 74.5 (C-4’), 71.2 (C-3’), 67.0 (C-5’), 54.9 (OMe) ppm, HRMS
(ESI-TOF) calcd for C11H15N2O7S [M+H]+ 319.0600 found 319.0604.
2ʹ,3ʹ-O-isopropylidene-2-thioorotidine methylester (6): To a solution of 2-thioorotidine 5'-
lactone 3 (462 mg, 1.42 mmol) in anhydrous MeOH (50 mL) was added 1.0 M NaOMe solution
(280 µL, 0.28 mmol) prepared by dissolving 1.17 g of sodium metal in 50.8 mL of dry MeOH
o
dropwise at room temperature and the reaction mixture was warmed to 30 C and stirred for
overnight, after which complete consumption of starting material was indicated by TLC. By
evaporating the solvent in rotary evaporator over silica gel, the residue was adsorbed on silica gel
and purified by column chromatography using CH2Cl2:acetone as eluent (95:5 to:20) to afford 6
as white foam. The product was further washed with diethyl ether to remove other impurities
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present. Rf = 0.24 (CH2Cl2:acetone, 8:2), Yield = 137 mg (27%), 1H NMR (600 MHz, DMSO-d6)
δ 6.77 (d, J = 3.1 Hz, 1H, H-1’), 6.33 (s, 1H, H-5), 5.12 (dd, J = 7.1 Hz, 3.0 Hz, 1H, OH, D2O
exchange-able), 5.01 (t, J = 5.5 Hz, H-2’) 4.49 (t, J = 6.7 Hz, 1H, H-3’), 3.85 (s, 3H, OMe), 3.81
(td, J = 6.3 Hz, 4.6 Hz, 1H, H-4’), 3.55 (ddd, J = 11.7 Hz, 5.5 Hz, 4.6 Hz, 1H, H-5’), 3.47 (ddd, J
= 11.8 Hz, 6.4 Hz, 5.4 Hz, 1H, H-5’) ppm. 13C{H} NMR (151 MHz, DMSO-d6) δ 176.9
(thiooro. C=S), 163.3 (thioorot.), 158.6 (thioorot.), 144.0 (thioorot.), 114.5 (quart. C), 109.7 (C-
5), 94.3 (C-1’), 85.4 (C-2’), 84.4 (C-4’), 79.5 (C-3’), 60.3 (C-5’), 53.6 (OMe), 27.1 (C-Me), 25.3
(C-Me) ppm, HRMS (ESI-TOF) calcd for C14H19N2O7S [M+H]+ 359.0913 found 359.0908.
Also, thioorotic acid methyl ester was isolated from the above column chromatography as pale-
yellow solid. Yield 108 mg (41%). Rf = 0.60 (CH2Cl2:acetone, 8:2).
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Methyl Thioorotic Acid (7): Thioorotic acid (2.0 g, 11.6 mmol) was suspended in 100 mL of
dry MeOH at room temperature and HCl gas (5 g, produced from 5 g, NaCl and 5 mL H2SO4)
was bubbled into the suspension. The reaction mixture was transfer to preheated oil bath at ~ 80
oC and refluxed for 24 h. The solvent was evaporated in rotary evaporator and to the residue was
added saturated aq. NaHCO3 and the aqueous layer was extracted with 3 x 100 mL of ethyl
acetate. Combined organic layers were collected and washed with sat. NH4Cl (aq.) and dried
over Na2SO4 and concentrated to get 7 as pale yellow solid. Yield 788 mg (36%). Rf = 0.60
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(CH2Cl2:acetone, 8:2). H NMR (600 MHz, DMSO-d6) δ 12.73 (bs, 1H, NH), 12.27 (bs, 1H,
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NH), 6.33 (s, 1H, H-5), 3.86 (s, 3H, OMe) ppm. C{H} NMR (151 MHz, DMSO-d6) δ 180.1
(thioorot. C=S), 166.3 (thioorot.), 163.9 (thioorot.), 153.2 (thioorot.), 104.6 (C-5), 52.0 (C-OMe)
ppm. HRMS (ESI-TOF) calcd for C6H7N2O3S [M+H]+ 187.0177 found 187.0176.
2ʹ,3ʹ-O-isopropylidene-2-thioorotidine sodium salt (8)
Method A: To a solution of acetonide protected 2-thioorotidine methyl ester, 6 (50 mg, 0.14
mmol) in 3 mL of ACN:H2O (1:1) mixture, was added crushed NaOH (11 mg, 0.28 mmol) at
room temperature, and the reaction was allowed to stir overnight. After complete consumption of
starting material as indicated by TLC, the solvent was evaporated in a rotary evaporator and the
residue thus obtained was purified by silica gel column chromatography using
CH2Cl2:MeOH:NH4OH (~30%) as eluent (95:5:0.1 to 80:20:0.2) to afford 8 as white solid, (Rf =
0.28 in 85:15:0.1 CH2Cl2:MeOH:NH4OH (~30%)) Yield = 42 mg (82%).
Method B: To a solution of 2-thioorotidine lactone, 4 (500 mg, 0.153 mmol) in 20 mL of
ACN:H2O (1:1) mixture, was added crushed NaOH (122 mg, 3.06 mmol) at room temperature,
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and the reaction was allowed to stir for overnight. After complete consumption of starting
material as indicated by TLC, the solvent was evaporated in a rotary evaporator and the residue
thus obtained was purified by silica gel column chromatography using CH2Cl2:MeOH:NH4OH
(~30%) as eluent (95:5:0.1 to 80:20:0.2) to afford 8 as white solid, (Rf = 0.28 in 85:15:0.1
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CH2Cl2:MeOH:NH4OH (~30%)) Yield = 492 mg (88%). H NMR (600 MHz, D2O) δ 6.78 (s,
1H, H-1’), 6.01 (s, 1H, H-5), 5.23 (dd, J = 7.6 Hz, 2.5 Hz, 1H, H-2’), 4.68 (m, 1H, H-3’), 4.00
(q, J = 6.4 Hz, 1H, H-4’), 3.83 (dd, J = 12.0 Hz, 7.2 Hz, 1H, H-5’), 3.75 (dd, J = 12.2 Hz, 4.6 Hz,
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1H, H-5’), 1.54 (s, 3H), 1.33 (s, 3H) ppm. C{H} NMR (151 MHz, D2O) δ 177.1 (thioorot.
C=S), 163.3 (thioorot.), 160.0 (thioorot.), 156.8 (thioorot.), 113.5 (quat. C), 102.3 (C-5), 94.4 (C-
1’), 86.0 (C-2’), 84.4 (C-4’), 79.0 (C-3’), 60.4 (C-5’), 27.3 (C-Me), 25.4 (C-Me) ppm, HRMS
(ESI-TOF) calcd for C13H16N2O7SNa [M+Na]+ 367.0576 found 367.0571.
2-Thioorotidine ammonium salt (9): To a small round bottom flask containing 200 mg of 2,3-
O-acetonide protected 2-thioorotidine sodium salt (8) was added 20 mL of 1 M HCl in MeOH at
room temperature and the reaction mixture was stirred for six hours. After complete consumption
of starting material as indicated by TLC, the reaction mixture was basified with the addition of
ammonium hydroxide solution to pH ~9-10. The solvent was then co-evaporated with ethanol (2
x 10 mL) and the residue thus obtained was dissolved in minimum amount of methanol and
absorbed onto silica gel, which was then purified by column chromatography using
dichloromethane: methanol:NH4OH (~30%) (2:1:0.1 to 1:1:0.1) as eluent to afford 9 as white
solid. Rf = 0.32 (iPrOH:H2O:NH4OH (~30%), 17.5:2:0.5). Yield 157 mg (91%). H NMR (600
1
MHz, D2O) δ 6.58 (d, J = 3.6 Hz, 1H, H-1’), 5.96 (s, 1H, H-5), 4.68-4.52 (m, 1H, H-2’), 4.16 (t,
J = 7.6 Hz, 1H, H-3’), 3.86 (dt, J = 8.4 Hz, 5.0 Hz, 1H, H-4’), 3.79 (d, J = 5.0 Hz, 2H, H-5’),
ppm. C{H} NMR (151 MHz, DMSO-d6) δ 177.1 (thioort. C=S), 167.3 (thioort. 7C=O), 162.4
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(thioort. 4C=O), 153.8 (thioorot.), 104.4 (C-5), 96.7 (C-1’), 83.9 (C-4’), 73.1 (C-3’), 68.2 (C-2’),
61.1 (C-5’) ppm, HRMS (ESI-TOF) calcd for C10H11N2O7SNa2 [M+2Na]+ 349.0082 found
349.0068.
N(3)-(2ʹ,3ʹ,5ʹ-tri-O-benzoyl-β-D-ribofuranosyl)-methyl thioorotate (11): To a suspension of
methyl thioorotate (7) (250 mg, 1.34 mmol) in dry acetonitrile (30 mL) was added BSA (N,O-
Bistrimethyl silylacetamide, 0.65 mL, 2.68 mmol) at room temperature. The reaction mixture
was then heated at 50 oC for 30 minutes and became homogeneous. After 30 minutes the reaction
mixture was cooled to room temperature. 1-O-Ac-2,3,5-O-Bz-β-D-ribofuranose (10) (677 mg,
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1.34 mmol) and TMSOTf (0.24 mL, 1.34 mmol) were added and the reaction was stirred for an
o
additional 3 h at 50 C. Progress of the reaction was monitored by TLC and after 3 h complete
consumption of sugar was observed, and the reaction was quenched by the addition of solid
NaHCO3 (100 mg). The solvent was concentrated in a rotary evaporator. The residue thus
obtained was purified by silica gel column chromatography using ethyl acetate:hexane as eluent
(1:5 to 1:1) to afford 11 as off-white foam. Rf = 0.28 (hexane:ethyl accetate, 1:1), Yield = 429
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mg (51%). H NMR (600 MHz, CDCl3) δ 8.12-8.10 (m, 2H, Bz), 8.04-8.02 (m, 2H, Bz), 7.94-
7.92 (m, 2H, Bz), 7.62-7.54 (m, 3H, Bz), 7.45 (q, J = 8.0 Hz, 4H, Bz), 7.38-7.36 (m, 2H, Bz),
6.98 (s, 1H, H-5), 6.42 (d, J = 3.0 Hz, 1H, H-1’), 6.11 (dd, J = 5.1 Hz, 3.1 Hz, 1H, H-2’), 6.03
(dd, J = 6.6 Hz, 5.1 Hz, 1H, H-3’), 4.83 (dt, J = 6.5 Hz, 3.9 Hz, 2H, H-4’), 4.79 (dd, J = 12.2 Hz,
13
3.6 Hz, 1H, H-5’), 4.61 (dd, J = 12.2 Hz, 4.1 Hz, 1H, H-5’), 3.80 (s, 3H, OMe) ppm. C{H}
NMR (151 MHz, CDCl3) δ 165.7, 164.8, 163.6, 163.4, 159.5, 152.2, 133.3, 133.2, 132.9, 129.5,
129.4, 129.3, 128.9. 128.3, 128.2, 128.1, 128.1, 128.0, 113.5, 85.9, 80.4, 75.2, 71.0, 62.9, 52.6
ppm, HRMS (ESI-TOF) calcd for C32H26N2O10S [M+Na]+ 653.1206 found 653.1206.
Iso-2-thioorotidine ammonium salt (12): 250 mg (0.40 mmol) of N(3)-(2ʹ,3ʹ,5ʹ-tri-O-benzoyl-
β-D-ribofuranosyl)-methyl thioorotate (10) was dissolved in 20 mL of 1:1 MeCN:H2O in a small
round-bottom flask at room temperature. Crushed NaOH (160 mg, 4 mmol) was added and the
reaction mixture was stirred for overnight. After complete consumption of starting material as
indicated by TLC, the solvent was then co-evaporated with ethanol (2 x 10 mL) and residue thus
obtained was dissolved in minimum amount of methanol and absorbed onto silica gel, which was
then purified by column chromatography using dichloromethane:methanol:NH4OH (~30%)
(2:1:0.1 to 1:1:0.1) as eluent to afford 12 as off-white solid. Rf = 0.28 (iPrOH:H2O:NH4OH,
17.5:2:0.5). Yield 71 mg (59%). 1H NMR (600 MHz, D2O) δ 6.45 (s, 1H, H-5), 5.96 (d, J = 4.2
Hz, 1H, H-1’), 4.30 (t, J = 4.6 Hz, 1H, H-2’), 4.23 (t, J = 5.3 Hz, 1H, H-3’), 4.03 (td, J = 5.3, 3.4
Hz, 1H, H-4’), 3.71 (dd, J = 12.6, 3.4 Hz, 1H, H-5’), 3.61 (dd, J = 12.7, 5.0 Hz, 1H, H-5’) ppm.
13C{H} NMR (151 MHz, DMSO-d6) δ 172.7 (thioort. C=S), 170.0 (thioort. 7C=O), 164.4
(thioort. 4C=O), 148.1 (thioorot.), 107.4 (C-5), 86.3 (C-1’), 84.2 (C-4’), 74.8 (C-3’), 70.1 (C-2’),
60.8 (C-5’) ppm, HRMS (ESI-TOF) calcd for C10H13N2O7S [M+H]+ 305.0443 found 305.0440.
Associated Content
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The supporting information is available free of charge on the ACS publication website at DOI:
10.021/acs.joc.xxxx
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Copies of H, C{H} NMR and HRMS spectra from pages S2-S26. Also, HPLC data from
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pages S26-S43.
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Author Information
Corresponding author
*E-mail: rkrishna@scripps.edu
ORCID
Ravi Kumar, https://orcid.org/0000-0001-8149-6498
Greg Springsteen, https://orcid.org/0000-0002-2020-281X
Ramanarayanan Krishnamurthy https://orcid.org/0000-0001-5238-610X
Author Contributions
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing interests.
Acknowledgements
This work was jointly supported by NSF and the NASA Astrobiology Program under the NSF
Center for Chemical Evolution, CHE-1504217.
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