Fluorescent labeling of s2T-incorporated DNA and m5s2U-modified RNA

Article abstract of DOI:10.1080/15257770.2021.1942044

We report herein comprehensive investigations of alkylation/sulfur exchange reactions of sulfur-containing substrates including nucleosides such as s²U, m⁵s²U, s⁴U, s²A and s²T-incorporated DNA enable by comprehensive screenings of the reagents (2a–2h). It has been proven that iodoacetamide (2a) displays the most promising feasibility toward sulfur-containing substrates including s²T, s²U, m⁵s²U, s⁴U and s²A. In sharp contrast, the alkylation process with S-benzyl methanethiosulfonate (BMTS, 2h) displays the best application potential only for s⁴U. Based on these results, the fluorescent labeling of s²T-incorporated DNA and m⁵s²U-modified RNA has been achieved. Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1942044.

Full text of DOI:10.1080/15257770.2021.1942044

Nucleosides, Nucleotides & Nucleic Acids
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Fluorescent labeling of s2T-incorporated DNA and
m5s2U-modified RNA
Ping Yu, Honglin Zhou, Yuanyuan Li, Zhifeng Du & Rui Wang
To cite this article: Ping Yu, Honglin Zhou, Yuanyuan Li, Zhifeng Du & Rui Wang (2021):
Fluorescent labeling of s2T-incorporated DNA and m5s2U-modified RNA, Nucleosides, Nucleotides
& Nucleic Acids, DOI: 10.1080/15257770.2021.1942044
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Nucleosides, Nucleotides aNd Nucleic acids
https://doi.org/10.1080/15257770.2021.1942044
Fluorescent labeling of s2T-incorporated DNA and
m5s2U-modified RNA
Ping Yu, Honglin Zhou, Yuanyuan Li, Zhifeng Du and
Rui Wang
Hubei Key laboratory of Natural Medicinal chemistry and Resource evaluation, school of Pharmacy,
tongji Medical college, Huazhong university of science and technology, Wuhan, Hubei, china
ARTICLE HISTORY
Received 3 March 2021
Accepted 31 May 2021
ABSTRACT
We report herein comprehensive investigations of alkylation/
sulfur exchange reactions of sulfur-containing substrates includ-
ing nucleosides such as s2U, m5s2U, s4U, s2A and s2T-incorpo-
rated DNA enable by comprehensive screenings of the reagents
(2a–2h). It has been proven that iodoacetamide (2a) displays
the most promising feasibility toward sulfur-containing sub-
strates including s2T, s2U, m5s2U, s4U and s2A. In sharp contrast,
the alkylation process with S-benzyl methanethiosulfonate
(BMTS, 2h) displays the best application potential only for s4U.
Based on these results, the fluorescent labeling of s2T-incorpo-
rated DNA and m5s2U-modified RNA has been achieved.
KEYWORDS
Sulfur modification;
s2T; m5s2U; alkylation;
sulfur exchange
1. Introduction
Nucleic acids play fundamental roles in living organism. In addition to
the four canonical nucleosides (adenosine, guanosine, cytosine and uridine),
nucleic acids use chemical modifications to diversify their structures and
functions.[1] Among the reported over 150 chemical modifications in a
wide range of RNA species, it has been revealed that thio-modifications
exist broadly in various cells (Fig. 1).[2] For instance, Liu et al. recently
disclosed that ges2U, mnm5ges2U and cmnm5ges2U are identified in living
organisms including Escherichia coli, Enterobacter aerogenes, Pseudomonas
aeruginosa and Salmonella enterico var. Typhimurium.[3,4] Considering the
roles of the sulfur-containing nucleotides of varied RNA species in regu-
lation and enhanced translation efficiency and fidelity, the transcriptom-
ics-based deep analysis of thio-modifications in nucleic acids show a great
potential for illustration of its molecular mechanism although it remains
elusive.[5,6] To that end, development of tools for selective labeling of
thio-modified nucleic acids is of highly urgent.
CONTACT Rui Wang
ez_rwang@hust.edu.cn
Hubei Key laboratory of Natural Medicinal chemistry and
Resource evaluation, school of Pharmacy, tongji Medical college, Huazhong university of science and technology,
Wuhan, Hubei 430030, china.
supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1942044.
© 2021 taylor & Francis Group, llc

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P. YU ET AL.
Figure 1. overall illustration of sulfur-containing nucleosides. Nucleosides (s2t and m5s2u) in
black dotted are presented as model substrates.
It has been proven that most of the thio group in nucleosides (s2U, s4U
and s2A derivatives, etc.) have very close reactivity’s with cysteine in pro-
teins. It is known that sulfur-containing derivatives of nucleic acids are
present in the tautomeric form thiol. Thio-modified nucleosides display
in two or more tautomeric forms which can further undergo nucleophilic
substitution reactions. To the regard, alkylation of thiolated nucleic acids
with various nucleophiles have been demonstrated successfully.[7]
In 1973, Söll and coworkers have reported that 4-thiouridine reacted
with the fluorescent reagent 4-bromomethyl-7-methoxy-2-oxo-2H-benzo-
pyran under physiological condition, the selective labeling of the 4-thiou-
ridine in Bacillus tRNA has been achieved.[8]
Later, s4U has been demonstrated successfully in metabolic labeling
studies. In more details, s4U incorporated RNA were selectively enriched
by use of streptavidin matrix HPDP-biotin (2-pyridylthio-activated biotin
disulfide) via sulfur exchange reaction, which have been served as a prac-
tical method for biotinylation of s4U-RNA.[9]
Duffy and coworkers have reported a chemically selective approach to
label and enrich s4U-modified RNA.[7] It has demonstrated that methan-
ethiolsulfonate (MTS) reagents reacted with s4U efficiently, leading to higher
yields and less biased enrichment than conventional HPDP-biotin. However,
although this method is useful, the application scope of other sulfur-con-
taining nucleosides such as s2T, s2U, m5s2U and s2A have not been clarified.
Other method that can be applied for the studies of highly selective labeling
or enrichment of sulfur-containing RNA/DNA molecules remain unknown.

NUcLEoSIDES, NUcLEoTIDES AND NUcLEIc AcIDS
3
Notably, mercury has also been used for labeling of thiolated RNA.[10–15]
It is noted that organomercury functionalized agarose and mercury con-
taining cellulose has been employed for the purification of thiolated RNA
since 1977. Interestingly, it has been showed that the degree of retardation
depends on the type of RNA modification with 4-thiouridine being more
retarded than 2-thiouridine or 5-methylamino-methyl-2-thiouridine
(mnm5s2U). Organomercury gels are now commonly used to study the
position 34 thiolated uridines in tRNAs.[16,17]
In general, thiols are strong nucleophiles that could react with a wide
range of electrophiles. For instance, iodoacetamides and bromoacetamides[18,19]
have been employed to label s2U- and s4U-incorporated nucleic acids.
However, reagents such as bromomethyl-coumarin[8] was reported to react
with s4U, the selectivity toward other thiolated nucleosides like s2U, s2A
or s2C remains unknown.
Therefore, intensive illustration of the intrinsic mechanism of thio-mod-
ified nucleosides is of highly urgent and necessary. We report here com-
prehensive investigations of nucleophiles used for highly selective labeling
and enrichment reagent for studies of thio-modified nucleosides such as
s2T, s2U, m5s2U, s2A, s4U and so forth. Based on preliminary screenings,
we demonstrate successfully the fluorescent labeling of s2T-incorporated
DNA and m5s2U-containing RNA.
2. Results and discussion
To evaluate reactivities of reagents used in the studies of sulfur-containing
nucleosides and further DNA and RNA molecules, we first chose thiouracil
as a model substrate. As depicted in Scheme 1, a series of alkylation
reagents including iodoacetamide (for 2a derivative, see Supporting
Information Figs. S1–S3), propargyl bromide (2b), prenyl bromide (2c),
geranyl bromide (2d), 4-methylcumarin bromide (2e), acrylonitrile (2f,
Supporting Information Figs. S4–S7),[20] vinyl sulfonate (2g, Supporting
[21]
Information Figs. S8–S11)
and S-benzyl methanethiosulfonate (BMTS,
2h, Supporting Information Figs. S12–S16) were gingerly investigated. It
was not surprising that nucleophiles such as reagents 2a and 2h displayed
unique dynamic properties. Comparing with allylic bromide (2c–2e,
Supporting Information Figs. S19–S21, Charts S5–S10) or alkynyl analogue
(2b, Supporting Information Fig. S18, Charts S3–S4), iodoacetamide (2a,
Supporting Information Fig. S17, Charts S1 and S2) and derivative dis-
played higher conversion rate in sulfur alkylation process. Unsaturated
alkenyl substrates such as acrylonitrile (2f, Supporting Information Figs.
S22, Chart S11), vinyl sulfonate (2g, Supporting Information Fig. S23,
Chart S12) had a very slow conversion rate toward those thiouracil. The

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Scheme 1. Model reaction investigations of thiouracil with various reagents. (a) overall reaction
illustration using various reagents (2a–2h). thiouracil (1.0 eq.) and reagent (2.0 eq.) in cd3od
(0.6 ml) were used for all experiments otherwise stated. (B) dynamic results of varied regents
(2a–2h) with thiouracil (1). it was demonstrated that 2a (blue bar) and 2h (black bar) showed
the best results. (c and d) dynamic details of reagents 2a or 2h with thiouracil (1). For
reagent 2a, the reaction proceeded rapidly in 70% conversion within 10 minutes, with the
extension of the reaction time, the conversion reached to 85% in 2 hours. For reagent 2h, the
reaction proceeded rapidly in 70% conversion in 10 minutes, with the extension of the reaction
time, the conversion did not increase.
neutral reaction condition may be accounted for the low nucleophilic
substituent reactivity. To our delight, the S-benzyl methanethiosulfonate
(BMTS, 2h, Supporting Information Figs. S24 and S25, Charts S13 and
S14) as well as allylic substrates (2c–2e) showed moderate to good con-
version rate, which was equal to iodoacetamide (2a, Scheme 1 and
Supporting Information Fig. S17, Charts S1 and S2). We next paid our
attentions toward these two reagents in the labeling studies of thio-con-
taining DNA and RNA. Monitoring of the reaction of thiouracil with
either iodoacetamide (2a) or S-benzyl methanethiosulfonate (BMTS, 2h,
Supporting Information Figs. S24 and S25) in situ showed different
dynamic. For instance, using of iodoacetamide (2a) resulted in a pseu-
do-first order kinetics with a half-time of approximate 50.8minutes (Scheme
1B,C, and Supporting Information Chart S1 and S2), while use of S-benzyl
methanethiosulfonate (BMTS, 2h) yielded a very new result (Scheme 1B,D,
and Supporting Information Chart S24 and S25), the conversion stayed at
a constant conversion of about 70% in 10 minutes, probably because this
process was partial reversible.
To determine feasibility of this method, alkylation of 5-methyl-2-thiou-
ridine (m5s2U) following this protocol was carried out. It was observed

NUcLEoSIDES, NUcLEoTIDES AND NUcLEIc AcIDS
5
that the alkylation using 2a or 2h was not so efficient. We tried to opti-
mize the reaction conditions to enhance its feasibility of implementation
(Supporting Information Figs. S26–S30). When reagent 2h was applied,
temperatures (40 or 60 °C) or concentrations (Supporting Information
Chart S14) trials has been screened, but this influence was minimal
(Supporting Information Fig. S26). Screenings of the reaction of m5s2U
with reagents 2h at different pH values and temperature failed, significantly
low conversion was observed. Interestingly, substrate s4U proceeded well
with reagent 2h (Supporting Information Figs. S27–S29) and 2a (Supporting
Information Figs. S30 and S31) in almost quantitative yields. In sharp
contrast, the reaction of m5s2U with 2a run well to produce alkylated
product in approximate 65% conversion at 40 °C in 12 hours (Supporting
Information Fig. S32). These results indicated the methyl has a crucial
influence on the reactivity toward sulfur-containing nucleosides. Multiple
parameters (pH values and temperatures) have also been evaluated
(Supporting Information Figs. S32–S35). It was anticipated that basic con-
dition would promote formation of sulfur anion, thus, accelerated the
nucleophile attachment process of sulfur-containing substrate with various
alkylation reagents, which indeed was observed in these experiments
(Supporting Information Figs. S34 and S35). In addition, the reaction of
the substrates s2U with 2a proceeded faster (Supporting Information Fig.
S37) than 2h (Supporting Information Fig. S36), and s2A (Supporting
Information Figs. S38–S40) displayed low activities toward reagents 2a or
2h. Moreover, the reactions of 2-thiouracil with reagent 2h under varied
conditions have also explored (Supporting Information Figs. S41 and S42).
It has demonstrated that m5s2U and 2-thiouracil had a similar reactivities
when reagent 2a was employed (Supporting Information Figs. S32–S35
versus S17), but the difference was obvious when using of 2h (Supporting
Information Figs. S26 versus S24). Thus, it has demonstrated that sul-
fur-modified sites have a significant influence on the reactivity toward
defined reagents such as iodoacetamide (2a) or S-benzyl methanethiosul-
fonate (2h). 2-Thio nucleosides have no activities in the absence or pres-
ence of 5-methyl substituent (Supporting Information Figs. S36, S26 and
S38) when the S-benzyl methanethiosulfonate (2h) was used.
Next, we aimed to investigate whether our approach could be utilized
into thio-modified oligos (Supporting Information Scheme 2 and Supporting
[22–24]
Information Figs. S43–S54).
The thio-modified base skeleton (3d)
was constructed[25] in 3 linear steps (23% overall yields, Scheme 2) from
aldehyde (3a). Subsequently, the Vorbrüggen nucleoside synthesis was
applied, the key intermediate 3e was obtained in relative low yield with
two isomers which was proven to be inseparable by flash chromatography
or high-performance liquid chromatography. Nevertheless, the deprotection

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Scheme 2. labeling studies of s2t-dNa. (a) synthesis details: (a) NaBH4, tHF, r.t., 12 hours,
57%; (b) socl2, r.t., 90%; (c) K2co3, acetone, refluxed, 46%; (d) N, O-bis(trimethylsilyl) acetamide,
cH2cl2; then, sncl4, used directly for next step; (e) NaoH (1.0 M), tHF-MeoH, 23% yield for 2
steps; (f) dMtcl, pyridine, r.t., 79%; (g) 2-cyanoethyl N, N-diisopropyl chlorophosphormidite,
diPea, cH2cl2, r.t., 2 hours, 90%. (B) Group1: native dNa; Group2: modified dNa; Group3:
modified dNa with Pia; Group4: modified-dNa + Pia + azido-Fitc; Group5: modi-
fied-dNa + Pia + azido-BodiPY. Method used here for the labeling of s2t-modified dNa: (i)
propargyl iodoacetamide (Pia), 90 °c, 5 minutes, NaoH (1.0 M, 1%); (ii) cuso4 (5.0 mM), tBta
(5.0 mM), sodium ascorbate (20.0 mM), tBuoH: H2o (3: 1); labeling approaches using azi-
do-BodiPY (1.8 mM) or azido-Fitc (1.8 mM) versus dNa (120 μM) via click reactions; (iii) Gel
electrophoresis assays and subsequent imaging under blue-light or uV-light illumination were
confirmed. 20% denature PaGe gel was utilized. left: fluorescent image (excitation wavelength
are 554 nm for BodiPY and 492 nm for Fitc). Right: GoldView dye was used.
of substrate (3e) proceeded well, leading to the deprotected nucleoside 3f
in 23% yield (Supporting Information Fig. S49). It was delighted to find
that when DMT group was employed for protection of 5′-OH function
in nucleoside 3f, 3g was obtained in 79% yield and the α/β isomers could

NUcLEoSIDES, NUcLEoTIDES AND NUcLEIc AcIDS
7
be isolated in over 95% purity based on the NMR data (Supporting
Information Figs. S50 and S51). Eventually, the DMT-protected nucleoside
3g reacted with 2-cyanoethyl N, N-diisopropylchlorophosphormidite
(CEP-Cl) under the optimized condition to give s2T-phosphormidite (3h,
Supporting Information Figs. S52–S54) in 90% yield. The synthesis of
2-thiothymidine (s2T)-containing oligo was carried out via solid-phase
synthesis (Supporting Information Table S1). After ammonium cleavage/
deprotection and HPLC purification, the full-length s2T-DNA product was
confirmed by HPLC and mass spectrum (Supporting Information Figs.
S55 and S56).
The circular dichroism (CD) spectra of double-strand of the native and
s2T-modified DNA were measured first (Supporting Information Fig.
S57.[26] As expected, both oligomers presented a spectrum typical of
B-DNA, which exhibited characteristic cross-over near 260 nm with a large
negative ellipticity at 250nm. In more details, in the U-A or s2U-A duplexes
spectrum (Supporting Information Fig. S57A) there was a strong positive
peak around 275 nm, a relatively weak negative peak around 245 nm.
Further Tm calculations based on the CD spectra have also been evaluated,
it was demonstrated the s2T-incorporated DNA comparing with its native
form resulted in a decrease temperature of 3.6 °C. The thio-modification
on 2-position perturbed the T-A base-pairing, leading to small biases of
the structure.
To evaluate the feasibility of this s2T-involved labeling and enrichment
of DNA as well as RNA molecules, we tried to further evaluate those
iodoacetamide (2a) or S-benzyl methanethiosulfonate (2h). It was observed
that the iodoacetamide (2a) derivative with propargyl handle proceeded
successfully under the optimized condition (95 °C, 5 minutes), which was
confirmed by HPLC (Supporting Information Figs. S58 and S59) and mass
spectra (Supporting Information Fig. S60). Notably, direct fluorescent
labeling of s2T-DNA (Supporting Information Scheme 3) using iodoacet-
amide-FITC (3) followed the optimized condition has also been performed
and further were confirmed by green fluorescence imaging (Supporting
Information Fig. S61) and mass spectra result (Supporting Information
Fig. S62 and Table S2).
Considering the propargyl handle installed on s2T-DNA, we next turned
to illustrate the possibility of labeling of s2T-DNA via click reaction. As
depicted in Supporting Information Scheme S3, the azido-incorporated
BODIPY were successfully inserted into the s2T-DNA which were further
analyzed via fluorescent spectroscopy and polyacrylamide gel electropho-
resis (Scheme 2B, see Supporting Information Scheme S3 for details).
Nucleic acid staining (right, Scheme 2B) or fluorescent detection (left,
Scheme 2B) of the gel revealed that selective labeling thio-modified DNA

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Scheme 3. synthesis of m5s2u-RNa and its fluorescent labeling study. (a) synthesis of m5s2u-in-
corporated RNa by use of transcriptional method. synthesis: (a) N, o-bis(trimethylsilyl)acetamide,
acetonitrile, 80 °c, 1 hour; trimethyl silyl triflate, 50 °c, 4 hours, 40%; (b) NaoMe, MeoH, 2 hours,
80%; (c) Proton sponge, trimethyl phosphate, 0 °c, 10 minutes; phosphorus oxychloride, 2 hours;
tributylamine, bis-(tributylammonium) pyrophosphate, 10% for 3 steps. (d) transcription pro-
tocol: eGFP dNa template (1.0 μg), t7 RNap (1.0 μl), buffer (2.0 μl) and rNtP mix: Group1
(a/c/G, 0.5 μl, 100 mM, m5s2utP, 2 μl, 100 mM), Group2 (a/c/G/u, 0.5 μl, 100 mM and m5s2utP,
2 μl, 100 mM), Group3 (a/c/G/u, 0.5 μl, 100 mM and m5s2utP, 4 μl, 100 mM), Group4 (a/c/G/u,
0.5 μl, 100 mM and m5s2utP, 8 μl, 100 mM). Final volume was 20 μl (dePc). (B) transcription
assays and analysis by gel electrolysis using GoldView dye. (c) Fluorescent labeling by use of
iodoacetamide-Fitc (3, 10.0 μM), 90 °c, 3 minutes. Fluorescent imaging was recorded via cy3
tunnel (ex.470 nm).
using our method have been achieved. Analysis of the mass spectroscopy
of the BODIPY-labeled product have successfully demonstrated the feasi-
bility of this method (Supporting Information Fig. S62 and Table S2).
Thus, through the optimized alkylation process directly with iodoacet-
amide-FITC (3), or with propargyl iodoacetamide along with the click
reaction, the fluorescent labeling of s2T-incorporated DNA has been
achieved.
In order to enhance feasibility of labeling of thio-modified nucleic acid
following this protocol, i.e., m5s2U-RNA, the m5s2U triphosphate was
synthesized in three steps (Scheme 3A and Supporting Information Figs.
S63–S68). Nucleoside 4b was synthesized first via the Vorbrüggen reaction

NUcLEoSIDES, NUcLEoTIDES AND NUcLEIc AcIDS
9
with two isomers which was inseparable by silica gel flash chromatography
(Supporting Information Fig. S63). Deprotection of 4b gave nucleoside 4c
in 80% yield, which was separated by HPLC and confirmed by proton
NMR (Supporting Information Figs. S64–S66). The one-pot m5s2U tri-
phosphate synthesis from unprotected 4c was carried out, m5s2U triphos-
phate was obtained in 10% yield (Supporting Information Figs. S67 and
S68). With this triphosphate in hand, we turned our attentions to corre-
sponding transcription assays to introduce m5s2U into RNA. We con-
structed a EGFP gene first (Supporting Information Fig. S69). After
concentration titration and optimization (Supporting Information Table
S3), it was revealed that when the triphosphate 4d reached to a concen-
tration of 20 mM, EGFP is efficiently transcribed to produce RNA with
m5s2U-modifications and band of this transcribed RNA on gel was obvious
(Scheme 3B, left, Group3, Supporting Information Fig. S70). Subsequently,
labeling of this m5s2U-modified transcript was performed using the opti-
mized condition in the absence of base. After installation of a propargyl
functionality into m5s2U-modified transcript with propargyl iodoacetamide
described above (Supporting Information Scheme S2), click reaction was
utilized with iodoacetamide-FITC reagents (3) in order to introduce a
FITC fluorophore. It seemed that when the transcript concentration reached
to 100 µM, and the concentration of iodoacetamide-FITC (3) (Supporting
Information Fig. S3) was 10µM, the fluorescent labeling of m5s2U-modified
RNA was effective (Scheme 3B, right and Supporting Information Fig.
S71). In a word, fluorescent labeling of thio-modified (m5s2U) newly
transcribed RNA has been achieved via alkylation by direct use of iodo-
acetamide FITC (3).
In previous studies, we demonstrated that alkylation with iodoacetamide
(2a) proceeded well for all substrates except s2A, and it has been proven
that s4U displayed the fastest kinetics. When reagent S-benzyl methanethio-
sulfonate (2h) was utilized, s4U exhibited great potential in the selective
alkylation process. However, it has been demonstrated that 2-thio substrates
exhibited low conversion rate toward those reagents (2h). To further expand
feasibility, the bio-orthogonality have also been evaluated. It has demonstrated
that reagent 2a or 2h were compatible for common amino acids including
lysine (Supporting Information Figs. S72 and S74), tyrosine (Supporting
Information Figs. S73 and S74) or cysteine analogue (Supporting Information
Fig. S75. In addition, it has been reported that s4U was easily transferred
into uridine when treated with hydro peroxide (Supporting Information Fig.
S79A. We try to explore whether this was also feasible for those 2-thio
nucleoside substrates (Supporting Information Fig. S79B). Interestingly, all
the thio-modified nucleosides were transformed to their native forms when
treated with hydro peroxide, s2U achieved the highest dynamics, while m5s2U

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P. YU ET AL.
and s2A displayed almost equal reactivities under the tested condition. Thus,
our investigations have shed fundamental light on the difference between
the reactivities of nucleosides including s2T, s2U, m5s2U, s4U and s2A.
3. Conclusions
We demonstrate herein comprehensive investigations of various sulfur-con-
taining nucleosides such as s2T, s2U, m5s2U, s4U, s2A, s2T-incorporated
DNA and m5s2U-modified RNA. It has been proven that iodoacetamide
(2a) display the most promising feasibility for sulfur-containing substrates
such as s2T, s2U, m5s2U and s4U. In sharp contrast, the alkylation with
S-benzyl methanethiosulfonate (2h) exhibit the greatest potential in the
studies of s4U. Based on optimized condition, we have successfully demon-
strated the fluorescent labeling of s2T-incorporated DNA and m5s2U-mod-
ified RNA.
Activity comparisons between iodoacetamide (2a) or S-benzyl methan-
ethiosulfonate (2h) with a wide range of sulfur-containing nucleosides
have also been evaluated. It seemed that alkylation with iodoacetamide
(2a) proceeded well for all substrates except s2A, and it has been proven
that s4U displayed the fastest kinetics. When reagent S-benzyl methaneth-
iosulfonate (2h) was utilized, s4U exhibited great potential in the selective
alkylation process. However, it has been demonstrated that 2-thio substrates
exhibited low conversion rate toward those reagents (2h). In addition, we
have also demonstrated that all the thio-modified nucleosides are able to
be transformed to their native forms when treated with hydro peroxide,
s2U achieved the highest dynamics, while m5s2U and s2A have almost
equal reactivities under the tested condition. Epitranscriptome analysis
based on this result is ongoing in my lab and will be reported in due course.
4. Experimental
4.1. General methods
Anhydrous solvents were used and redistilled using standard procedures.
All solid reagents were dried under a high vacuum line prior to use. Air
sensitive reactions were carried out under argon. Analytical TLC plates
pre-coated with silica gel F254 (Yantai Chemical Industry Research Institute)
were used for monitoring reactions and visualized by UV light and/or
PMA or KMnO4. Flash column chromatography was performed using
silica gel (32–63 μm, Qingdao Marine Chemical Inc., China), or high-res-
1
olution liquid chromatography. All H- and 13C-NMR spectra were recorded
1
on a Bruker AM-400 spectrometer (400 MHz), and the H and 13C NMR
chemical shifts were referenced to the solvent or solvent impurity peaks

NUcLEoSIDES, NUcLEoTIDES AND NUcLEIc AcIDS
11
for CDCl3 at δ1H 7.24 and δ13C 77.23. High resolution mass spectra were
recorded on a Bruker microTOF II spectrometer using electrospray ion-
ization (ESI) achieving at Huazhong University of Science and Technology.
The Supporting Information is available free of charge at https://www.
tandfonline.com/
Acknowledgment
We thank Prof. Chaomei Xiong for NMR experiment and Prof. Lingkui Meng for mass
spectrum.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
R. Wang thanks the starting fund from Huazhong University of Science and Technology
for financial support. NSFC (21904044 to Z.F.D) is appreciated for financial support.
ORCID
Rui Wang
http://orcid.org/0000-0003-2553-620X
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