Synthesis and Substrate Evaluation of (E)-5-[(3-Selenophene-2-Carboxamido)Prop-1-en-1-yl]-Uridine-5′- O -Triphosphate for RNA Polymerase

Article abstract of DOI:10.1080/15257770.2015.1081941

Design, synthesis and T7 RNA polymerase substrate evaluation of (E)-5-[(3-selenophene-2-carboxamido)prop-1-en-1-yl]-uridine-5′-O-triphosphate is reported. The title compound is shown to be a good substrate for RNA polymerase by RNA labeling through in vitro transcription. pTRI-plasmid DNA with β-actin gene sequence (～300 base pairs) with T7 promoter was used as a template for the in vitro transcription. Transcribed product is characterized for incorporation by gel assay and for integrity, full length and size by bioanalyzer. The title compound will be very useful in biophysical techniques to obtain information on dynamics and recognition properties in real time as well as 3D structure of nucleic acids.

Full text of DOI:10.1080/15257770.2015.1081941

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and Substrate Evaluation of (E)-5-[(3-
Selenophene-2-Carboxamido)Prop-1-en-1-yl]-
Uridine-5′-O-Triphosphate for RNA Polymerase
Anilkumar R. Kore, Bo Yang, Samaraj S. Thiyagarajan & Balasubramanian
Srinivasan
To cite this article: Anilkumar R. Kore, Bo Yang, Samaraj S. Thiyagarajan & Balasubramanian
Srinivasan (2015) Synthesis and Substrate Evaluation of (E)-5-[(3-Selenophene-2-
Carboxamido)Prop-1-en-1-yl]-Uridine-5′-O-Triphosphate for RNA Polymerase, Nucleosides,
Nucleotides and Nucleic Acids, 34:12, 866-876, DOI: 10.1080/15257770.2015.1081941
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Date: 14 December 2015, At: 01:26

Nucleosides, Nucleotides and Nucleic Acids, 34:866–876, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2015.1081941
SYNTHESIS AND SUBSTRATE EVALUATION OF
(E)-5-[(3-SELENOPHENE-2-CARBOXAMIDO)PROP-1-EN-1-YL]-
URIDINE-5ꢀ-O-TRIPHOSPHATE FOR RNA
POLYMERASE
Anilkumar R. Kore, Bo Yang, Samaraj S. Thiyagarajan, and Balasubramanian
Srinivasan
Life Sciences Solutions Group, Thermo Fisher Scientific, Austin, TX, USA
Design, synthesis and T7 RNA polymerase substrate evaluation of (E)-5-[(3-selenophene-2-
2
carboxamido)prop-1-en-1-yl]-uridine-5ꢁ-O-triphosphate is reported. The title compound is shown to
be a good substrate for RNA polymerase by RNA labeling through in vitro transcription. pTRI-
plasmid DNA with β-actin gene sequence (∼300 base pairs) with T7 promoter was used as a
template for the in vitro transcription. Transcribed product is characterized for incorporation by gel
assay and for integrity, full length and size by bioanalyzer. The title compound will be very useful
in biophysical techniques to obtain information on dynamics and recognition properties in real time
as well as 3D structure of nucleic acids.
Keywords Modified AA-UTP; selenophene containing UTP; In Vitro transcription
INTRODUCTION
Ribonucleic acid involves in several key cellular processes such as catal-
ysis, and regulation of genetic information by interacting with proteins, nu-
cleic acids and small molecule metabolites. These interactions are governed
by intrinsic conformational dynamics resulting from diverse secondary and
tertiary structures.[1] To mention a few, techniques such as fluorescence,
nuclear magnetic resonance, electron paramagnetic resonance and elec-
trophoresis are commonly used to advance our understanding on the struc-
tural dynamics and recognition properties of nucleic acids.[2] In addition,
high resolution X-ray structure of functional RNA motifs offers fundamen-
tal understanding on correlation of RNA structure with their function at
molecular level.[3] Three-dimensional structural determination of nucleic
Received 22 December 2014; accepted 7 August 2015.
Address correspondence to Anilkumar R. Kore, Director, Chemistry, Life Sciences Solutions
Group, Thermo Fisher Scientific, 2130 Woodward Street, Austin, Texas 78744-1832, USA. E-mail:
anil.kore@thermofisher.com
866

Synthesis and Substrate Evaluation
867
acids using X-ray crystallography has heavily relied on heavy atom derivatiza-
tion of nucleic acids for phase determination. Single-wavelength anomalous
dispersion (SAD) phasing or multi-wavelength anomalous dispersion (MAD)
phasing are the conventional techniques in structural determination which
involves the anomalous scattering from a bromine or iodine atom of bromi-
nated or iodinated nucleic acids.[4] Halogenated oligonucleotides, being
light sensitive, undergo dehalogenation upon X-ray exposure causing fail-
ures in phasing. To overcome this issue, the use of selenium atom that
displays superior anomalous scattering property in protein X-ray crystallog-
raphy has also been extended to nucleic acid X-ray crystallography.[5] Huang
et al used Se-MAD phasing technique successfully to report the x-ray structure
of selenium modified DNA and RNA oligonucleotides.[6] These selenium
heavy atom derivatization methodologies have also been successfully used in
the X-ray structural determination of nucleic acids,[7] nucleic acid-protein
and nucleic acid-small molecule complexes.[8] To broaden our understand-
ing on how RNA structure complements its function, it will be attractive to
design a dual function label that allows obtaining information on dynamics
and recognition properties in real time as well as 3D structure of nucleic acids
at the same time. For example, an anomalous scattering heavy atom (sele-
nium) containing conformation-sensitive fluorescent selenophene label on
the nucleosides would facilitate such structure-function correlation analysis
by both X-ray crystallography and fluorescence.[9] Aminoallyl-RNA can be ob-
tained by the incorporation of 5-(3-aminoallyl)-uridine-5’-triphosphate into
RNA by T7 RNA polymerase. The aminoallyl functionality in the aminoallyl
nucleotides serves as an anchor point to install various labels, tags and probe
molecules such as biotin, fluorescent dyes etc by conjugation via either pre-
labeling of aminoallyl nucleotides or post-labeling of product RNA.[10] Com-
monly, these labeled aminoallyl probes are used in microarrays for simul-
taneous analysis of multiple gene expression levels.[11] In this manuscript,
we describe the utilization of the aminoallyl handle present in (E)-5-[3-
Aminoallyl]-uridine-5ꢁ-triphosphate, AA-UTP to attach a dual function se-
lenophene label to obtain (E)-5-[(3-selenophene-2-carboxamido)prop-1-en-
1-yl]-uridine-5ꢁ-O-triphosphate. This selenophene labeled AA-UTP will find
applications in biophysical techniques to obtain information on dynamics
and recognition properties in real time as well as 3D structure of nucleic
acids.
RESULTS AND DISCUSSION
An anomalous scattering heavy atom (selenium) containing
conformation-sensitive fluorescent selenophene label on the nucleosides
would facilitate structure-function correlation analysis by both X-ray crystal-
lography and fluorescence. Pawar et al.[9] showed usefulness of selenophene

868
A. R. Kore et al.
label as a fluorescence probe by synthesizing UTP containing (selenophen-
2-yl)prymidine core for monitoring binding of aminoglycoside antibiotics
to the bacterial ribosomal decoding A-site. Selenophene-conjugated uri-
dine analogue was shown to display emission in the visible region and
very good fluorescence solvatochromism.[9] This work is essentially based
on the earlier development of fluorescent assay involving nucleoside ana-
logues bearing a furan moiety at 5-position to detect the presence of DNA
abasic sites and the binding of aminoglycoside antibiotics to RNA.[12] The ad-
vantage of replacing furan with selenophene is the incorporation of anoma-
lous heavy atom for 3D structural determination in addition to generat-
ing minimally perturbing emissive nucleobase analogue. Selenium contain-
ing nucleobases were shown to be the good substrates for polymerases.[13]
Aminoallyl nucleotides aid enzymatic introduction of functional groups
into nucleic acid targets. Aminoallyl-RNA can be easily obtained by the
incorporation of (E)-5-(3-aminoallyl)-uridine-5’-triphosphate (AA-UTP) by
RNA polymerases. The aminoallyl handle present in (E)-5-[3-Aminoallyl]-
uridine-5ꢁ-triphosphate, AA-UTP 4 allows facile attachment of dual bio-
physical functional selenophene label to obtain (E)-5-[(3-selenophene-2-
carboxamido)prop-1-en-1-yl]-uridine-5ꢁ-O-triphosphate 5.
In view of above facts and in continuation of our efforts in synthe-
sizing various biologically useful phosphorylated nucleosides,[14] we are
much interested to synthesize and evaluate the selenophene labeled AA-
UTP as substrate for RNA polymerase. The reaction pathway leading to
selenophene tagged AA-UTP, (E)-5-[(3-selenophene-2-carboxamido)prop-
1-en-1-yl]-uridine-5ꢁ-O-triphosphate is depicted in Scheme 1. The chem-
istry essentially involves coupling of activated ester, 2,5-dioxopyrrolidin-1-
yl selenophene-2-carboxylate 3 to aminoallyl moiety of AA-UTP 4. The re-
quired selenophene-2-carboxylic acid N-hydroxy succinimidyl activated ester
was synthesized in two steps starting from selenophene-2-carbaldehyde 1.
The first step is the oxidation of carbaldehyde to carboxylic acid and the
second step is the coupling of N-hydroxysuccinimide to the carboxylic
acid. Though there are many reagents available for the oxidation step,
silver (I) oxide was reported to work very well for heterocycle carbalde-
hyde oxidation.[15] Silver (I) oxide mediated oxidation of selenophene-2-
carbaldehyde 1 in aqueous sodium hydroxide solution resulted in the for-
mation of selenophene-2-carboxylic acid 2 in 97% yield. DCC mediated
coupling of N-hydroxysuccinimide to selenophene-2-carboxylic acid in dry
acetonitrile as the solvent afforded 2,5-dioxopyrrolidin-1-yl selenophene-2-
carboxylate 3 in 96% yield. This step does not require any purification
other than filtering to remove the precipitated side product dicyclohexyl
urea. As the activated ester of selenophene-2-carboxylic acid is ready, the
next step would be the preparation of the coupling partner AA-UTP 4. The
common and popular method to make aminoallyl nucleoside involves mer-
curation of uridine triphosphates using mercuric acetate and subsequent

Synthesis and Substrate Evaluation
869
palladium-catalyzed reaction with allylamine.[16] This method heavily use
no greener, toxic and hazardous mercuric acetate and obtaining mercury-
free allylamino nucleoside triphosphates is challenging. Hence we synthe-
sized AA-UTP 4 via palladium-catalyzed Heck coupling of 5-iodouridine
triphosphates with allylamine, a highly concise and mercury free method
recently reported.[17–19] With AA-UTP in hand, it is then coupled with 2,5-
dioxopyrrolidin-1-yl selenophene-2-carboxylate 3 in the presence of triethyl
amine as the base and anhydrous dimethylformamide as the solvent. The
reaction mixture was reduced under vacuum, reconstituted in 0.1 M aque-
ous triethylammonium bicarbonate (TEAB) buffer, pH 7.8, pH adjusted
to 6.5 using dilute acetic acid and loaded on a DEAE Sepharose column.
The desired product was eluted using a linear gradient of 0–1 M TEAB and
the fractions containing the product was pooled, evaporated and coevap-
orated with water. The triethylammonium salt of (E)-5-[(3-selenophene-2-
carboxamido)prop-1-en-1-yl]-uridine-5ꢁ-O-triphosphate 5 thus obtained was
subjected to ion-exchange with sodium perchlorate in acetone for twice to
afford the sodium salt of 5 in 76% yield with 98% purity by HPLC. Fluores-
cence of 100 μmolar aqueous solution of selenophene labeled AA-UTP 5
was compared with the same concentrations of other NTPs (C, G, A and U)
commonly used in the polymerase mediated in vitro transcription reactions.
Selenophene labeled AA-UTP 5 displays strong fluorescence signal (∼3 or-
der) at its emission maximum (454 nm) upon excitation at 375 nm where as
CTP, GTP, UTP and ATP do not display any fluorescence at the same mea-
surement conditions. This observation is in accordance with earlier similar
observations on UTP probe having (selenophen-2-yl)pyrimidine core.[9]
SCHEME
1 Synthesis
of
(E)-5-[(3-selenophene-2-carboxamido)prop-1-en-1-yl]-uridine-5ꢁ-O-
triphosphate 5.
Modified RNA oligonucleotides can be obtained by enzymatic methods
using RNA polymerases and ligases. Here we performed in vitro transcrip-
tion reactions in the presence of SP6, T3 and T7 phage RNA polymerases
enzyme mixture to incorporate (E)-5-[(3-selenophene-2-carboxamido)prop-
1-en-1-yl]-uridine-5ꢁ-O-triphosphate 5 into RNA by using MAXIscript(TM) kit
(Life Technologies Corporation, Carlsbad, CA, USA) following manufac-
turer’s protocol. The RNA polymerase enzyme mixture includes ribonucle-
ase inhibitor protein and hence protects the newly transcribed RNA from

870
A. R. Kore et al.
degradation by RNases, if any. Transcription reactions have been carried
out in the presence of promoter pTRI-β-actin-Mouse plasmid DNA template
having ∼300 base pairs. The template contains several adenosine residues
to facilitate the incorporation of selenophene labeled AA-UTP. RNA poly-
merase first binds to its double-stranded DNA promoter, then it separates
the two DNA strands, and uses the 3ꢁ to 5ꢁ strand as a template to synthesize
a complementary 5ꢁ to 3ꢁ at the end of the DNA template. All transcription
reactions were performed in triplicates with following components: pTRI-β-
actin-Mouse plasmid DNA template; reaction buffer, 1X; T7 RNA polymerase
enzyme mix; 0.5 mM of each ATP, CTP and GTP; and selenophene labeled
AA-UTP and/or UTP (total final concentration 0.5 mM). In control tran-
scription reaction, no selenophene labeled AA-UTP 5 was added. Two other
reaction were set up with the selenophene labeled AA-UTP at different ratios
as below indicated. In another reaction, a mixture of 0.2 mM of selenophene
labeled AA-UTP and 0.3 mM UTP (ratio of 40:60) was added. This reaction
was designated as “Mixed”. In third reaction, 0.5 mM of selenophene labeled
AA-UTP was added (represnting 100% for UTP) and no unlabeled UTP was
added. This reaction was designated as “Labeled”. Transcription reactions in
triplicate were incubated at 37◦C. In order to hydrolyze the remaining plas-
mid DNA, turbo DNase was added and the reaction mixture was further incu-
bated at 37◦C for 15 min. Purifications of the RNA from these transcription
reactions were done by using the MEGAclear(TM) kit (Life Technologies Cor-
poration, Carlsbad, CA, USA) as per manufacturer’s protocol. The transcrip-
tion yield of RNA was calculated by measuring absorbance at 260 nm. The
transcript yields indicates that (E)-5-[(3-selenophene-2-carboxamido)prop-
1-en-1-yl]-uridine-5ꢁ-O-triphosphate 5 is a good substrate for SP6, T3 and T7
phage RNA polymerases. Assessing the yield of all three reactions, shows con-
trol transcription reaction with no selenophene labeled AA-UTP 5 and the
mixed reaction (40:60) have similar yield. This indicates the T7 Polymerase
efficiency is not reduced by the presence of selenophene labeled AA-UTP
5 in in vitro Transcription reaction. However, in the reaction that contains
100% selenophene labeled AA-UTP 5 showed a 20% reduction in the yield.
Low yield of in vitro transcription reaction, in which 100% selenophene
labeled AA-UTP 5 could be due to fidelity and recognization of T7 RNA
polymerase enzyme with the modified compound 5.[20,21]
The products of transcription reactions were analyzed by gel analysis.
Because the total yield of RNA produced is high in non-limiting NTP con-
centration conditions, unlabelled, and nonisotopically labeled transcripts
can be visualized in gels. To perform gel assay, aliquot of transcription reac-
tions were mixed with gel loading buffer II containing formaldehyde. 10 μL
of this mixture were loaded on to 2% agarose gel precasted with Ethidium
bromide stain. Gel bands were visualized using AlphaImager (Figure 2). The
gel picture clearly indicates the incorporation of the probe in the RNA tran-
script as seen in the lane B and C, Irrespective of all three transcripts having

Synthesis and Substrate Evaluation
871
FIGURE 1 SP6, T3 and T7 phage RNA polymerases transcription yields (quantified at A260 nm by using
NanoDrop): A) control transcription reaction, no selenophene labeled AA-UTP 5, but 0.5 mM UTP was
used; B) Labeled transcription reaction, 0.2 mM of selenophene labeled AA-UTP 5 and 0.3 mM UTP
was used (mixed in ratio of 40:60); C) labeled transcription reaction, 0.5 mM of selenophene labeled
AA-UTP 5 (ratio of 100%) and no UTP was used.
the same size, the labeled reactions in the lane B and C had slower movement
compared to the control sample in lane A. This is due the molecular weight
difference between unmodified and modified selenophene labeled AA-UTP
5, which retards the shift as compared to control lane A. Additionally, due
to complete incorporation of selenophene labeled AA-UTP 5 and removal
of unincorporated NTPs from the in vitro transcription reactions, there is
no additional bands observed in the gel. Transcript RNAs from all the tran-
scription reactions were analyzed for their integrity, full length and size using
Bioanalyzer (Agilent). The promoter pTRI-β-actin-Mouse plasmid DNA tem-
plate used in the transcript reaction has ∼300 base pairs. The control, mixed
and labeled transcript reactions resulted in the full length RNA transcript
formation (with high integrity) with ∼ 339, 328, and 330 base pairs respec-
tively. Bioanlayzer profile indicates the incorporation of the selenophene
labelled AA-UTP 5 in the RNA transcript. The selenophene labeled AA-UTP
5 labeled generate signficantly stronger signal due to the additional fluores-
cence from the selenophene labeled AA-UTP 5. This can be accessed from
the bioanalyzer profile for the selenophene labeled AA-UTP 5, both mixed
FIGURE 2 Gel analysis of transcription reactions. A) control transcription reaction, no selenophene
labeled AA-UTP 5, but 0.5 mM UTP was used; B) mixed transcription reaction, 0.2 mM of selenophene
labeled AA-UTP 5 and 0.3 mM UTP was used; C) labeled transcription reaction, 0.5 mM of selenophene
labeled AA-UTP 5 and no UTP was used.

872
A. R. Kore et al.
FIGURE 3 Integrity and size anlaysis (using Bioanalyzer) of product transcript RNA from A) control
transcription reaction, no selenophene labeled AA-UTP 5, but 0.5 mM UTP was used; B) mixed tran-
scription reaction, 0.2 mM of selenophene labeled AA-UTP 5 and 0.3 mM UTP was used; C) labeled
transcription reaction, 0.5 mM of selenophene labeled AA-UTP 5 and no UTP was used. (FU-Fluoresecent
Units, nt-nucleotides)
and labeled have twice the fluorescent units compared to the unlabeled
control reaction.[22] (Figure 3).
In summary, (E)-5-[(3-selenophene-2-carboxamido)prop-1-en-1-yl]-
uridine-5ꢁ-O-triphosphate 5 synthesized from AA-UTP serves as a good
substrate for RNA polymerases. This observation hold great promise on the
design of this probe molecule because selenophene label can be attached
later also to aminoallyl modified RNA in a post-labeling fashion using
2,5-dioxopyrrolidin-1-yl selenophene-2-carboxylate 3. Selenophene labeled
AA-UTP 5 will find applications in biophysics filed as this probe contains
both anomalous scattering heavy atom (selenium) as well as conformation-
sensitive fluorescent selenophene label in its structure. Selenophene
derivatization of nucleic acids is of great importance because of its dual
probe nature and could provide new avenues to study structure-function
relationships in biomacromolecules.
EXPERIMENTAL
All of the commercial reagents and solvents are used as such without
further purification. Nucleosides were purchased from ChemGenes Corpo-
ration. (Wilmington, MA, USA). 1H NMR spectra were recorded in D2O on a
Bruker 400 MHz and 31P NMR were recorded on a Bruker 162 MHz. Chem-
ical shifts are reported in ppm, and signals are described as s (singlet), d
(doublet), t(triplet), q (quartet), and m (multiplet). ESI mass was recorded
on Applied Biosystems/Sciex MDX API 150 model. HPLC was run on a
Waters 2996 (Waters Corporation, Milford, MA, USA) using Hypersil SAX
column. FPLC (fast protein liquid chromatography) was performed on a

Synthesis and Substrate Evaluation
873
AKTA purifier (GE Healthcare, Piscataway, NJ, USA) using DEAE Sepharose
column.
Synthesis of selenophene-2-carboxylic acid (2) To a solution of NaOH
(1.95 g, 48.75 mmol) in water (20 mL) was added Ag2O (4.37 g, 18.86 mmol)
in water (20 mL) at room temperature. After stirring for 15 min,
selenophene-2-carbaldehyde 1 (2 g, 12.57 mmol) was added and the re-
action mixture was stirred at room temperature for 0.5 h, and at 50◦C for
4 h. A crystalline precipitate formed was filtered off and washed with wa-
ter. The filtrate and washings were combined, poured into chilled 10% HCl
(10 mL) and extracted with ethyl acetate. The ethyl acetate extract was
washed successively with water and brine; and dried over anhydrous sodium
sulfate. The organic solution was evaporated under reduced pressure to give
selenophene-2-carboxylic acid 2 in 97% yield (2.14 g, 12.2 mmol). The char-
acterization data for 2 was in agreement with that reported earlier.[23] 1
H
NMR (CDCl3, 400 MHz) δ (ppm) 8.24–8.27 (m, 1H), 8.02–8.05 (m, 1H),
7.35–7.38 (m, 1H).
Synthesis of 2,5-dioxopyrrolidin-1-yl selenophene-2-carboxylate (3) A
mixture of selenophene-2-carboxylic acid 2 (2 g, 11.4 mmol), N-hydroxy
succinimide (1.32 g, 11.4 mmol) and N,Nꢁ-dicyclohexyl carbodimide (DCC)
(2.35 g, 11.4 mmol) in dry acetonitrile (50 mL) was stirred at room tem-
perature for 14 h. The reaction mixture was filtered to remove dicyclohexyl
urea and the filtrate was evaporated and dried under vacuum to afford
2,5-dioxopyrrolidin-1-yl selenophene-2-carboxylate 3 in 96% yield (2.98 g,
10.94 mmol). The product was used as such for the next step.
Synthesis
of
(E)-5-[(3-selenophene-2-carboxamido)prop-1-en-1-yl]-
mixture of 2,5-dioxopyrrolidin-1-yl
uridine-5ꢁ-O-triphosphate (5)
A
selenophene-2-carboxylate 3 (1 g, 3.66 mmol), (E)-5-[3-Aminoallyl]-
uridine-5ꢁ-triphosphate, AA-UTP 4 (2.16 g, 3.66 mmol) and triethylamine
(2 mL, 14.33 mmol) in anhydrous dimethylformamide (50 mL) was stirred
at room temperature for 15 h. The reaction mixture was reduced under
vacuum, reconstituted in 0.1 M aqueous triethylammonium bicarbonate
(TEAB) buffer, pH 7.8 (200 mL), pH adjusted to 6.5 using dilute acetic acid
and loaded on a DEAE Sepharose column. The desired product was eluted
using a linear gradient of 0–1 M triethylammonium biocarbonate (TEAB)
and the fractions containing the product was pooled, evaporated and
coevaporated with water (3 × 100 mL). The triethylammonium salt of (E)-5-
[(3-selenophene-2-carboxamido)prop-1-en-1-yl]-uridine-5ꢁ-O-triphosphate
5 thus obtained was subjected to ion-exchange with sodium perchlorate
(2 g) in acetone (50 mL) for twice to afford the sodium salt of 5 in 76%
yield (2.08 g, 2.78 mmol). 1H NMR (D2O, 400 MHz) δ (ppm) 8.42–8.44 (m,
1H), 7.99–8.03 (m, 1H), 7.45–7.48 (m, 1H), 6.54–6.60 (m, 1H), 6.41–6.45
(m, 1H), 6.02–6.04 (d, J = 5.2 Hz, 1H), 4.47–4.49(m, 2H), 4.29–4.33 (m,
3H), 4.11–4.13 (m, 1H), 1.66–1.69 (m, 1H), 1.36–1.42 (m, 1H); 31P NMR
(D2O, 162 MHz) δ −6.93 (d, J = 19.8 Hz, 1P), −10.34 (d, J = 20.9 Hz, 1P),

874
A. R. Kore et al.
−21.33 (t, J = 20.3 Hz, 1P); MS (ESI, m/z): calc. for C17H22N3O16P3Se
[M-H]−: 695.9, found 696.5.
RNA Labeling Assay Through In vitro Transcription
RNA polymerase in vitro transcription was performed by using MAXIs-
cript(TM) kit (Life Technologies Corporation, Carlsbad, CA, USA) following
manufacturer’s protocol. All transcription reactions were performed in a
20 μL final volume at the following final concentrations of components:
pTRI-β-actin-Mouse plasmid DNA template, 0.5 mg/mL (1 μg total); re-
action buffer, 1X; RNA polymerase enzyme mix,T7 15 U/μL; 0.5 mM of
each ATP, CTP and GTP; and selenophene labeled AA-UTP and/or UTP
(total final concentration 0.5 mM). In control reaction, no selenophene
labeled AA-UTP was added. In another reaction, a mixture of 0.2 mM of
selenophene labeled AA-UTP and 0.3 mM UTP was added. In third reac-
tion, 0.5 mM of selenophene labeled AA-UTP (and no UTP) was added.
Transcription reactions in triplicate were incubated at 37◦C for 4 hours. In
order to hydrolyze the remaining plasmid DNA, 1 μL of turbo DNase was
added and the reaction mixture was further incubated at 37◦C for 15 min.
Purifications of the RNA from these transcription reactions were done by
using the MEGAclear(TM) kit (Life Technologies Corporation, Carlsbad, CA,
USA) as per manufacturer’s protocol. The transcription yield of RNA was as-
sessed using NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific,
Wilmington, DE, USA) by measuring absorbance at 260 nm. To perform gel
assay, 10 μL of transcription reactions were mixed with 10 μL of gel loading
buffer II (contains formaldehyde). 10 μL of this mixture were loaded in 2%
agarose gel [precast Ethidium bromide gel; ReliantTM GELS (2%SKG,TBE,
EB,2×12W) (Lonza, Basel, Switzerland)] and quantified using AlphaImager
(ProteinSimple, Santa Clara, California, USA).
REFERENCES
1. (a) Hermann, T.; Patel, D.J. RNA bulges as architectural and recognition motifs. Structure 2000,
8, R47–R54. (b) Leulliot, N.; Varani, G. Current topics in RNA−protein recognition: control of
specificity and biological function through induced fit and conformational capture. Biochemistry
2001, 40, 7947–7956. (c) Al-Hasani, H.M.; Walter, N.G. RNA dynamics: it is about time. Curr. Opin.
Struct. Biol. 2008, 18, 321–329. (d) Aitken, C.E.; Petrov, A.; Puglisi, J.D. Single ribosome dynamics
and the mechanism of translation. Ann. Rev. Biophys. 2010, 39, 491–513.
2. (a) Ranasinghe, R.T.; Brown, T. Fluorescence based strategies for genetic analysis. Chem. Commun.
2005, 5487–5502. (b) Asseline, U. Development and applications of fluorescent oligonucleotides.
Curr. Org. Chem. 2006, 10, 491–518. (c) Marti, A.A.; Jockusch, S.; Stevens, N.; Ju, J.; Turro, N.J.
Fluorescent hybridization probes for sensitive and selective DNA and RNA detection. Acc. Chem.
Res. 2007, 40, 402–409. (d) Bardaro, M.F. Jr.; Varani, G. Examining the relationship between RNA
function and motion using nuclear magnetic resonance. WIREs RNA, 2012, 3, 122–132. (e) Nguyen,
P.; Qin, P.Z. RNA dynamics: perspectives from spin labels. WIREs RNA, 2012, 3, 62–72.
3. (a) Holbrook, S.R. Structural principals from large RNAs. Ann. Rev. Biophys. 2008, 37, 445–464. (b)
Serganov, A.; Patel, D.J. Molecular recognition and function of riboswitches. Curr. Opin. Struct. Biol.
2012, 22, 279–286.

Synthesis and Substrate Evaluation
875
4. (a) Ennifar, E.; Carpentier, P.; Ferrer, J.-L.; Walter, P.; Dumas, P. X-ray-induced debromination of
nucleic acids at the Br K absorption edge and implications for MAD phasing. Acta Crystallogr. 2002,
D58, 1262–1268. (b) Dibrov, S.; Mclean, J.; Hermann, T. Structure of an RNA dimer of a regulatory
element from human thymidylate synthase mRNA. Acta Crystallogr. 2011, D67, 97–104.
5. (a) Egli, M.; Pallan, P.S. Insights from crystallographic studies into the structural and pairing prop-
erties of nucleic acid analogs and chemically modified DNA and RNA oligonucleotides. Ann. Rev.
Biophys. Biomol. Struct. 2007, 36, 281–305. (b) Sheng, J.; Huang, Z. Selenium derivatization of nucleic
acids for X-ray crystal-structure and function studies. Chem. Biodivers. 2010, 7, 753–785.
6. (a) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C.J.; Egli, M.; Huang, Z. Internal derivatization of
oligonucleotides with selenium for X-ray crystallography using MAD. J. Am. Chem. Soc. 2002, 124,
24–25. (b) Wilds, C.J.; Pattenayek, R.; Pan, C.; Wawrzak, Z.; Egli, M. Selenium-assisted nucleic acid
crystallography: use of phosphoroselenoates for MAD phasing of a DNA structure. J. Am. Chem. Soc.
2002, 124, 14910–14916. (c) Teplova, M.; Wilds, C.J.; Wawrzak, Z.; Tereshko, V.; Du, Q.; Carrasco,
N.; Huang, Z.; Egli, M. Covalent incorporation of selenium into oligonucleotides for X-ray crystal
structure determination via MAD: Proof of principle. Biochimie 2002, 84, 849–858.
7. (a) Salon, J.; Gan, J.; Abdur, R.; Liu, H.; Huang, Z. Synthesis of 6-Se-guanosine RNAs for structural
study. Org. Lett. 2013, 15, 3934–3937. (b) Hassan, A.E.A.; Sheng, J.; Jiang, J.; Zhang, W.; Huang,
Z. Synthesis and crystallographic analysis of 5-Se-thymidine DNAs. Org. Lett. 2009, 11, 2503–2506.
(c) Sheng, J.; Jiang, J.; Salon, J.; Huang, Z. Synthesis of a 2ꢁ-Se-thymidine phosphoramidite and its
incorporation into oligonucleotides for crystal structure study. Org. Lett. 2007, 9, 749–752. (d) Salon,
J.; Chen, G.; Portilla, Y.; Germann, M.W.; Huang, Z. Synthesis of a 2’-Se-uridine phosphoramidite
and its incorporation into oligonucleotides for structural study. Org. Lett. 2005, 7, 5645–5648. (e)
Buzin, Y.; Carrasco, N.; Huang, Z. Synthesis of selenium-derivatized cytidine and oligonucleotides
for X-ray crystallography using MAD. Org. Lett. 2004, 6, 1099–1102.
8. (a) Serganov, A.; Keiper, S.; Malinina, L.; Tereshko, V.; Skripkin, E.; Hobartner, C.; Polonskaia, A.;
Phan, A.T.; Wombacher, R.; Micura, R.; Dauter, Z.; Jaschke, A.; Patel, D.J. Structural basis for Diels-
Alder ribozyme-catalyzed carbon-carbon bond formation. Nat. Struct. Mol. Biol. 2005, 12, 218–224.
(b) Freisz, S.; Lang, K.; Micura, R.; Dumas, P.; Ennifar, E. Binding of aminoglycoside antibiotics to
the duplex form of the HIV-1 genomic RNA dimerization initiation site. Angew. Chem. Int. Ed. 2008,
47, 4110–4113. (c) Salon, J.; Jiang, J.; Sheng, J.; Gerlits, O.O.; Haung, Z. Derivatization of DNAs with
selenium at 6-position of guanine for function and crystal structure studies. Nucleic Acids Res. 2008,
36, 7009–7018. (d) Olieric, V.; Rieder, U.; Lang, K.; Serganov, A.; Schulze-Briese, C.; Micura, R.;
Dumas, P.; Ennifar, E. A fast selenium derivatization strategy for crystallization and phasing of RNA
structures. RNA 2009, 15, 707–715. (e) Sun, H.; Sheng, J.; Hassan, A.E.A.; Jiang, S.; Gan, J.; Huang,
Z. Novel RNA base pair with higher specificity using single selenium atom. Nucleic Acids Res. 2012,
40, 5171–5179.
9. Pawar, M.G.; Nuthanakanti, A.; Srivatsan, S.G. Heavy atom containing fluorescent ribonucleoside
analog probe for the fluorescence detection of RNA-ligand binding. Bioconjugate Chem. 2013, 24,
1367–1377.
10. Vaghefi, M. Nucleoside Triphosphates and their Analogues; CRC Press, Taylor and Francis Droup, Boca
Raton, 2005.
11. (a) Brown, P.O.; Botstein, D. Exploring the new world of the genome with DNA microarrays. Nature
Genet. 1999, 21, 33–37. (b) Lockhart, D.J.; Winzeler, E.A. Genomics, gene expression and DNA arrays.
Nature 2000, 405, 827–836.
12. (a) Srivatsan, S.G.; Tor, Y. Fluorescent pyrimidine ribonucleotide: synthesis, enzymatic incorpora-
tion, and utilization. J. Am. Chem. Soc. 2007, 129, 2044–2053. (b) Greco, N.J.; Tor, Y. Simple fluo-
rescent pyrimidine analogues detect the presence of DNA abasic sites. J. Am. Chem. Soc. 2007, 127,
10784–10785.
13. (a) Zhang, W.; Huang, Z. Synthesis of the 5ꢁ-Se-thymidine phosphoramidite and convenient labeling
of DNA oligonucleotide. Org. Lett. 2011, 13, 2000–2003. (b) Sun, H.; Jiang, S.; Caton-Williams, J.;
Liu, H.; Huang, Z. 2-Selenouridine triphosphate synthesis and Se-RNA transcription. RNA 2013, 19,
1309–1314.
14. (a) Kore, A.R.; Yang, B.; Srinivasan, B. Ionic-tag-assisted synthesis of nucleoside triphosphates. Tetrahe-
dron Lett. 2014, 55, 5088–5091. (b) Kore, A.R.; Yang, B.; Srinivasan, B. Efficient synthesis of terminal
4-methylumbelliferyl labeled 5-fluoro-2ꢁ-deoxyuridine-5ꢁ-O-tetraphosphate (Um-PPPP-FdU): a po-
tential probe for homogenous fluorescent assay. Tetrahedron Lett. 2014, 55, 4822–4825. (c) Kore, A.R.;

876
A. R. Kore et al.
Yang, B.; Srinivasan, B. Concise and efficient synthesis of 3ꢁ-O-triphosphates of 2ꢁ-deoxyadenosine and
2ꢁ-deoxycytidine. Tetrahedron Lett. 2014, 55, 1573–1576. (d) Kore, A.R.; Yang, B.; Srinivasan, B. Concise
synthesis of 5-methyl-, 5-formyl, and 5-carboxy analogues of 2ꢁ-deoxycytidine-5ꢁ-triphosphate. Tetra-
hedron Lett. 2013, 54, 5325–5327. (e) Kore, A.R.; Yang, B.; Srinivasan, B. Fluorous-assisted synthesis
of (E)-5-[3-Aminoallyl]-uridine-5ꢁ-triphosphate. Tetrahedron Lett. 2013, 54, 6264–6266. (f) Kore, A.R.;
Senthilvelan, A.; Srinivasan, B.; Shanmugasundaram, M. Facile protection-free one-pot synthesis of
7-deaza-2ꢁ-deoxyguanosine-5ꢁ-triphosphate – a versatile molecular biology probe. Can. J. Chem. 2013,
91, 718–720. (g) Kore, A.R.; Srinivasan, B. Recent advances in the syntheses of nucleoside triphos-
phates. Curr. Org. Synth. 2013, 10, 903–934. (h) Kore, A.R.; Senthilvelan, A.; Srinivasan, B.; Shanmu-
gasundaram, M. Facile protection-free one-pot chemical synthesis of nucleoside-5’-tetraphosphates.
Nucleosides Nucleotides Nucleic Acids 2013, 32, 411–420. (i) Kore, A.R.; Srinivasan, B. Efficient syn-
thesis of 3-cyanovinylcarbazole-1ꢁ-β-deoxyriboside-5ꢁ-triphosphate: a reversible photo-cross-linking
probe. Tetrahedron Lett., 2012, 53, 4012–4014. (j) Kore, A.R.; Senthilvelan, A.; Shanmugasundaram, M.
Highly chemoselective palladium-catalyzed Sonogashira coupling of 5-iodouridine-5ꢁ-triphosphates
with propargylamine: a new efficient method for the synthesis of 5-aminopropargyl-uridine-5ꢁ-
triphosphates. Tetrahedron Lett., 2012, 53, 3070–3072. (k) Kore, A.R.; Senthilvelan, A.; Shanmuga-
sundaram, M. A new, facile, and protection-free one-pot chemical synthesis of 2ꢁ-deoxynucleoside-
5ꢁ-tetraphosphates. Tetrahedron Lett., 2012, 53, 5868–5870.
15. Noguchi, T.; Hasegawa, M.; Tomisawa, K.; Mitsukuchi, M. Synthesis and structure–activity relation-
ships of 5-phenylthiophenecarboxylic acid derivatives as antirheumatic agents. Bioorg. Med. Chem.
2003, 11, 4729–4742.
16. (a) Langer, P.R.; Waldrop, A.A.; Ward, D.C. Enzymatic synthesis of biotin-labeled polynucleotides:
novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 6633–6637. (b) Schoetzau,
T.; Langner, J.; Moyroud, E.; Roehl, I.; Vonhoff, S.; Klussmann, S. Aminomodified nucleobases:
functionalized nucleoside triphosphates applicable for SELEX. Bioconjug. Chem. 2003, 14, 919–926.
17. Kore, A.R.; Shanmugasundaram, M. Highly stereoselective palladium-catalyzed Heck coupling of
5-iodouridine-5ꢁ-triphosphates with allylamine: a new efficient method for the synthesis of (E)-5-
aminoallyl-uridine-5ꢁ-triphosphates. Tetrahedron Lett., 2012, 53, 2530–2532.
18. Kore, A.R.; Senthilvelan, A.; Shanmugasundaram, M.; Sandoval, D.; and Pardo, A. A new effi-
cient stereoselective method for the synthesis of (E)-5-aminoallyl-pyrimidine-5-triphosphates using
palladium-catalyzed Heck reaction. Nucleosides, Nucleotides and Nucleic Acids 2015, 34, 221–228.
19. Dadova, J.; Vidlakova, P.; Pohl, R.; Havran, L.; Fojta, M.; and Hocek, M. Aqueous Heck cross-coupling
preparation of acrylate-modified nucleotides and nucleoside triphosphates for polymerase synthesis
of acrylate-labeled DNA. J. Org. Chem. 2013, 78, 9627–9637.
20. Vaish, N.K.; Fraley, A.W.; Szostak, J.W.; McLaughlin, L.W. Expanding the structural and functional
diversity of RNA: analog uridine triphosphates as candidates for in vitro selection of nucleic acids.
Nucleic Acid Res. 2000, 28, 3316–3322.
21. Pawar, M.G.; Srivatsan, S.G. Photophysical Characterization, and Enzymatic Incorporation of a
Microenvironment-Sensitive Fluorescent Uridine Analog. Org. Lett. 2011, 13, 1114–1117.
22. Analysis of Cy5-labeled cRNAs and cDNAs using the Agilent 2100 Bioanalyzer and the RNA 6000
ꢀR
LabChip kit.
23. Sakamoto, T.; Kondo, Y.; Uchiyama, M.; Yamanaka, H. Concise synthesis of CC-1065/duocarmycin
pharmacophore using the intramolecular Heck reaction. J. Chem. Soc., Perkin Trans. 1993, 1941–1942.