Synthesis of a protected ribonucleoside phosphoramidite-linked spin label via an alkynyl chain at the 5′ position of uridine

Article abstract of DOI:10.1080/00397911.2018.1545033

New, spin-labeled nucleosides, and an efficient synthetic route for a modified uridine amidite, were developed. The spin-labeled part was the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) group, which was linked via an alkynyl chain at the 5 position of uridine. Three typical protecting groups, the t-butyldimethylsilyl (TBDMS) group at 2′, the dimethoxytrityl (DMTr) group at 5′, and the phosphoramidite group at 3′, were introduced to produce an automated nucleic acid synthesizer. The TEMPO group at the 5 position in the uridine structure affected introduction of bulky protecting groups, such as the DMTr group at the 5′ position and the TBDMS group at the 2′ position. The electron paramagnetic resonance (EPR) data revealed a nitroxyl radical in the structure of synthetic nucleoside compounds; however, RNA produced by automated synthesis using a TEMPO-linked uridine phosphoramidite building block was EPR silent.

Full text of DOI:10.1080/00397911.2018.1545033

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Synthesis of a protected ribonucleoside
phosphoramidite-linked spin label via an alkynyl
chain at the 5′ position of uridine
Akihiko Hatano, Nanae Terado, Yuichi Kanno, Toshikazu Nakamura & Gota
Kawai
To cite this article: Akihiko Hatano, Nanae Terado, Yuichi Kanno, Toshikazu Nakamura
& Gota Kawai (2019): Synthesis of a protected ribonucleoside phosphoramidite-linked spin
label via an alkynyl chain at the 5′ position of uridine, Synthetic Communications, DOI:
10.1080/00397911.2018.1545033
To link to this article: https://doi.org/10.1080/00397911.2018.1545033
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https://doi.org/10.1080/00397911.2018.1545033
Synthesis of a protected ribonucleoside phosphoramidite-
linked spin label via an alkynyl chain at the 50 position
of uridine
Akihiko Hatanoa, Nanae Teradoa, Yuichi Kannoa, Toshikazu Nakamurab, and
Gota Kawaic
aDepartment of Chemistry, Faculty of Engineering, Shibaura Institute of Technology, Saitama, Japan;
bDepartment of Materials Molecular Science, Institute for Molecular Science, Okazaki, Japan;
cDepartment of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology,
Chiba, Japan
ABSTRACT
ARTICLE HISTORY
Received 17 September 2018
Accepted 1 November 2018
New, spin-labeled nucleosides, and an efficient synthetic route for a
modified uridine amidite, were developed. The spin-labeled part was
the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) group, which was
linked via an alkynyl chain at the 5 position of uridine. Three typical
protecting groups, the t-butyldimethylsilyl (TBDMS) group at 20, the
dimethoxytrityl (DMTr) group at 50, and the phosphoramidite group
at 30, were introduced to produce an automated nucleic acid synthe-
sizer. The TEMPO group at the 5 position in the uridine structure
affected introduction of bulky protecting groups, such as the DMTr
group at the 50 position and the TBDMS group at the 20 position.
The electron paramagnetic resonance (EPR) data revealed a nitroxyl
radical in the structure of synthetic nucleoside compounds; however,
RNA produced by automated synthesis using a TEMPO-linked uridine
phosphoramidite building block was EPR silent.
KEYWORDS
Electron paramagnetic
resonance (EPR); RNA; spin
label; 2,2,6,6-
tetramethylpiperidine-1-oxyl
(TEMPO); unnat-
ural nucleoside
GRAPHICAL ABSTRACT
Introduction
Magnetic resonance techniques are standard tools for analyzing biomolecules’ structure
and function.[1] Spin labels have attracted widespread attention as probes for examining
biomolecules’ structure, dynamics, and complexes by nuclear magnetic resonance
(NMR)[2,3] and electron paramagnetic resonance (EPR).[4–7] Sigurdsson et al. reported a
CONTACT Akihiko Hatano
a-hatano@sic.shibaura-it.ac.jp
Department of Chemistry, Faculty of Engineering,
Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama, 337-8570, Japan.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2019 Taylor & Francis Group, LLC

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A. HATANO ET AL.
rigid spin label for nucleosides.[8–13] The flexibility of the spin-labeled part in nucleoti-
des decreases EPR measurements’ accuracy.[5,14] These labeled molecules contain a
nitroxyl-free radical fused to the nucleobase. The labeled nucleosides can be immobi-
lized in a nucleic acid duplex by forming a base pair derived from hydrogen bonding
and base stacking.[15–20]
Paramagnetic relaxation enhancement (PRE) NMR has recently proven to be an
extremely powerful tool for determining solution structure.[3,4,21–23] PRE is caused by
magnetic dipolar interactions between a nucleus and a paramagnetic center’s unpaired
electrons, which increase the nuclear magnetization relaxation rate.[24] Spin-labeled radi-
cals are used to determine long-range distance restraints for the solution structure of
RNA domains and the intermolecular dynamics and interactions between nucleic acids
and proteins.[25–27] PRE NMR has been used to reveal the ultra-weak interaction
between nonspecific protein and DNA. This analysis involves preparing an EDTA-
derived deoxythymidine to incorporate into oligo-DNA sequences using automated syn-
thesis methods at the target position. The EDTA part of deoxythymidine can bind to a
Mn2þ ion to afford a stable, chelated complex in solution, and Mn2þ has an unpaired
electron. Previous studies have incorporated modified DNA into this dT–EDTA, yield-
ing an EDTA–Mn2þ complex to examine DNA–protein interactions by PRE. There is
no need to prepare (or synthesize) modified nucleosides that can tightly bind to the
unpaired electron in the molecular structure for PRE NMR measurements.
A uridine derivative, which was linked to a nitroxyl-free radical via a flexible alkynyl
chain at the 5 position of the nucleobase uracil, was synthesized. A Pd-catalyzed
Sonogashira coupling was used to introduce 2,2,6,6-tetramethylpiperidine-1-oxyl
(TEMPO) into a 5-iodouridine derivative easily and efficiently (Figure 1). To introduce
a spin-labeled nucleoside into the sequence for using a nucleic acid synthesizer, three
OH groups of the spin-labeled nucleoside needed to be substituted using specific pro-
tecting and functional groups. Bulky dimethoxytrityl (DMTr) and t-butyldimethylsilyl
(TBDMS) groups protect the ribosyl hydroxyl groups at the 50 and 20 positions, respect-
ively. The spin-labeled nucleoside, linking the nitroxyl radical via the alkynyl chain at
the 5 position of uridine, was then designed. Since this spin-labeled group was con-
nected with uridine at the 5 position of the base via the alkenyl linker, we expected this
synthesized nucleoside to enable base-pairing via hydrogen bonds with A and base-
stacking in the RNA duplex. We expected the 5 position of the nucleobase uracil part
to be located toward the outside of the RNA duplex through the major groove.
Therefore, this uridine derivative would not hinder the change in the RNA structure.[28]
And the linker connecting the nucleobase with the radical moiety need not be a rigid
bond for PRE NMR. The introduced DMTr and TBDMS to the ribosyl hydroxyl groups
might be affected by the steric hindrance of 5-position-modified uridine, which is linked
by the TEMPO spin label.
Results and discussion
Scheme 1 shows a synthetic route to the TEMPO-linked nucleoside. First (route 1), a
Pd-catalyzed Sonogashira coupling between 5-iodouridine 1 and (TEMPO-4-oxy)-5-hex-
yne 3 afforded reaction product 2 in moderately high yield (71%). Additionally, 20-

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Figure 1. Synthetic route of spin-labeled nucleoside.
deoxyuridine-linked TEMPO at the 5 position was synthesized starting from 20-deoxy-5-
iodouridine using the same coupling method (15, 68%). To produce the standard DNA/
RNA automated synthesizer, 50-O-dimethoxytritylation, 20-O-t-butyl dimethylsilylation,
and 30-O-phosphoramidation of the three OH groups of TEMPO-linked uridine 2 were
performed. To achieve site-selective protection of the 20OH of ribose by TBDMS,
nucleoside 2 was treated with 1.1 equiv. of di-t-butylsilyl bis(trifluoromethanesulfonate)
in DMF at 0 ꢀC, yielding 30,50-O-silyl-protected intermediate 4. Site-specific silylation of
the 20OH group of the intermediate 4 was performed using TBDMSCl.[29] The di-t-
butylsilyl protecting group of ribose was removed using HF/pyridine in CH2Cl2 at 0 ꢀC,
affording a product yield reaching 95%. Slightly undesirable migration of the silyl group
from the 20-isomer 6 to the 30-isomer was observed. It was difficult to introduce the
DMTr group at the 50OH (7, 8.6%) position of the 20-TBDMS-protected nucleoside-
linked TEMPO 6 because TEMPO is a large, flexible group, and the nucleophilicity of

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A. HATANO ET AL.
Scheme 1. Synthetic route to a protected phosphoramidite nucleoside-linked TEMPO. (a) (1-Oxyl-
2,2,6,6-tetramethylpiperidine-4-oxy)-5-hexyne (3), Pd(PPh3)4, CuI, DIPEA in DMF at rt, (b) di-t-butylsilyl
bis(trifluoromethanesulfonate) in DMF at rt, (c) TBDMSCl and imidazole in DMF at rt, (d) HF in pyri-
dine/CH2Cl2 at 0 ꢀC, (e) DMTrCl in pyridine at rt, and (f) 2-cyanoethyldiisopropylamino chlorophosphor-
amidite and DIPEA in CH2Cl2 at rt.
the 50OH of the nucleoside was decreased due to the steric hindrance of TEMPO and
the large modified nucleoside’s reduced mobility.
The synthetic route to target-protected TEMPO nucleoside phosphoramidite 14 was
changed, allowing chemical RNA synthesis on DNA/RNA automated synthesizers. In

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Scheme 2. Synthetic route to the deoxyuridine linked TEMPO (16) and uracil linked TEMPO (21). (a)
Each alkyne unit, Pd(PPh3)4, CuI, DIPEA in DMF at rt, (b) TBAF, in THF at rt, (c) TsCl in pyridine at rt,
(d) 4-hydroxy-TEMPO free radical, t-BuOK in DMF at rt.
route 2, shown in Scheme 1, the 50OH group of 1 was protected using DMTrCl (8,
60%) before introducing reactant 8 into the 20 position of the TBDMS silyl ether using
TBDMSCl and imidazole, affording moderate product yields (20OTBDMS: 44% and
30OTMDMS: 19%, 20:30 = 2.3:1.0). Previous studies reported the ribonucleoside’s site-
selective protection by silylation using TBDMS.[30–32] The TBDMS group was more
favorably introduced at the 20OH ribosyl form compared to the 30OH form. Compound
9 was expected to be coupled with 3 at the 5 position by Pd catalysis, such as
Sonogashira coupling, without steric hindrance. This coupling reaction led to 7 in good
yield (from 9 to 7, 92%). The total yield of this route was satisfactory (24%), requiring
three simple steps to achieve 7 synthesis. Synthesis of 20OTBDMS 9 from 8 might
increase. The low steric hindrance effect was observed during the introduction of all the
necessary groups. For efficient synthesis, after tritylation of 1 using the DMTr group,
produced compound 8 could be introduced into the TEMPO part by Pd catalysis, such
as Sonogashira coupling, leading to a good product yield (71%, route 2, bottom). The 20
position of compound 10 was protected using TBDMS, leading to a low product yield
of 22%. The total yield of this route was 9.4%, starting from compound 1 to 7 via 10,
and the total yield of this route 2 (bottom) via 10 is lower than route 2 (upper) via
compound 9.
The highest total yield was observed via route 3: from 1 to 7, the final product was
obtained in 28% yield. However, the synthesis via route 3 comprised five steps, and
deprotection of the bis(t-butyl) group required the use of toxic HF. The subsequent 30-
phosphitylation of 7 in the presence of 2-cyanoethyl-N,N0-diisopropyl chlorophosphora-
midite and diisopropylethylamine afforded 14 (63%), which was used to incorporate the
TEMPO nucleoside into RNA oligonucleotides via solid-phase automated synthesis.

6
A. HATANO ET AL.
Figure 2. CW electron paramagnetic resonance (EPR) spectra of ribouridine (2), deoxyuridine (16),
and uracil (21), where each compound is linked to a spin label via an alkyl chain in MeOH at 294 K.
Figure 2 shows continuous-wave (CW) EPR spectra of the spin-labeled nucleosides
and their base. Three equally typical signals, derived from the nitroxyl-free radicals,
were observed for all compounds. Compounds 2, 16, and 21 contain nucleobase-linked
TEMPO at the 5 position via the alkynyl chain (Scheme 2). Compound 2 should be a
powerful tool for determining structures in solution, i.e., RNA and RNA–protein com-
plexes, by PRE NMR.
Nitroxyl radicals are moderately sensitive under the oxidation and detritylation condi-
tions employed in standard solid-phase synthesis.[6,10] Oligonucleotides synthesized via
an automated synthesis were EPR silent, and the LC-ESI mass spectral analysis of the
ꢁ
rU7rU rU7 oligonucleotide revealed that synthetic oligonucleotides exhibit a mixture of
five peaks (rU shows TEMPO-linked uridine 2; see section 4 of Supporting
ꢁ
Information). Peak A’s first retention time corresponded to the end-capped rU7–OAc
oligomer, which did not combine with the completely protected uridine linked by
alkynyl TEMPO 14. Peak A’s spectrum gave a molecular ion peak of 2081.30 by LC-ESI
mass analysis, and this molecular weight (MW) completely fitted the MW of rU7–OAc.
ꢁ
Most of the peak corresponding to synthesized rU7rU rU7 exhibited a MW of 15, which
was lower than the value expected for the spin-labeled 15 mer of rU (peaks B and D

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are shown in the LC-ESI mass data in Figure S4 in the Supplementary Information). A
ꢁ
small LC peak, corresponding to rU7rU rU7’s expected MW when incorporating an
unnatural nucleoside with a spin label at the central position, was observed, similar to
peaks C and E. These results suggested that nitroxyl radicals N-Oꢂ are lost in most syn-
thetic oligonucleotides and converted to amino groups as NH during the detritylation
using TFA of the automated synthesizer.[6,11,33,34]
ꢁ
To incorporate rU (compound 2) into the RNA sequence with the automated syn-
thesizer, we tested the stability of the nitroxyl radicals of compound 2 in the presence
of acid for deprotection (by trifluoroacetic acid, TFA) and oxidation reagents for phos-
phorus (by iodine, I2).[9] After incubating compound 2 in iodine and methyl ethyl
ketone peroxide oxidation solutions for 24 h, nitroxyl radicals were not reduced to the
NH form by these reagents (91% of compound 2 remained with iodine and 100% with
methyl ethyl ketone peroxide, see section 5 of Supporting Information). We also tested
the stability of compound 2 in 3% TFA and dichloroacetic acid (DCA) to remove the
DMTr group during oligomer synthesis. When using TFA, 28% of compound 2 was
reduced to the corresponding amine after 1 h, and compound 2 was reduced to 38%
NH form after 24 h. We tested the weaker acid, DCA. No decomposition of compound
ꢁ
2 was observed upon incubation in DCA solution, even after 24 h. The rU was incor-
porated into 34 mer RNA by the automated synthesizer using optimized reaction condi-
tions. However, the ESI-MS analysis of this RNA showed that almost all the synthesized
RNA’s nitroxyl radicals were reduced to the undesired RNA amine.
Conclusions
In conclusion, a novel, nitroxyl-free, radical-labeled uridine was synthesized at the 5
position as a spin probe. Compound 2 had a flexible TEMPO spin label, which was
linked via the alkynyl chain at the 5 position of uridine. To synthesize an oligonucleo-
tide-containing spin label, we attempted to introduce three typical protecting groups,
i.e., a TBDMS group at 20, a DMTr group at 50, and a phosphoramidite group at 30, in
5-iodouridine or 5-[(1-oxyl-2,2,6,6-tetramethylpiperidine-4-oxy)-5-hexyne-6-yl] uridine
(2). The large spin-labeled group at the 5 position of uridine inhibited introducing pro-
tecting groups at the 20 and 50 positions (Figure 1, route 1). To introduce TEMPO after
protecting ribosyl OH at 20 and 50, the total yields obtained from routes 2 and 3 were
greater than that obtained from route 1. Most of the short synthetic method was route
2; however, the most effective method was route 3, comprising five steps. The EPR data
revealed a nitroxyl radical in these synthetic compounds’ structures. However, the RNA
ꢁ
rU7rU rU7 oligomer was EPR silent. Furthermore, the LC-ESI mass data revealed that
ꢁ
the MW of most of the synthetic rU7rU rU7 containing spin-labeled uridine was 15,
ꢁ
which is lower than the expected value for the spin-labeled rU7rU rU7. These results
revealed that the radical in the oligomer is quenched via the automated synthetic cycle
of oligonucleotides.

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A. HATANO ET AL.
Experimental
All reactions were performed under an Ar atmosphere. Reagent-grade solvents and
reagents were used without further purification, and the yields of isolated compounds
were reported. TLC analysis was performed on silica gel 60 F254 1.05554 (Merck).
Column chromatography was performed using silica gel 60 N (Kanto Chemical Co.,
1
Inc.). The H, 13C, and COSY NMR spectra were recorded on a JEOL ECS 400 spec-
1
trometer (400.0 MHz for H and 100.4 MHz for 13C). The spectra were referenced to
TMS in chloroform-d, CD3OD-d4, and DMSO-d6. The chemical shifts d were recorded
in ppm, and the multiplicity is indicated as follows: singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), and broad (br). The coupling constant J is reported in Hz.
NMR signals were assigned based on 2D NMR (HH-COSY and HMQC) experiments.
Signals corresponding to the TEMPO part for compounds 2, 3, 4, 5, 6, 7, 10, 14, 16,
1
and 21 were not completely observed via H NMR, and the other signals became broad.
MS of small organic compounds was performed on an AccuTOF TLC JMS-T200TD
(JEOL) instrument. The ESI-LC mass spectra of RNAs were recorded on an AXIMA
Assurance MS system (Shimadzu).
General procedure for the synthesis of 5-[(1-oxyl-2,2,6,6-tetramethylpiperidine-4-
oxy)-5-hexyne-6-yl]-20-O-t-butyldimethylsilyl-50-O-(4,40-dimethoxytrityl) uridine (7)
Synthesis of compound 7 by route 2 (upper) (from compounds 9 to 7): (PPh3)4Pd
(44 mg, 0.038 mmol), CuI (14 mg, 0.074 mmol), and diisopropylethylamine (147 mg,
196 mL, 1.14 mmol) were added to a solution of compound 9 (298 mg, 0.379 mmol) in
3.5 mL of DMF under an Ar atmosphere. The mixture was stirred for 5 min at room
temperature. TEMPO-1-oxy-5-hexyne 3 (119 g, 0.474 mmol) was added to this solution.
The reaction mixture was stirred at room temperature for 1 h and monitored via TLC.
Most of the DMF evaporated under reduced pressure, and the residue was purified by
silica-gel column chromatography (hexane:AcOEt =1:1, plus 1% triethylamine), yielding
target compound 7 as an orange oil (316 mg, 92%).
1H NMR (400 MHz, CDCl3) d: 8.11 (br), 7.82 (br), 7.35 (br), 7.20 (br), 6.85 (br), 6.01
(br), 4.50 (br), 4.25 (br), 4.17 (br), 4.13(br), 3.80 (br), 3.47 (br), 3.36 (br), 2.67 (br), 2.05
(br), 1.62 (br), 1.26 (br), 1.08 (br), 0.12 (s, 9H), and 0.13 (s, 6H).
13C NMR (100 MHz, CDCl3) d: 161.4, 158.8, 149.4, 144.5, 141.7, 135.3, 130.2, 128.3,
127.0, 127.2, 117.8, 113.6, 101.6, 95.1, 87.6, 87.4, 84.2, 76.3, 72.8, 70.8, 63.6, 59.3, 55.6,
43.1, 28.9, 25.9, 25.5, 24.8, 20.8, 19.3, 18.2, 4.45.
HRMS (ESI) m/z [M þ Na]þ calcd. for C51H68N3NaO10Si: 933.4572, found 933.4500.
Funding
This work was supported by JSPS KAKENHI Grants-in-Aid for Scientific Research [Grant
Number 18K05360].

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