Synthesis and Multiple Incorporations of 2′-O-Methyl-5-hydroxymethylcytidine, 5-Hydroxymethylcytidine and 5-Formylcytidine Monomers into RNA Oligonucleotides

Article abstract of DOI:10.1002/cbic.201700492

The synthesis of 2′-O-methyl-5-hydroxymethylcytidine (hm⁵Cm), 5-hydroxymethylcytidine (hm⁵C) and 5-formylcytidine (f⁵C) phosphoramidite monomers has been developed. Optimisation of mild post-synthetic deprotection conditions enabled the synthesis of RNA containing all four naturally occurring cytosine modifications (hm⁵Cm, hm⁵C, f⁵C plus 5-methylcytosine). Given the considerable interest in RNA modifications and epitranscriptomics, the availability of synthetic monomers and RNAs containing these modifications will be valuable for elucidating their biological function(s).

Full text of DOI:10.1002/cbic.201700492

Accepted Article
Title: Synthesis and multiple incorporations of 2′-O-methyl-5-
hydroxymethyl-, 5-hydroxymethyl- and 5-formylcytidine
monomers into RNA oligonucleotides
Authors: Shankar Balasubramanian and Arun Ankush Tanpure
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201700492
Link to VoR: http://dx.doi.org/10.1002/cbic.201700492
A Journal of
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10.1002/cbic.201700492
ChemBioChem
COMMUNICATION
Synthesis and multiple incorporations of 2ʹ-O-methyl-5-
hydroxymethyl-, 5-hydroxymethyl- and 5-formylcytidine
monomers into RNA oligonucleotides
Arun A. Tanpure and Shankar Balasubramanian*
Abstract: The synthesis of 2ʹ-O-methyl-5-hydroxymethylcytidine
(hm5Cm), 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C)
phosphoramidite monomers has been developed. Optimization of
mild post-synthetic deprotection conditions enabled the synthesis of
RNA containing all four naturally occurring cytosine-modifications
(hm5Cm, hm5C, f5C plus 5-methylcytosine). Given the considerable
interest in RNA modifications and epitranscriptomics, the availability
of synthetic monomers and RNAs containing these modifications will
be valuable to elucidate their biological function(s).
translational initiation and elongation sites of mRNA.10 Chemical
labeling coupled with liquid chromatography-mass spectrometry
(LC-MS) analysis revealed the existence of 5-carboxycytidine
(ca5C in mouse liver tissue, albeit with
a
very low
concentration.11 Recently, we discovered the existence of 2ʹ-O-
methyl-5-hydroxymethylcytidine (hm5Cm), a second methylated
metabolite of m5C, in RNA of higher organisms (Figure 1).12
Formation of hm5Cm from m5C by stepwise oxidation via hm5C
as an intermediate supports the dynamic nature and complexity
of these cytosine-modifications in RNA.12-13 An efficient synthesis
of oligonucleotides (ONs) containing these modifications is
essential to elucidate the chemistry and function of RNA
cytosine derivatives. For example, site-specific incorporation of
these cytosine-modifications in RNA will enable the development
of sequencing methods to decode the modification14 and also
help identify the reader proteins to understand the cellular
functions of these modifications.8
Post-transcriptional chemical modifications in RNA are more
diverse and complex than epigenetic modifications in DNA and
in histones. So far more than 140 chemically distinct RNA
modifications have been identified in various species. The
majority of these modifications involve methylation, of which 2ʹ
ribose sugar methylation is the most abundant.1 These
modifications were initially considered as static and stable marks,
however, recent studies have revealed their dynamic nature and
involvement in important gene regulatory functions.2 For
instance, N6-methyladenosine (m6A) a predominant internal
modification in eukaryotic messenger RNA (mRNA) can be
oxidatively converted to adenosine by demethylase such as fat
mass and obesity-associated protein (FTO) and alkB homologue
5 (ALKBH5).3 This reversible adenosine methylation is proposed
to be involved in RNA maturation, protein translation and gene
expression.4 5-Methylcytidine (m5C), is another important
methylated ribonucleoside that exists in transfer RNA (tRNAs),
ribosomal RNA (rRNAs), and in the untranslated regions of
mRNAs.2,5 We and others have demonstrated that m5C can be
oxidatively metabolised to produce 5-hydroxymethylcytidine
(hm5C) and 5-formylcytidine (f5C) (Figure 1).6,7 Additionally, it is
proposed that m5C is essential for mRNA export and post-
transcriptional regulation.8 Significant enrichment of hm5C in
polyadenylated RNA compared to total RNA further suggests
that biosynthesis of hm5C might be a part of a dynamic
regulatory mechanism of RNA.6b,9 The f5C modification is
prevalent at the wobble position of an anticodon loop of
mitochondrial methionine tRNA in many species including
human. It was observed that f5C provides flexibility to the loop of
tRNA and affords an ability to decode both AUG and AUA in
Figure 1. A) Chemical structure of major cytosine-modifications known to exist
in eukaryotic RNA. B) Corresponding phosphoramidite monomers synthesized
in this report.
A phosphoramidite monomer used for the synthesis of m5C
containing RNA ONs is commercially available, while syntheses
of f5C and hm5C monomers have been reported in the
literature.10,15 We set out to develop a synthesis of a hm5Cm
monomer compatible with solid-phase RNA ONs synthesis,
which has not yet been described. In addition, the reported
monomer for the synthesis of f5C containing RNA ONs requires
a multi-step synthesis via 2′-bis(2-acetoxyethoxy)methyl (ACE)
orthoester and 5′-O-benzhydroxy-bis (trimethylsiloxy)silyl (BZH)
protection.10 A drawback of this monomer is the presence of a
free formyl group, which is susceptible to oxidation and
nucleophilic attack encountered during ON assembly and post-
synthetic resin cleavage respectively.10,16 Consequently, multiple
incorporations using the existing f5C phosphoramidite is difficult,
especially in the synthesis of longer oligomers and in the
Dr. A. A. Tanpure, Prof. S. Balasubramanian
Department of Chemistry, University of Cambridge Lensfield Road,
Cambridge, CB2 1EW (UK)
E-mail: sb10031@cam.ac.uk
Prof. S. Balasubramanian
Cancer Research, UK, Cambridge Institute, Li Ka Shing Centre
University of Cambridge Robinson Way, Cambridge, CB2 0RE (UK)
and
School of Clinical Medicine, University of Cambridge Cambridge,
CB2 0SP (UK)
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700492
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combination with other cytosine-modifications such as hm5C and
hm5Cm containing a nucleophilic moiety. To overcome these
practical limitations we designed an alternative f5C monomer in
which the reactive formyl group is appropriately masked. Riml et
al. reported the synthesis and incorporation of hm5C monomer
into RNA ONs, their route provides this monomer in a 3% overall
yield in eight steps from 5-hydroxymethyluridine (hm5U),15 which
itself is obtained in a three step synthesis from uridine and
totalling the number of steps to 11.17 We therefore, considered
the development of a faster and more efficient route to this
monomer at the same time. The presence of the 2ʹ hydroxyl
group, makes the synthesis and incorporation of functionally-
described
for
synthesizing
an
analogous
2'-deoxy
phosphoramidite.21 Fully protected uridine analogue 16 was then
treated with TPS-Cl, followed by ammonolysis to give cytidine
analogue 17. Acetylation of the exocyclic amino group was
carried out using acetic anhydride in pyridine to provide 18.
modified
challenging,
ribonucleoside
compared
phosphoramidites
to the
particularly
analogous DNA
phosphoramidite.10,15,18 Moreover, an orthogonal protection
strategy developed for DNA ON synthesis does not necessarily
work for RNA due to its inherently more labile nature.18a Herein,
we describe the development of phosphoramidite building blocks
of hm5Cm, f5C and hm5C (Figure 1) and demonstrate the
incorporation of all these cytosine-modifications into RNA ONs
on multiple positions and combinations by solid-phase RNA
synthesis.
To synthesize hm5Cm phosphoramidite 1 we decided to
protect the 5-hydroxymethyl with an acetyl group, which is
compatible with the incorporation of hm5C into RNA ONs.15
Starting with commercially available 2'-O-methyl-5-methyluridine
4 we protected the 5ʹ-hydroxyl with a 4,4'-dimethoxytrityl (DMT)
and the 3ʹ-hydroxyl with a tert-butyldimethylsilyl (TBS) group
(scheme 1).19 The DMT and TBS protected uridine 6 was then
subjected to azobisisobutyronitrile (AIBN) catalyzed bromination
of 5-methyl group using N-bromosuccinimide (NBS).20 The crude
bromo-derivative was treated with potassium acetate to yield the
fully protected uridine analogue 7 in moderate yield.19 The
reaction of 7 with 2,4,6-triisopropylbenzenesulfonyl chloride
(TPS-Cl) resulted into regioselective O4-trisylation, which was
readily converted into cytidine analogue 8 upon ammonolysis.
Acetylation of the exocyclic amino group was achieved using
acetic anhydride in pyridine to provide nucleoside 9. Cleavage of
the 3′-OTBS group with TBAF and acetic acid in THF gave
precursor 10. Finally, phosphitylation of the 3′-hydroxyl with 2-
cyanoethyl-N,N-diisopropylchloro-phosphoramidite (CEP-Cl) in
Scheme 1. Synthesis of hm5Cm phosphoramidite 1. (DMT: 4,4′-
dimethoxytrityl;
bromosuccinimide;
TBS-Cl:
AIBN:
tert-butyldimethylsilyl
chloride;
NBS:
N-
2,4,6-
azobisisobutyronitrile;
TPS-Cl:
triisopropylbenzenesulfonyl chloride; DMAP: 4-dimethylaminopyridine; CEP-
Cl: 2-cyanoethyl-N,N-diisopropylchloro-phosphoramidite).19
the
presence
of
N,N-diisopropylethylamine
afforded
phosphoramidite 1 in a 6.7% overall yield in seven steps from 4
(Scheme 1).19
In DNA synthesis, acetal chemistry has been successfully
utilized to protect the formyl group.21 To explore the analogous
protection of f5C for RNA, we synthesized phosphoramidite
building block 2 as depicted in scheme 2.19 Starting with 5-
methyluridine 11, this time we protected the 3'- and 5'-hydroxyl
with the cyclic di-tert-butylsilylene and subsequently 2'-hydroxyl
with TBS group.22 Nucleoside 12 was selectively brominated at
the C5-methyl group followed by acylation yielded uridine-
derivative 13 in good yield. Hydrolysis of 13 provided 5-
hydroxymethyl derivative 14, which was then converted into
aldehyde 15 by Dess–Martin periodinane (DMP) oxidation.23
Next, the formyl group was protected as a 1,3-dioxane using
propane-1,3-diol in presence of TiCl4, following the procedure
Scheme 2. Synthesis of f5C phosphoramidite 2.19
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10.1002/cbic.201700492
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COMMUNICATION
Selective deprotection of 3', 5'-O-di-tert-butylsilylene group was
achieved by treating 18 with hydrogen fluoride in pyridine.
Finally, 5'-OH DMT protection and subsequent phosphitylation
yielded target monomer 2. Starting from 5-methyluridine and
following this ten step route we obtained monomer 2 in a good
10.5 % overall yield (Scheme 2).19 Compared to the previously
reported synthesis for an f5C phosphoramidite, we have
significantly improved the overall yield and more importantly, we
have now deployed a formyl protective group.10 We anticipated
that this fully protected monomer would allow us to incorporate
f5C at multiple positions in longer ONs and also in the
combination with the other cytosine-modifications m5C, hm5C
and hm5Cm.
CPG solid support tethered to TEG-Biotin. Modified
phosphoramidites were incorporated into RNA ONs following a
standard solid-phase RNA ON synthesis protocol. Incorporations
of modified phosphoramidite substrates 1−3 were performed
with a coupling time of 10 min, with 80−90% coupling efficiency,
based on a trityl release assay. Oligonucleotides were cleaved
from the solid support with 20 % Ethanol in NH4OH solution. 2'-
O-TBS deprotection was performed by 1:1 mixture of anhydrous
DMSO and triethylamine trihydrofluoride. RNA ONs 1 and 3
were purified by HPLC at this stage, while RNA ONs 2 and 4
were each subjected to deprotection of the acetal moiety to
reveal the formyl group.19 Conditions previously used for the
acetal deprotection of fdC in DNA led to only partial deprotection
of acetal group for RNA ONs 2 and 4, along with precipitation of
RNA. On screening several deprotection conditions we found
that treatment of RNA ON with 20% aqueous acetic acid results
in complete and clean scission of acetal group without cleavage
of the RNA (Figure S1). The purity and integrity of all modified
RNA ONs was confirmed by LC-MS analysis (Figure 2 and
Figure S1, S2).19
Next, we synthesized the hm5C phosphoramidite 3 (Scheme
3). During the synthesis of the previous two monomers, we
noted that the methyl group of appropriately protected 5-
methyluridine could be functionalized efficiently. Hence, we
started our synthesis with 5-methyluridine 11 followed by
protecting 5ʹ-hydroxyl with DMT (21) and the protection of the 2ʹ-
and 3ʹ-hydroxyls with TBS. Conversion of the 5-methyl group of
22 into the 5-acetyloxymethyl group was the key step in this
synthesis, which we achieved by bromination and subsequent
acylation (Scheme 3).19 From here onwards we followed the
route reported by Riml et al. and obtained the monomer 3 in 8
steps with 6.4 % overall yield (Scheme 3).15,19 During the
preparation of this manuscript the Micura group improved their
initial synthesis and reported an alternative method, starting
from cytidine to obtained this monomer in 8 steps with 9.2%
overall yield.24
Figure 2. A) Sequences of modified RNA ONs synthesised in this study. RNA
ONs 1-3 contains hm5C, f5C, hm5Cm respectively at three different positions
whereas, RNA ON 4 contains all four cytosine-modifications. The 3ʹ end of
each RNA ON is tagged with TEG-Biotin (B*). B) Representative LC trace of
RNA ON 4. C) Corresponding ESI-MS spectrum of RNA ON 4.19
Figure 3. LC-MS chromatogram of RNA ON 2 before (A) and after (B) post-
synthetic chemical reaction with ethoxyamine hydrochloride at pH 5.0.24
Sequence of the RNA ON 5 is given at the bottom and structure of the
modified nucleoside (X) formed upon selective condensation reaction between
f5C and ethoxyamine is shown in chromatogram (B).19
Scheme 3. Synthesis of hm5C phosphoramidite 3.19
To evaluate the utility of our building blocks to obtain ONs
containing various modifications we synthesised a small series
of RNA ONs (1–4) (Figure 2A). To further confirm the
robustness of these monomers we synthesised the RNA ONs 1–
3 containing each single modification at three different positions,
and RNA ON 4 contains all of four cytosine-modifications. To
ascertain the compatibility of these monomers in the presence of
a biochemical tag we also performed the RNA ON synthesis on
To further demonstrate the intactness of the modified
ribonucleosides upon RNA synthesis and deprotection beyond
mass spectroscopy, we perform a post-synthetic chemical
functionalization of the RNA. Firstly, we subjected the RNA ON 2
containing a reactive formyl group for nucleophile addition with
ethoxyamine hydrochloride at pH 5.0 in the presence of
anisidine.(24) All three f5C nucleotides present in RNA 2 each
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In summary, we have demonstrated the efficient syntheses
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RNA ONs containing these modifications at multiple positions in
excellent yield and purity. The availability of these monomers
and the capacity to prepare RNA oligomers with any
combination, in desired positions, will be vital for studying the
function(s) of cytosine modifications in biology.
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We acknowledge support from University of Cambridge and
Cancer Research UK program. The Balasubramanian laboratory
is supported by core funding from Cancer Research UK
(C14303/A17197). S.B. is a Senior Investigator of the Wellcome
Trust (grant no. 099232/z/12/z).
W. Huang, M-D. Lan, C-B. Qi, S-J. Zheng, S-Z. Wei, B-F. Yuan, Y-Q.
Feng, Chem. Sci. 2016, 7, 5495–5502.
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Keywords: RNA epigenetic • 5-methylcytosine • 5-hydroxy-
methylcytosine • 5-formylcytosine • 2ʹ-O-methyl-5-hydroxy-
methylcytidine
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Arun A. Tanpure and Shankar
Balasubramanian*
Page No. – Page No.
Synthesis and multiple incorporations
of 2ʹ-O-methyl-5-hydroxymethyl-, 5-
hydroxymethyl- and 5-formylcytidine
monomers into RNA oligonucleotides
Text for Table of Contents
Synthesis and multiple incorporations of 2ʹ-O-methyl-5-hydroxymethylcytidine (hm5Cm), 5-hydroxymethylcytidine (hm5C) and 5-
formylcytidine (f5C) phosphoramidite monomers into RNA ONs have been developed. Optimization of mild post-synthetic
deprotection conditions enabled the preparation of RNA oligomers containing all four of the cytosine-modifications at multiple
positions and in combination.
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