Synthesis and biophysical characterization of RNAs containing 2′-fluorinated northern methanocarbacyclic nucleotides

Article abstract of DOI:10.1021/acs.orglett.8b04153

2′-Fluorinated Northern methanocarbacyclic (2′-F-NMC) nucleosides and phosphoramidites, based on a bicyclo[3.1.0]hexane scaffold bearing all four natural nucleobases (U, C, A, and G), were synthesized to enable exploration of this novel nucleotide modification related to the clinically validated 2′-deoxy-2′-fluororibonucleotides (2′-F-RNA). Biophysical properties of the 2′-F-NMC-containing oligonucleotides were evaluated. A duplex of 2′-F-NMC-modified oligonucleotide with RNA exhibited thermal stability similar to that of the parent RNA duplex, 2′-F-NMC-modified oligonucleotides had higher stability against 5′- A nd 3′-exonucleolytic degradation than the corresponding oligonucleotides modified with 2′-F-RNA, and 2′-F-NMC-modified oligonucleotides exhibited higher lipophilicity than the corresponding RNA oligonucleotides as well as those modified with 2′-F-RNA.

Full text of DOI:10.1021/acs.orglett.8b04153

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis and Biophysical Characterization of RNAs Containing
2′‑Fluorinated Northern Methanocarbacyclic Nucleotides
Masaaki Akabane-Nakata,† Pawan Kumar,† Rajat S. Das,† Namrata D. Erande,† Shigeo Matsuda,†
Martin Egli,‡ and Muthiah Manoharan*,†
†Alnylam Pharmaceuticals, 300 Third Street, Cambridge, Massachusetts 02142, United States
‡Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232, United States
S
* Supporting Information
ABSTRACT: 2′-Fluorinated Northern methanocarbacyclic (2′-F-NMC) nucleo-
sides and phosphoramidites, based on a bicyclo[3.1.0]hexane scaffold bearing all
four natural nucleobases (U, C, A, and G), were synthesized to enable
exploration of this novel nucleotide modification related to the clinically
validated 2′-deoxy-2′-fluororibonucleotides (2′-F-RNA). Biophysical properties
of the 2′-F-NMC-containing oligonucleotides were evaluated. A duplex of 2′-F-
NMC-modified oligonucleotide with RNA exhibited thermal stability similar to
that of the parent RNA duplex, 2′-F-NMC-modified oligonucleotides had higher
stability against 5′- and 3′-exonucleolytic degradation than the corresponding
oligonucleotides modified with 2′-F-RNA, and 2′-F-NMC-modified oligonucleo-
tides exhibited higher lipophilicity than the corresponding RNA oligonucleotides
as well as those modified with 2′-F-RNA.
herapeutics based on RNA interference (RNAi) have
Tgreat potential for treating human diseases. Both US FDA
and EMA have recently approved the first small interfering
RNA (siRNA) drug, ONPATTRO (patisiran), for treatment of
polyneuropathy caused by hereditary transthyretin-mediated
amyloidosis.1 The oligonucleotides used in oligonucleotides
based therapeutics require use of unnatural nucleotide building
blocks to stabilize the agents against nuclease degradation, to
enhance cell-membrane permeability, and to limit immune
responses.2 The 2′-deoxy-2′-fluororibonucleotide (2′-F-RNA,
Figure 1A) and 2′-O-methyl (2′-OMe) modifications have
been used to modify siRNAs,3−5 antisense oligonucleotides,6,7
aptamers,8 microRNAs,9 and ribozymes.10 Incorporation of 2′-
F-RNA and 2′-O-Me residues stabilizes an A-form RNA
duplex.4,11 Specifically, 2′-F ribo-substitution preorganizes the
sugar into a C3′-endo or North conformation and hence
reduces the entropic penalty for the formation of the A-form
duplex, and increases base stacking and Watson−Crick
hydrogen-bond stabilities due to its electron-withdrawing
Figure 1. (A−C) Structures of (A) 2′-F-RNA, (B) NMC, and (C) 2′-
power.11 Furthermore, 2′-F-RNA-modified siRNAs have
F-NMC. (D) Model of a hybrid duplex between 2′-F-NMC and RNA
reduced immune stimulation and improved activity in vitro
constructed using the program UCSF Chimera; 2′-F-NMC residues
and in vivo compared to unmodified siRNA,4 and are in clinical
have an idealized C2′-exo pucker with an axial 2′-fluorine.
development. The 2′-F-RNA-modified oligonucleotides are,
however, more sensitive to nucleolytic degradation than other
improve pharmacological properties of oligonucleotides. A
number of fluorine-containing building blocks have been
2′-modified oligonucleotides; moreover, these monomers are
recognized, albeit poorly, by human RNA polymerases at high
concentrations.12,13 Hence, chemically modified building
blocks that retain the advantages of 2′-F-RNA with additional
features to overcome these limitations have the potential to
Received: December 31, 2018
© XXXX American Chemical Society
A
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synthesized and evaluated in the context of oligonucleotide-
based therapeutics.14−29 However, to the best of our
knowledge, none of these modifications have been made
with all four nucleobases. As nucleotide pharmacology in a
therapeutic oligonucleotide like siRNA depends on both
sequence and position, it is necessary to have all four
nucleosides with same ribosugar modification to evaluate its
therapeutic potential.30
Scheme 1. Synthesis of 2′-F-NMC U and C Amidites
Conformationally restricted nucleic acids bearing bicyclic or
tricyclic scaffolds that exhibit high affinity for complementary
RNA are also being widely explored as potential therapeutic
oligonucleotide modifications.31,32 Nucleosides with a carbo-
cyclic bicyclo[3.1.0]hexane system, here referred to as
Northern methanocarbacyclic (NMC) nucleosides, are con-
strained to an RNA-like sugar pucker (Figure 1B).33−38 The
bicyclic NMC sugar is predicted to adopt a pseudoboat C2′-
exo conformation due to the methylene bridge between the
C4′ and C6′ positions. Consequently, NMC-modified
oligonucleotides form more stable duplexes with RNA than
do unmodified DNA.
Jung and co-workers recently synthesized thymidine analogs
2′-F-NMC T (Figure 1C) and ara-2′-F-NMC T and
oligonucleotides containing these building blocks.18 Deoxy-
oligonucleotides containing 2′-F-NMC T have higher RNA
binding affinity than oligonucleotides containing 2′-deoxy-2′-
fluoro uridine or nonfluorinated NMC thymidine.19 This
duplex stabilization is presumed to result from stabilization of
Watson−Crick hydrogen-bonding and base-stacking interac-
tions due to the 2′-fluoro incorporation and demonstrates the
potential of 2′-F-NMC analogs in oligonucleotide-based
therapeutics.
Our laboratory has systematically evaluated the role of
chemical modifications in siRNA activity. To assess 2′-F-NMC
analogs in terms of their sequence- and position-dependent
RNAi activity, we required all RNA nucleobase analogs. Here,
we report the synthesis of 2′-F-NMC phosphoramidites
bearing the four natural RNA nucleobases (i.e., A, U, G, C)
from a common starting material in a convergent approach.
We also report the binding affinities to a target RNA and
susceptibilities to exonuclease degradation of oligonucleotides
containing 2′-F-NMC.
yield of the reaction for this adenine precursor was low, and
synthesis of 2′-F-NMC purine analogs was not pursued.18 This
prompted us to explore alternate routes. Nencka et al. reported
a one-pot build-up procedure leading to 6-chloro- or 2-amino-
6-chloropurines, which are, respectively, adenosine and
guanosine precursors, from a primary amine.41 Marquez et
al. also achieved excellent yields of bicyclo[3.1.0]hexane
carbocyclic nucleosides, which involve 6-chloropurines on a
primary amine, using a microwave reactor.42 Therefore, we
evaluated the synthesis of the 2′-F-NMC purine phosphor-
amidites from the starting amine 1.18 As shown in Scheme 2,
the reaction of 1 with 4,6-dichloro-5-formamidopyrimidine
followed by cyclization of the formamido intermediate 10 in
the presence of diethoxymethyl acetate gave the 6-
chloropurine derivative 11 in 54% yield over two steps.
Ammonolysis of the compound 11 using a microwave reactor
The 2′-F-NMC U and 2′-F-NMC C phosphoramidites were
synthesized as depicted in Scheme 1. The starting amine 1 was
prepared according to the procedure reported by Jung et al.18
and was coupled with 3-methoxyacryloyl isocyanate and
cyclized under acidic conditions to afford the uridine derivative
2.39,40 The 5′-OH group of the nucleoside 2 was protected
with 4,4′-dimethoxytriphenylmethyl chloride (DMTrCl) to
give compound 3. Subsequent phosphitylation of the 3′-OH
group of 3 gave the desired phosphoramidite 4. For the
synthesis of 2′-F-NMC C, fully protected nucleoside 5 was
obtained by tert-butyldimethylsilyl (TBS) protection of 3.
Compound 5 was then converted into the cytidine derivative 6
by reacting with 1,2,4-triazole in the presence of Et3N and
POCl3, followed by treatment with aqueous NH3. The
exocyclic amine of 6 was benzoylated using benzoyl chloride
(BzCl), and the resulting protected cytidine derivative 7 was
treated with tetra-n-butylammonium fluoride (TBAF) to
obtain alcohol 8. Phosphitylation of 8 gave the desired
phosphoramidite 9.
Scheme 2. Synthesis of 2′-F-NMC A Amidite
To synthesize the purine analogs, Jung et al. attempted to
introduce the N-benzoyladenine base into the unsubstituted
enone derivative of the NMC scaffold by 1,4-addition. The
B
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produced the desired adenine nucleoside 12 in 91% yield. The
amino group was then protected with BzCl to furnish the
dibenzoyl derivative 13 (19%) and monobenzoyl derivative 14
(65%). Compound 13 was readily converted to 14 by
treatment with aqueous NH3 in THF. The silyl protection
was then removed by treating 14 with Et3N·3HF at 55 °C, to
obtain diol 15 in 98% yield. Subsequent dimethoxytritylation
of the 5′-OH followed by phosphitylation of 16 yielded the
desired phosphoramidite 17 (82%).
Scheme 3. Synthesis of 2′-F-NMC G Amidite
For the synthesis of 2′-F-NMC G, various conditions were
evaluated for the preparation of the 2-amino-6-chloropurine
intermediate 19 using the microwave-assisted reaction of the
amine 1 with 2-amino-4,6-dichloro-5-formamidopyrimidine
(Table 1). The reaction in 1,4-dioxane at 100 °C led to only
Table 1. Construction of 2′-F-NMC G Precursor 19
slight improvements in the thermal stability. These experi-
ments demonstrate that the 2′-F-NMC modification is well
tolerated in an RNA duplex. The slight loss in thermal stability
may prove advantageous in RNAi-based applications where
high binding affinities in the seed region can induce
undesirable off-target effects.43 Importantly, a 10-mer duplex
with six 2′-F-NMC nucleotides in the center displayed thermal
stability comparable to that of the unmodified dsRNA (entry
5) suggesting that this modification is very well accommodated
in an RNA:RNA duplex.
yield (%)
entry
solvent
temp (°C)
time (h)
18
19
1
2
3
1,4-dioxane
toluene
n-BuOH
100
160
160
12
2
2
88
70
59
trace
24
40
Table 2. UV Melting Temperatures of Modified Duplexes
the intermediate 18 as a mixture of rotamers (entry 1). By
heating at 160 °C in toluene, the intermediate 18 and the
desired product 19 were obtained in 70% and 24% yield,
respectively (entry 2). The yield of 19 was improved to 40% by
changing the solvent to n-BuOH (entry 3). However, longer
reaction times resulted only in substitution of 6-Cl groups by
n-BuOH. Attempts to cyclize intermediate 18 in 1,4-dioxane
by heating under microwave conditions resulted in disappoint-
ingly low yields (17%). The presence of the bulky 5′-O-TBS
group may hinder imidazole ring closure, resulting in poor
yields. Attempts to use diethoxymethyl acetate for the
cyclization yielded a complex mixture, including 19, as
shown by LC-MS (data not shown). 2′-F-NMC G was
eventually synthesized from the 2-amino-6-chloropurine
derivative 19 by reacting with 3-hydroxypropionitrile in the
presence of NaH (Scheme 3). Standard protection of exocyclic
amine using isobutyryl chloride afforded protected nucleoside
21 (95%). Desilylation using Et3N·3HF gave diol 22 in 98%
yield. Dimethoxytritylation gave DMTr-protected compound
23 (66%), which was converted to the corresponding
phosphoramidite 24 in 61% yield.
To gauge the effect of modified nucleotides on RNA affinity,
the 2′-F-NMC modifications were incorporated into 10-mer
and 12-mer oligoribonucleotides via standard solid-phase
synthesis (see Supporting Information). The 2′-F-NMC-
modified oligonucleotides were mixed with complementary
RNA in PBS buffer, and the melting temperatures (Tm) were
determined (Table 2). Modified duplexes containing a single
2′-F-NMC nucleotide at the center showed a decrease of at
most 1.3 °C in the melting temperature compared to the
unmodified duplex (Table 2, Entry 1−4). In a similar context,
incorporation of 2′-F-RNA-modified nucleotides resulted in
a
Tm values were obtained in PBS (pH 7.4) using 2.0 μM
concentrations of each strand. Red letters indicate sites of
modification. ΔTm is the difference in melting temperature between
the duplex with the modified strand and the unmodified reference
b
duplex (5′-UACAGUCUAUGU-3′:3′-AUGUCAGAUACA-5′, Tm
=
c
53.6 °C). Unmodified reference duplex for entry 5 is 5′-GCGAU-
CUCAC-3′:3′-CGCUAGAGUG-5′ with Tm = 57.6 °C.
Next, the thermal stability of duplexes carrying 2′-F-NMC
base pairs was determined (Table S2). Interestingly, in this
context the UF‑NMC−AF‑NMC base pair was better accommo-
dated (a drop of only 0.4 °C compared to unmodified RNA)
than a CF‑NMC−GF‑NMC base pair (a drop of 2.2 °C compared
to unmodified RNA).
The global conformations of RNA duplexes with one or two
2′-F-NMC nucleotides were evaluated using circular dichroism
(CD) spectroscopy. The CD spectra of modified duplexes
were comparable to that of the unmodified RNA and featured
a strong positive band at around 260 nm and a negative band
C
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at around 210 nm characteristic of an A-form duplex (Figure
2). This indicates that the 2′-F-NMC modification does not
significantly distort the global geometry of an RNA:RNA
duplex.
half-life of the 2′-F-NMC-modified oligonucleotide was around
90 min, whereas the half-life of dT19CF was 15 min. The 3′-
terminal dT in the oligonucleotide with the 2′-F-NMC residue
at the penultimate position relative to the 3′ end
(dT18CF‑NMCdT) was lost within an hour; however,
dT18CF‑NMC had a half-life of 170 min, whereas dT18CFdT
and dT19CF had half-lives of around 15 min.
Oligonucleotides modified at the 5′ ends were incubated
with the 5′-exonuclease phosphodiesterase II (PDE-II). Unlike
the 3′-end-modified dT18CF‑NMCdT, removal of the 5′-terminal
dT was not observed when dTCF‑NMCdT18 was incubated with
PDE-II. Rather, the full-length 20-mer oligonucleotide with a
penultimate CF‑NMC was very stable with a half-life of 5 h
(Figure 3B). Surprisingly, CF‑NMCdT19 was degraded rapidly
with a half-life of 12 min. This indicates that 2′-F-NMC
modification improves stability at the penultimate position.
Nevertheless, the 2′-F-NMC-modified oligonucleotides were
more stable than 2′-F-RNA-modified oligonucleotides, as
CFdT19 and dTCFdT18 were completely degraded by the first
time point (1 h) after addition of PDE-II.
In summary, we present efficient routes for convergent
synthesis of the four 2′-F-NMC ribonucleosides. 2′-F-NMC
residues are well accommodated in double-stranded RNAs and
do not alter the global structure of the duplex. 2′-F-NMC
oligonucleotides are more resistant to nuclease degradation
than are oligonucleotides modified with 2′-F-RNA. The higher
lipophilicity of an oligonucleotide containing 2′-F-NMC
residues compared to that with 2′-F-RNA residues (Figure
S2) may result in improved cellular uptake and endosomal
release of RNAs.44 Having access to all four phosphoramidites
will allow exploration of the full potential of 2′-F-NMC
modification in therapeutic oligonucleotides such as siRNAs.
These studies are ongoing in our laboratories.
Figure 2. CD spectra of 2′-F-NMC-modified RNA and comple-
mentary RNA (cRNA) duplex at 15 °C in PBS (pH 7.4). See Table
S3 for the details of the duplexes.
To determine the effect of the 2′-F-NMC modification on
degradation of oligonucleotides by exonucleases, we incorpo-
rated 2′-F-NMC C (CF‑NMC) or 2′-F-RNA C (CF) at the
terminus or at the penultimate position of a dT oligonucleo-
tide. The oligonucleotide modified at the 3′ end with 2′-F-
NMC (dT19CF‑NMC) was more resistant to degradation by
snake venom phosphodiesterase (SVPD) than was the
oligonucleotide modified with 2′-F-RNA (Figure 3A). The
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b04153.
Experimental, compound characterization, and assays
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mmanoharan@alnylam.com.
ORCID
Pawan Kumar: 0000-0001-7936-4613
Rajat S. Das: 0000-0003-3169-6218
Martin Egli: 0000-0003-4145-356X
Muthiah Manoharan: 0000-0002-7931-1172
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We dedicate this work to our long-time advisor, Professor
Michael E. Jung (UCLA), for his numerous contributions to
chemistry, medicinal chemistry, and nucleic acid chemistry. We
are grateful to the Alnylam Research Group for their support,
Drs. Ivan Zlatev and K.G. Rajeev for critical reading of the
manuscript, Dr. Anne Noronha (Concordia University) for
Figure 3. HPLC quantification of indicated full-length oligonucleo-
tide after incubation with (A) SVPD and (B) PDE-II as a function of
time. For dT18CF‑NMCdT, the percentage of 19-mer dT18CF‑NMC
remaining is plotted.
D
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DOI: 10.1021/acs.orglett.8b04153
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