Conception and Synthesis of Oxabicyclic Nucleoside Phosphonates as Internucleotidic Phosphate Surrogates in Antisense Oligonucleotide Constructs

Article abstract of DOI:10.1021/acs.orglett.8b02233

The stereocontrolled synthesis of a novel oxabicyclic nucleoside phosphonate comprising a perhydrofuropyran core unit was achieved. It was incorporated in an oligonucleotide sequence as a 5′-3′ phosphonate-phosphate insert, and the stability properties of the resulting duplex were measured. The oxabicyclic nucleoside framework was designed so as to restrict rotation around angles ?, ?, and ? of a natural nucleoside.

Full text of DOI:10.1021/acs.orglett.8b02233

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Conception and Synthesis of Oxabicyclic Nucleoside Phosphonates
as Internucleotidic Phosphate Surrogates in Antisense
Oligonucleotide Constructs
Juan C. Salinas,† Jeff Yu,‡ Michael Østergaard,‡ Punit P. Seth,*,‡ and Stephen Hanessian*,†
†
́
́
́
Department of Chemistry, Universite de Montreal, P.O. Box 6128, Downtown Station, Montreal, QC H3C 3J7, Canada
‡Department of Medicinal Chemistry, Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, California 92010, United States
S
* Supporting Information
ABSTRACT: The stereocontrolled synthesis of a novel oxabicyclic nucleoside
phosphonate comprising a perhydrofuropyran core unit was achieved. It was
incorporated in an oligonucleotide sequence as a 5′−3′ phosphonate−phosphate
insert, and the stability properties of the resulting duplex were measured. The
oxabicyclic nucleoside framework was designed so as to restrict rotation around angles
γ, δ, and ε of a natural nucleoside.
odified nucleic acids are used extensively for applica-
Mtions in biotechnology, diagnostics, and as nucleic acid-
based therapeutics.1 Conformationally restricted nucleic acids
are of particular interest as they can enhance duplex stability or
modulate hydrophobicity, which can impact their biological
profile. Some notable examples of conformationally restricted
nucleic acid analogs include locked nucleic acids (LNA),2
tricycloDNA (tcDNA),3 and α,β-constrained nucleic acids
(α,β-CNA).4 LNA and related analogs lock the furanose ring
in the RNA-like C3′-endo sugar pucker.5 tcDNA restricts rota-
tion around the C4′−C5′ exocyclic bond,6 while α,β-CNA
restricts rotation around torsion angles α and β of the phos-
phodiester linkage.7
We have previously evaluated the duplex stabilizing prop-
erties of dual constrained nucleic acid analogs as components
of antisense nucleoside sequences (ASOs) for applications in
nucleic acid therapeutics.8 Multiple modes of conformational
restriction are enforced in such analogs to understand the
contributions of individual constraint strategies toward overall
duplex stability. As part of this effort, we reported the effect of
Figure 1. Perhydrofuropyran core in nucleoside A restricts conforma-
tional freedom around torsion angles γ, δ, and ε.
restricting rotation around the C4′-C5′ exocyclic bonds in
LNA and α-L-LNA.9 Here, we report the synthesis and bio-
carbon framework of the intended bicyclic nucleoside would
be secured. We opted for this second approach.
physical evaluation of oligonucleotides modified with a novel
perhydrofuropyran based nucleoside scaffold, which simulta-
neously restricts rotation around torsion angles γ, δ, and ε
(Figure 1).
In considering strategies to construct the perhydrofuropyran
nucleoside phosphonate ester motif A, we envisaged two
approaches.10 Starting with thymidine would secure the desired
stereochemistry of the nucleosidic part but would require
the formation of the fused perhydrofuropyran including the
stereocontrolled C−C branching at C-3′. The second approach
would start with the readily available diacetone-D-glucose as a
chiron, and after stereocontrolled branching at C-3, the entire
We started with the transformation of the commercially
available diacetone-D-glucose 1 to the known11 hydroxymethyl
analog 2, followed by protection as a 2-naphthylmethyl ether
(Scheme 1). At this juncture, we decided to exchange the 5,6-
isopropylidene acetal for a more convenient pair of protecting
groups. Thus, acid-mediated cleavage, selective protection of
the primary hydroxyl group as the TBDPS ether was followed
by benzylation and acetolysis. The installation of the nucleobase
Received: July 17, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b02233
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. Synthesis of Nucleoside Phosphonate 10
was successfully achieved using a Vorbru
̈
ggen reaction12
ester 10 as the nucleophile and a 5′-activated nucleoside as the
nucleofugal partner, we could obtain the desired phosphonate
dinucleotide 12 (Scheme 2). After extensive optimization,22
we found that warming a mixture of nucleosides 10 and 11 in
DMF in the presence of Cs2CO3 gave dinucleoside hemi-
phosphonic ester (not shown).23 However, these conditions
resulted in the hydrolysis of the methyl ester of the phospho-
nate, which was re-esterified using freshly prepared diazo-
methane to give 12 affording a 1:1 mixture of P-diastereomers.
Hydrogenolysis of 12 in the presence of Pearlman’s catalyst24
followed by protection of the 5′-hydroxyl group as the levu-
linate ester25 and deprotection of the TBDPS ether using
catalytic amounts of CsF26 in a mixture of DMSO/MeOH
afforded 13 as 1:1 mixture of phosphonate P-diastereomers in
65% yield. A phosphitylation reaction provided the phospho-
nate phosphoramidite 14. The GGATGTTCTCGA oligonu-
cleotide incorporating 14 was then paired with matched and
mismatched RNAs where it showed a slightly enhanced ability
to stabilize the duplex and similar ability to discriminate mis-
matches as natural DNA (Table 1).
To help understand these observations, we created a struc-
tural model that depicts the preferred conformation around the
internucleosidic phosphodiester linkage observed in RNA
duplexes (Figure 2).
The O3′-P-O5′-C5′-H bonds form the outline of a six-
membered chair, and this conformation is stabilized by anomeric
effects through the phosphorus atom.27 Presumably, these
nonbonding interactions serve to preorganize the backbone in
nucleic acid duplexes. Our results show that the perhydrofur-
opyran ring in A can mimic conformational preferences around
γ, δ, and ε in nucleic acid duplexes..
affording nucleoside 4. Direct and selective alkylation of the
2′-OH of an unprotected nucleoside is a difficult task.13 As an
alternative, we chose to protect the thymine base as a N-3
t-butyl-carbamoyl derivative.14 Subsequent base mediated
cleavage of the 2′-OAc allowed methylation of the 2′-OH in
the presence of NaH and iodomethane, followed by cleavage of
the 2-naphthylmethyl ether with DDQ15 releasing the primary
alcohol 5 in excellent overall yield. Swern oxidation followed
by a Pudovik−Abramov reaction16 with dimethyl H-phos-
phonate led to the α-hydroxyphosphonate 6 as a single isomer.
Cleavage of the TBDPS group was achieved by treatment of
6 with 3HF·NEt3 to give a mixture of compounds 7 and 8.
The configuration of the newly formed stereogenic center of
the α-hydroxyphosphonate 6 was confirmed to be (R) by
single crystal X-ray analysis of compound 8. The exclusive
Re-face addition of the dimethoxy H-phosphonate anion appears
to be sterically controlled by the 2′-OMe group of the furanose
ring since the addition gave the same (R)-isomer as the major
product in a model substrate.17 Initial attempts to form the
oxabicyclic tetrahydropyran via mesylation or tosylation of the
primary alcohol in either 7 or 8 followed by DBU-mediated
cyclization led to low conversion. Gratifyingly, the desired
oxabicyclic compound 9 was obtained using the triflate ester of
7 in pyridine. Protection of N-3 in the nucleobase with a BOM
group8 was successfully achieved and was followed with the
cleavage of the phosphonate dimethyl ester to give the hemi-
ester 10 in 85% yield over two steps.
Our original plan for the formation of phosphonate-linked
dinucleotide 12 required the activation of the acidic −OH in
bicyclic nucleoside 10, then a traditional ester coupling with
5′-OH of a 3′-OTBDPS-protected thymidine. In our attempts
to optimize this process, we found that commonly utilized
conditions such as the Mitsunobu reaction18 or ester formation
in the presence of BOP,19 HATU,20 or DEPBT21 led only to
traces of product. We reasoned that by using hemiphosphonic
In conclusion, we report the synthesis and the biophysical
evaluation of a novel perhydrofuropyran nucleoside phospho-
nate analog that restricts conformational preferences around
torsion angles γ, δ, and ε. The modification showed a slight
enhancement in duplex stability as compared to unmodified
B
DOI: 10.1021/acs.orglett.8b02233
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
DNA in the sequence evaluated. However, additional work
using multiple sequences is required to make a complete
assessment. Recent work has highlighted the ability of sugar
and backbone modifications on the ASO to modulate inter-
actions with cellular proteins, which can have profound implica-
tions on ASO behavior in biological systems.28 Further biological
evaluation of this and related oligonucleotide modifications
will reveal their potential utility for applications in antisense
therapeutics.
Scheme 2. Synthesis of Phosphoramidite 14
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.8b02233.
Experimental details and characterization data for the
products (PDF)
Accession Codes
CCDC 1827233 and 1850195 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: pseth@ionisph.com.
*E-mail: stephen.hanessian@umontreal.ca.
ORCID
Stephen Hanessian: 0000-0003-3582-6972
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The Montreal group thanks Ionis Pharmaceuticals and NSERC
for financial support.
■
Table 1. Duplex Stabilizing and Mismatch Discrimination
Properties of the Oligonucleotide Incorporating the
Phosphonate Dinucleoside 13 (TT)
a
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DOI: 10.1021/acs.orglett.8b02233
Org. Lett. XXXX, XXX, XXX−XXX