Straightforward Synthesis of Purine 4′-Alkoxy-2′-deoxynucleosides: First Report of Mixed Purine-Pyrimidine 4′-Alkoxyoligodeoxynucleotides as New RNA Mimics

Article abstract of DOI:10.1021/acs.orglett.5b01430

Purine and pyrimidine 4′-alkoxy-2′-deoxynucleosides were efficiently prepared from nucleoside 4′-5′-enol acetates in three steps by N-iodosuccinimide promoted alkoxylation, hydrolysis, and reduction followed by conversion to phosphoramidite monomers for t

Full text of DOI:10.1021/acs.orglett.5b01430

Letter
pubs.acs.org/OrgLett
Straightforward Synthesis of Purine 4′-Alkoxy-2′-deoxynucleosides:
First Report of Mixed Purine−Pyrimidine 4′-
Alkoxyoligodeoxynucleotides as New RNA Mimics
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Magdalena Petrova,* Ondrej Pav, Milos Budesínsky, Eva Zborníkova, Pavel Novak, Sarka Rosenbergova,
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Ondrej Paces, Radek Liboska, Ivana Dvorakova, Ondrej Simak, and Ivan Rosenberg*
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Department of Bioorganic and Medicinal Chemistry, Institute of Organic Chemistry and Biochemistry Academy of Sciences of the
Czech Republic v.v.i., Flemingovo nam. 2, 166 10 Praha 6, Czech Republic
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S
* Supporting Information
ABSTRACT: Purine and pyrimidine 4′-alkoxy-2′-deoxynu-
cleosides were efficiently prepared from nucleoside 4′-5′-enol
acetates in three steps by N-iodosuccinimide promoted
alkoxylation, hydrolysis, and reduction followed by conversion
to phosphoramidite monomers for the solid-phase synthesis of
the oligonucleotides. Fully modified 4′-alkoxyoligodeoxynu-
cleotides, which are characterized by a prevalent N-type (RNA-
like) conformation, exhibited superior chemical and nuclease resistance as well as excellent hybridization properties with a strong
tendency for RNA-selective hybridization, suggesting a potential application of 4′-alkoxy-oligodeoxynucleotides in antisense
technologies.
Scheme 1. Routine Synthesis of 4′-Methoxynucleosides
hemically modified antisense oligonucleotides (AOs) are
C
powerful tools for the regulation of gene expression.1 AO
modifications, which improve their affinity for cognate RNA by
the conformational restriction of the sugar−phosphate backbone
in the RNA-like C3′-endo conformation2 (e.g., 2′-O-Me, 2′-O-
MOE, and LNA3), are a powerful tool for antisense drug design,
which target metabolic and cardiovascular diseases as well as
cancer.4 In addition, progress in AO chemistry is closely related
to the development of RNAi technologies and the advance of
siRNAs5 and miRNAs6 from basic research to therapeutic
applications.7
A very simple modification of the sugar part of 2′-deoxy-
nucleosides (dNs) was accomplished by 4′-alkoxy substitution,
where β-D-erythro epimers preferentially adopt a C3′-endo
conformation.8 In 2011, we reported the synthesis of 4′-alkoxy
oligothymidylates, whose hybridization properties with a
complementary rA15 counterpart were superior to those of
unmodified dT15. Furthermore, the observed Tm enhancement
was of similar magnitude to the regioisomeric 2′-O-Me
oligoribothymidylate reference.9 Recently, oligomers consisting
of a 2′-O,4′-C-ethyleneoxy bridged 5-methyluridine derivative,
which significantly stabilized duplex and triplex formation, were
reported.10
The routine synthesis of epimeric 4′-methoxy dNs 2 is based
on the transient epoxidation of 4′,5′-exo-methylene nucleoside
precursors 1 with m-chloroperbenzoic acid followed by opening
with methanol (MeOH) (Scheme 1).8,9,11 Although the 4′-
alkoxy thymidine derivatives were prepared in good yields, only
trace amounts of the purine derivatives were produced.11 The 4′-
epimeric mixture of 2′-deoxy-4′-methoxyadenosine was alter-
natively obtained by the radical photolysis of a 4′-phenylselenide
compound.12 Therefore, for the preparation of mixed purine−
pyrimidine 4′-methoxy oligodeoxynucleotides (MONs), a new,
more robust synthetic approach that preferentially yields β-D-
erythro epimers of purine derivatives would be highly useful.
The concept of utilizing nucleoside 4′,5′-enol acetates as
unsaturated derivatives to preserve the oxygen function at C5′
was introduced by Cook and Secrist in the late 1970s.13
However, their attempts at direct alkoxylation failed. In this
Letter, we disclose the true potential of 4′,5′-enol acetates for the
introduction of alkoxy substituents, as demonstrated in the dNs
series.
A modified14 Moffat oxidation of N-acyl and 3′-O-tert-
butyldiphenylsilyl (TBDPS) protected 2′-deoxyadenosine 3a
and 2′-deoxyguanosine 3b afforded 5′-aldehydes 4a (88%) and
4b (81%), respectively, which were converted by reaction with
acetic anhydride in the presence of K2CO3 in acetonitrile13b to
4′,5′-enol acetates 5a (61%) and 5b (43%), respectively. In
consistency with the literature data,13b enol acetates 5 with Z
configuration were exclusively formed. Surprisingly, the
conversion of 5 using N-iodosuccinimide (NIS) and anhydrous
Received: May 15, 2015
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.5b01430
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Organic Letters
Letter
MeOH as the solvent readily afforded the corresponding 5′-
acetoxy-5′-iodo-4′-methoxy intermediates 6, which were directly
subjected to hydrolysis (2 M aqueous triethylammonium
bicarbonate−N,N-dimethylformamide mixture, TEAB−DMF,
1:10). The obtained complex mixture of acetals 7 and
hemiacetals 8 was treated with sodium borohydride in a
DMF−MeOH mixture to yield the desired 4′-methoxy products
9βa (76%) and 9βb/9αb (2/1, 66%) (Scheme 2). Similar results
Scheme 2. Synthesis of 4′,5′-Enol Acetates 5 and 4′-Methoxy
dNs 9−12
Figure 1. (a) 4′-Alkoxy substituted dNs 13−16 prepared in the present
study. (b) Calculated conformation of 2′-deoxy-4′-methoxyadenosine
13a.
The assumed preferential C3′-endo conformation of 2′-deoxy-
4′-methoxyadenosine 13a was confirmed by NMR spectroscopy
(Figure 1b). The configuration at carbon C4′ and the preferred
orientation of adenine were determined from the observed NOE
contacts between the base and pentose protons. The absence of
H-4′ does not allow for the use of a complete pseudorotational
conformation calculation for the pentose ring. The application of
the empirical relationship16 for the population of C2′-endo and
C3′-endo (C2′-endo = [17.8 − (J(1′,2″) + J(2″,3′))]/10.9; C3′-
endo = [(J(1′,2″) + J(2″,3′)) − 6.9]/10.9) and the observed
vicinal coupling constants (J(1′,2″) = 7.6 and J(2″,3′) = 9.2 Hz)
yields a population ratio for C2′-endo/C3′-endo of 9:91%. The
use of J(1′,2″) and J(2″,3′) for guanine derivative 13b (7.8 and
9.2 Hz) and cytosine derivative 13c (7.9 and 9.1 Hz) gives the
same ratio for C2′-endo/C3′-endo of 7:93%, which is in
agreement with the data reported earlier for thymine derivative
13d.8
Surprisingly, 4′-methoxy dNs exhibit exceptional stability in
acidic media. Depurination of adenine and guanine derivatives
13a and 13b in 0.05 M HCl at 40 °C was monitored by the
decreasing UV absorbance at wavelengths corresponding to the
highest differences between the extinction coefficients of the
appropriate nucleoside/nucleobase pairs. It was characterized by
the initial depurination rate (IDR), expressed as the first
derivative of time-dependent nucleobase cleavage curve at the
initial time point zero, with obtained values 1.29 × 10−3 for 2′-
deoxyadenosine compared to 1.29 × 10−4 for 13a at λ = 256 nm,
and 2.04 × 10−3 for 2′-deoxyguanosine compared to 2.32 × 10−4
for 13b at λ = 260 nm (Figure 2). Concerning the mechanism of
acidic hydrolysis of nucleosides, there are two mechanisms
described in the literature.17 The first mechanism assumes
protonation of the furanose 4′-oxygen followed by breaking the
C−O bond, attack of the water molecule on the newly formed
Schiff base, and subsequent release of the nucleobase and
(deoxy)ribose. A second, more recently claimed A-1 mechanism
assumes protonation of the nucleobase, followed by slow
breaking the C−N bond and formation of an oxocarbenium
ion. The strong stabilizing effect of 2′-hydroxy and 2′-methoxy
groups, as well as less stabilizing effect of 3′-hydroxy and 3′-
methoxy groups are well established and explained in terms of
were obtained for pyrimidine dNs: 86% yields for both 5′-
aldehydes 4c and 4d; 59% and 52% for 4′,5′-enol acetates 5c and
5d, respectively; and 38%, 2.5%, 58%, and 5% for 4′-methoxy
9βc, 9αc, 9βd, and 9αd, respectively (Scheme 2).
The general usefulness of 4′,5′-enol acetates 5 as key
intermediates in the synthesis of various 4′-alkoxy substituted
dNs was demonstrated by the preparation of protected 4′-
methoxyethoxy 10 (44%), 4′-allyloxy 11 (44%), and 4′-
propargyloxy 12 (35%) derivatives of 2′-deoxyadenosine. The
initial reaction with NIS also proceeds in a diluted mixture of
alcohol and methylene chloride (1:4), and the final reduction is
conveniently accomplished using sodium cyanoborohydride in
MeOH at a pH of ∼4 (Scheme 2).
To obtain free nucleosides 13, the 3′-O-TBDPS protecting
group from 9β was removed with a 0.5 M solution of
tetrabutylammonium fluoride (TBAF) in tetrahydrofuran
(THF) followed by the removal of the N-acyl protecting groups
from the nucleobases by heating in a 33% solution of
methylamine in ethanol at 50 °C for 1−2 h (total yields for the
two steps 66% 13a, 86% 13b, 57% 13c; 92% 13d only for the
TBAF treatment). Similar results were obtained for the 4′-
methoxyethoxy 14, 4′-allyloxy 15, and 4′-propargyloxy 16
derivatives of 2′-deoxyadenosine (51% each) (Figure 1a).
B
DOI: 10.1021/acs.orglett.5b01430
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Scheme 3. Synthesis of Phosphoramidite Monomers 19
the duplex stability with varied potency. The results are
summarized in Table 1.
Table 1. Duplex Thermal Stability of Modified
Oligonucleotides Compared to Their DNA and RNA
a
Complements
Tm (ΔTm per residue) °C
b
c
entry
sequence (5′−3′)
vs DNA
36.9
34.5
vs RNA
34.9
46.1
1
2
3
4
5
6
7
d(GCA TAT CAC)
r(GCA UAU CAC)
d(GCA TAT CAC)
d(GCA UAU CAC)
d(GCA TAT CAC)
d(GCA UAU CAC)
PS-d(GCA TAT CAC)
43.6 (+0.7)
39.9 (+0.3)
52.2 (+1.9)
50.4 (+1.7)
d
61.8 (+2.8)
Figure 2. UV-monitored stability of 2′-deoxy-4′-methoxypurines in 0.05
M HCl at 40 °C. (a) 2′-Deoxy-4′-methoxyadenosine 13a vs 2′-
deoxyadenosine; λ = 256 nm. (b) 2′-Deoxy-4′-methoxyguanosine 13b
vs 2′-deoxyguanosine; λ = 260 nm.
e
54.2 (+1.9)
f
38.0 (+0.1)
47.5 (+1.4)
a
Tm values were measured in 50 mM sodium phosphate buffer (pH
7.2) containing 100 mM NaCl and 1 mM EDTA at a total strand
b
concentration 4 μM. Complementary DNA: 3′-d(CGT ATA GTG).
c
d
Complementary RNA: 3′-r(CGU AUA GUG). Complementary
the negative inductive effect of the substituents.17a A similar
effect of 2′- and/or 3′-fluoro substituents is also attributed to the
inductive effect leading to the destabilization of the oxocarbe-
nium ion.18 However, 4′-fluoro substituent led to a significant
labilization of the glycosidic linkage.19 Thus, there is a strong
difference between the stability of nucleosides having 4′-alkoxy
and 4′-fluoro substituents. This may conceivably be attributed to
different electronegativity of these groups and their capability to
increase or reduce, respectively, electron density of the furanose
ring, i.e., to hamper or enhance, respectively, the formation of an
oxocarbenium ion and the elimination of a nucleobase.
To prepare phosphoramidite monomers 19, the dimethoxy-
trityl (DMT) group was introduced at the 5′-hydroxyl of 9β,
followed by the removal of the 3′-O-TBDPS group from the 5′-
O-DMT derivatives 17. After purification of 3′-hydroxy
derivatives 18 on reverse phase, reaction with 2-cyanoethoxy-
N,N-diisopropylaminochlorophosphine in THF in the presence
of diisopropylethylamine (DIPEA) gave 3′-phosphoramidites
19. Because the complete removal of the chlorophosphine
reagent residues using standard silica gel chromatography failed,
the procedure based on RP chromatography using a CH3CN
gradient in TEAA buffer followed by rapid extraction with DCM
upon cooling has been employed as the final purification step.
Protected monomers were prepared in good yields (19a 71%,
19b 59%, and 19c 75%) (Scheme 3).
e
strand 3′-d(CGT ATA GTG). Complementary strand 3′-d(CGU
AUA GUG). Fully phosphorothioate.
f
Relative to natural oligomers, which form more stable DNA/
DNA or RNA/RNA duplexes than hybrid DNA/RNA (entries 1
and 2), the modified oligonucleotides hybridized better with
complementary RNA strands (entries 3, 4, and 7). These results
correlate with the preferential RNA-type conformation of the
sugar moieties in 4′-methoxy dNs. The thymine-containing
oligomers (entries 3 and 5) exhibited a higher thermal stability
than the uracil congeners (entries 4 and 6). The highest thermal
stability was observed when both complementary strands were
modified and contained thymine nucleobases (entry 5). The
combination of fully 4′-methoxy and phosphorothioate mod-
ifications (entry 7) led to a significant decrease in duplex stability
compared to DNA, with a Tm value comparable to the natural 9-
mer (entry 7 vs entry 1). The duplex stabilization compared to
RNA decreased only slightly and remained similar to that for the
4′-MONs with a phosphodiester backbone (entry 7 vs entries 3
and 4).
Fully phosphodiester 9-mer 5′-d(GTG ATA TGC) exhibited
exceptional stability against cleavage by nucleases. The experi-
ments were carried out under conditions in which the half-time
cleavage of the natural DNA counterpart was less than 1 min.
Complete stability was observed in the presence of a 10,000 g rat
liver homogenate, phosphodiesterase II (EC 3.1.16.1), nuclease
P1 (EC 3.1.3.16), and micrococcal nuclease (EC 3.1.31.1) during
a 2 h incubation. Considerable stability (half-time cleavage of
approximately 40 min) against cleavage was observed for
phosphodiesterase I (EC 3.1.4.1).
The ability of 4′-methoxyoligodeoxynucleotides (4′-MONs)
to stabilize duplexes with complementary DNA and RNA was
measured using fully modified 9-mers that were synthesized by a
phosphoramidite method on solid phase.9 Two types of
sequences were evaluated: DNA-like nonamers featuring two
4′-methoxythymidine units (entries 3, 5, and 7), as well as RNA-
like nonamers having two 2′-deoxy-4′-methoxyuridine units
instead (entries 4 and 6). All of the studied oligomers enhanced
In summary, we have developed a simple, β-D-erythro-
preferential synthetic procedure for the previously unavailable
C
DOI: 10.1021/acs.orglett.5b01430
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purine 4′-methoxy dNs, and this method is fully applicable to the
pyrimidine dNs series. The preferred C3′-endo conformation of
the β-D-erythro epimers was confirmed by NMR (>90% for 13a,
13b, and 13c). Fully modified oligonucleotides were synthesized
on solid phase and exhibited superior chemical and nuclease
stabilities. Moreover, their excellent hybridization properties
with a remarkable tendency toward RNA-selective hybridization
suggest the potential application of the 4′-MONs for the
construction of AOs. In addition, we have demonstrated the
general applicability of this synthetic procedure for 4′-alkoxy
dNs. The use of a variety of 4′-alkoxy substituents in AOs is
currently under investigation, and the results will be reported in
due course.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
Full experimental details, H, 13C, and 31P NMR spectra of all
new compounds, HPLC charts and MALDI-TOF-MS spectra of
new oligonucleotides, and representative UV melting and
nuclease stability data. The Supporting Information is available
free of charge on the ACS Publications website at DOI: 10.1021/
acs.orglett.5b01430.
(14) Petrova,
2010, 51, 6874−6876.
́ ̌ ̌
́
M.; Budesínsky, M.; Rosenberg, I. Tetrahedron Lett.
(15) Representative procedure (synthesis of compound 9βa): To a stirred
solution of 5a (1.60 g, 2.5 mmol) in absolute MeOH (25 mL) at −20 °C
under argon, light-protected NIS was added (731 mg, 3.25 mml, 1.25
equiv.), and the mixture was allowed to stand as the temperature
gradually increased to rt. After 2 h, the mixture was concentrated, and
the residue was dissolved in DMF (25 mL). Upon cooling in an ice bath,
2 M TEAB was added (2.5 mL, 5 mmol, 2 equiv.). The mixture was
stirred at rt overnight, concentrated and dissolved in DMF (25 mL).
Absolute MeOH was added (1 mL), the mixture was cooled in an ice
bath, and NaBH4 was added in portions (315 mg, 7.25 mmol, 3.3 equiv.
in total). The mixture was maintained at a low temperature overnight
before being diluted with CHCl3 and washed with brine. The dried
organic layer was concentrated, and the residue was purified by flash
chromatography (CHCl3-10% EtOH) to afford β-D-erythro 9βa (1.18 g,
1.90 mmol, 76%) as a white foam.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: petrova@uochb.cas.cz.
*E-mail: rosenberg@uochb.cas.cz.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Czech Science Foundation (13-
26526S and 13-24880S) and the Technology Agency of the
Czech Republic (TA03010598) under the IOCB research
project RVO:61388963.
(16) Rinkel, L. J.; Altona, C. J. J. Biomol. Struct. Dyn. 1987, 4, 621−649.
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(b) Srivastava, P. C.; Robins, R. K.; Meyer, R. B., Jr. In Chemistry of
Nucleosides and Nucleotides Vol. 1; Townsend, L. B., Ed.; Springer US:
Boston, MA, 1988; p 265.
(18) Chong, Y.; Gumina, G.; Mathew, J. S.; Schinazi, R. F.; Chu, Ch. K.
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