Efficient functionalization of oligonucleotides by new achiral nonnucleosidic monomers

Article abstract of DOI:10.1021/ol500668n

A novel synthetic strategy has been designed for preparation of achiral nonnucleosidic phosphoramidite monomers for automated solid-phase oligonucleotide synthesis. It is based on O-DMTr-protected 4-(2-hydroxyethyl)- morpholine-2,3-dione as the key compound and a family of building blocks obtained by its ring-opening by primary aliphatic amines. A series of nonnucleosidic phosphoramidites containing various side-chain functionalities was synthesized, and corresponding oligodeoxyribonucleotides incorporating modified units in single or multiple positions along the chain were prepared.

Full text of DOI:10.1021/ol500668n

Letter
pubs.acs.org/OrgLett
Efficient Functionalization of Oligonucleotides by New Achiral
Nonnucleosidic Monomers
Maxim S. Kupryushkin,† Mikhail D. Nekrasov,†,‡ Dmitry A. Stetsenko,† and Dmitrii V. Pyshnyi*,†,‡
†Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev
Avenue, Novosibirsk 630090, Russia
‡Novosibirsk State University, 2 Pirogova Street, Novosibirsk 630090, Russia
S
* Supporting Information
ABSTRACT: A novel synthetic strategy has been designed for
preparation of achiral nonnucleosidic phosphoramidite mono-
mers for automated solid-phase oligonucleotide synthesis. It is
based on O-DMTr-protected 4-(2-hydroxyethyl)-morpholine-
2,3-dione as the key compound and a family of building blocks
obtained by its ring-opening by primary aliphatic amines. A series
of nonnucleosidic phosphoramidites containing various side-
chain functionalities was synthesized, and corresponding oligodeoxyribonucleotides incorporating modified units in single or
multiple positions along the chain were prepared.
ligonucleotides with pendant chemical functionalities are
Oused widely as research tools for life sciences and as
therapeutic or diagnostic agents.1 Automated solid-phase
synthesis by the 2-cyanoethyl phosphoramidite method is a
We have applied our new building block strategy for the
preparation of achiral phosphoramidites bearing various side-
chains (Scheme 1, Table 1). The approach requires two
separately functionalized building blocks. One is a key block
that introduces the two hydroxyls needed for incorporation into
DNA chain. Another is a functional block tagged with side-
chain groups to convey specific properties to oligonucleotides.
The key block is a common reagent for the preparation of a
general chemical method for preparing polynucleotides.2
A
majority of known chemical modifications could be conven-
iently tagged onto oligonucleotide sequences by solid-phase
synthesis via either nucleosidic or nonnucleosidic phosphor-
amidites with tethered functional groups.3 Nonnucleosidic
monomers have an additional advantage of being independent
of the oligonucleotide sequence since they are not usually
involved in complementary binding. Sometimes a monomer is
designed to mimick an acyclic nucleoside and thus maintains a
five-atom distance between the P(V) atoms.4 A majority of the
available nonnucleosidic synthons could be split into two
groups: racemic5 and homochiral.6 The former suffer from the
disadvantage of producing diastereomeric oligonucleotides,
which sometimes complicate isolation of the product.7 The
latter are normally derived from a homochiral natural precursor
that is often costly.5a,b Frequently, preparation of a monomer
bearing the required functionality may be accomplished only by
multistep chemical synthesis in low yield.8 Thus, the develop-
ment of convenient, versatile, and efficient methods for
preparation of nonnucleosidic phosphoramidites equipped
with desired side-chain functionalities remains an important
task.
Scheme 1. Synthesis of a Family of Achiral Nonnucleosidic
a
Phosphoramidites and Oligonucleotides Thereof
We describe herein a novel strategy for the design of
nonnucleosidic phosphoramidites for nucleic acid functionaliza-
tion, which is a merger of two approaches: one utilizing the
ring-opening of lactones by aliphatic amines9 and another that
uses activated oxalate esters for oligonucleotide derivatization.10
Also, it takes into account our previous experience in the
synthesis and application of oligonucleotides with non-
nucleosidic inserts.11
a
Key: CE, 2-cyanoethyl; DIEA, N,N-diisopropylethylamine; DMTr,
4,4′-dimethoxytrityl.
Received: April 1, 2014
Published: May 12, 2014
© 2014 American Chemical Society
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Letter
a
Table 1. Structure and Properties of Oligonucleotide Derivatives
a
b
c
d
For experimental details, see the Supporting Information. A: buffer B 50% MeCN. B: buffer B 90% MeCN. R1: Y = CF3CO, R3: Y = H.
family of synthons with different side-chains. In each case
oligonucleotide modification is accomplished by employing an
appropriately functionalized monomer or combination of
monomers during standard solid-phase synthesis.
successfully tested in solid-phase oligonucleotide synthesis
according to standard phosphoramidite protocol, except that a
higher concentration of 0.15 M and longer coupling time of 7
min were used. We were able to introduce up to three
consecutive modifications into any position of choice within
oligonucleotide chain in good overall yield.13
The copper(I)-catalyzed azide−alkyne cycloaddition
(CuAAC)14 has found widespread use for bioconjugation as a
widely popular example of click chemistry.15 A number of
methods for preparation of alkyne derivatives of oligonucleo-
tides were published.16 However, a reliable method for side-
chain alkynyl group attachment in the middle of the chain
would still be useful.17 We have tagged an alkyne onto the side-
chain of a nonnucleosidic monomer via either a shorter (5a) or
longer five-atom (5b) linker. Both monomers were successfully
applied to make alkynyl oligonucleotides by solid-phase
synthesis. The potential of the alkyne derivatives for click
chemistry was demonstrated by facile coupling of an azido Cy3
dye to an alkynyl oligonucleotide under CuAAC conditions
(see the Supporting Information).
Pyrene is a robust fluorophore that has been widely used to
sense subtle changes in nucleic acid geometry.18 We have
attached pyrene via its aminomethyl derivative to the side-chain
of a nonnucleosidic monomer 5d. The reagent was successfully
used for solid-phase synthesis of oligonucleotides labeled with
one or more pyrene residues in various positions. When one or
two pyrenyl groups were attached to the 5′-end, a marked
stabilization of complementary complex resulted, which
correlates well with previous observations.19
We have selected diethanolamine as an inexpensive starting
material that provides both hydroxyl groups required for
incorporation into the oligonucleotide chain and a secondary
amino group for attachment of a side-chain. N-Substituted
diethanolamine derivatives12 were described previously as
precursors for nonnucleosidic phosphoramidites, but a difficulty
of selective protection of only one of its two identical hydroxyls
had significantly lowered yields of the products. We have solved
this problem by simultaneous protection and activation of only
one of the two hydroxyls as a cyclic oxalamide ester. The
reaction of diethanolamine with inexpensive diethyl oxalate
gives lactone 1 in high yield and purity.
It could be O-dimethoxytritylated in good yield affording
DMTr-lactone 2 as our key block, which was used in all
subsequent reactions with functional blocks. Those are primary
aliphatic monoamines 3a−g, which are either commercially
available or are easily synthesized. The structure of the side-
chain of a functional block (Table 1, R1) defines specific
property that is conveyed to a modified oligonucleotide: a
bioconjugation site (a, b), a hydrophobic aromatic (c) or
aliphatic (d, g) moiety, or a positive charge (e, f). Ring-opening
of the key dimethoxytritylated lactone 2 by a functional primary
amino block 3a−g followed by phosphitylation yields a family
of nonnucleosidic phosphoramidite monomers 5a−g tagged
with diverse side-chains (Scheme 1). The monomers were
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Poor cell uptake is a problem that currently limits therapeutic
application of oligonucleotide and their analogues.20 Function-
alization with lipids is known to increase intracellular delivery of
oligonucleotides.21 We have designed a nonnucleosidic
monomer 5d for incorporation of dodecyl groups into synthetic
oligonucleotides at one or several positions within their chain.
It has resulted in a marked rise in lipophilicity of the
oligonucleotides with the increase of the number of dodecyl
groups (Figure 1).
of properties useful for design of new diagnostic nucleic acid
probes and potential antisense therapeutic agents with
improved cell delivery.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, NMR spectra for monomers and
precursors, HPLC, PAGE and MALDI-TOF data for
oligonucleotides, and thermal denaturation for pyrene-labeled
complexes. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: pyshnyi@niboch.nsc.ru.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Figure 1. RP-HPLC traces of oligonucleotides functionalized with
one, two, or three dodecyl groups; R3 = dodecyl, X marks the position
of the modification.
We thank Dr Alexander A. Lomzov for carrying out thermal
denaturation experiments and Marat F. Kassakin for recording
MALDI-TOF spectra. This work was supported by RFBR
Grant 13-04-01176, the Russian Government support for
research projects implemented under supervision of world’s
leading scientists (Agreement No. 14.B25.31.0028 with Sidney
Altman as the leading scientist), and partly by an interdiscipli-
nary grant from the Siberian Branch of Russian Academy of
Sciences.
Nucleic acids are polyanions and as such they do not readily
cross cell membranes. Introduction of positively charged groups
into oligonucleotides may improve their cell uptake as well as
result in better target binding due to partial neutralization of
negatively charged phosphate groups. We have designed two
nonnucleosidic monomers incorporating aliphatic amino
groups that should be positively charged under physiological
conditions. One (5e) has a tertiary dimethylaminopropyl
group, and another (5f) has a primary amino group on a 13-
atom hydrophilic poly(ethylene glycol) linker. In addition, the
latter reagent could potentially be employed for label
attachment to, or bioconjugation of, modified oligonucleotides
via the primary amino group.
It was shown that cholesterol attachment can improve
cellular permeability of oligonucleotides and their analogues.22
The property is especially important for therapeutic antisense
agents. We have applied our new method for cholesterol
attachment via aminohexyl carbamate side-chain. The mono-
mer 5g proved to be sparingly soluble in acetonitrile, so 1:1
mixture of acetonitrile with THF was used, which resulted in
smooth incorporation of the cholesterol group into an
oligonucleotide chain in good yield.
In conclusion, we have described a novel, versatile and
efficient strategy for the design and synthesis of a family of side-
chain functionalized nonnucleosidic phosphoramidite mono-
mers for automated solid-phase oligonucleotide assembly. The
strategy is based on a simple protection-activation reaction of
diethanolamine to produce a key lactone that could be ring-
opened by a series of primary aliphatic amines. The monomers
were successfully employed to synthesize oligonucleotides
functionalized by one or more pendant groups that convey
hydrophobicity, positive charge, or click chemistry potential.
The new chemistry relies on simple, inexpensive, and easily
obtainable starting materials and good-yielding convergent
chemical syntheses. It should be readily applicable to a range of
backbone modifications such as RNA or other biologically
important 2′-substituted analogues. Our method could be
conveniently used to obtain oligonucleotides with diverse sets
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