The synthesis of diene-containing nucleoside phosphoramidites and their use in the labeling of oligonucleotides

Article abstract of DOI:10.1081/NCN-200060085

A variety of furan-modified nucleoside phosphoramidite monomers has been prepared and efficiently incorporated into oligonucleotides. These take part in Diels-Alder reactions with fluorescent maleimides to give fluorescent-labeled oligonucleotides. This r

Full text of DOI:10.1081/NCN-200060085

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THE SYNTHESIS OF DIENE-CONTAINING NUCLEOSIDE
PHOSPHORAMIDITES AND THEIR USE IN THE LABELING
OF OLIGONUCLEOTIDES
Emma Anderson a & Douglas Picken a
a Link Technologies Ltd. , Scotland, UK
Published online: 15 Nov 2011.
To cite this article: Emma Anderson & Douglas Picken (2005) THE SYNTHESIS OF DIENE-CONTAINING NUCLEOSIDE
PHOSPHORAMIDITES AND THEIR USE IN THE LABELING OF OLIGONUCLEOTIDES, Nucleosides, Nucleotides and Nucleic Acids,
24:5-7, 761-765, DOI: 10.1081/NCN-200060085
To link to this article: http://dx.doi.org/10.1081/NCN-200060085
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):761–765, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060085
THE SYNTHESIS OF DIENE-CONTAINING NUCLEOSIDE
PHOSPHORAMIDITES AND THEIR USE IN THE
LABELING OF OLIGONUCLEOTIDES
5
Emma Anderson and Douglas Picken
Link Technologies Ltd., Scotland, UK
5
A variety of furan-modified nucleoside phosphoramidite monomers has been prepared and efficiently
incorporated into oligonucleotides. These take part in Diels-Alder reactions with fluorescent maleimides
to give fluorescent-labeled oligonucleotides. This represents a strategy for oligonucleotide labeling that is
orthogonal to amine-based methods.
INTRODUCTION
The need to detect DNA sequences with high sensitivity[1] has seen the
development of a plethora of techniques in molecular biology such as fluorogenic
5’ nuclease assays, molecular beacons, and scorpion probes. Central to the use of
all these techniques is the ability to synthesize oligonucleotides modified with
suitable reporter groups at specific positions. While the chemistry for the synthesis
of oligonucleotides is now very well established, the incorporation of some
desirable labels is problematic because of their incompatibility with the phos-
phoramidite method, most especially the deprotection conditions. While tech-
niques have been developed to overcome these difficulties by using amino
modifications for post-synthetic labeling, some significant drawbacks remain.
In this work we have set out to expand the repertoire of available techniques for
the labelling of oligonucleotides by the synthesis of a variety of phosphoramidite
monomers containing electron-rich dienes that can participate in a Diels-Alder
reaction with electron-deficient alkenes. Many fluorophores are available as
maleimide derivatives, which are suitable dienophiles.
Address correspondence to Douglas Picken, Link Technologies Ltd., 3 Mallard Way, Strathclyde Business
Park, Bellshill, Lanarkshire, ML4 3BF, Scotland, UK.
761
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E. Anderson and D. Picken
MONOMER SYNTHESIS
The furan ring has been widely used as an electron-rich diene in the Diels-
Alder reaction. Due to the convenience of its introduction and relative stability we
have chosen to concentrate on this modification.
As a first approach to the modification of oligonucleotides with furan residues,
we chose to prepare phosphoramidite 1, where the modification is at the N4-
position of a deoxycytidine residue. The synthetic route to this compound is shown
in Scheme 1.
While it was felt that this particular monomer was useful in the preparation of
terminally modified oligonucleotides, it would be desirable to have a method of
modification that would not interfere with Watson-Crick hydrogen bonding.
One of the most widely used positions for base modification is the 5-position of
pyrimidine residues and we have prepared two different compounds bearing furan
groups at this position.
The synthesis of monomer 2 starts from 5’-DMTr-5-bromo-deoxyuridine (see
Scheme 2), which is transformed by reaction with liquid ammonia[3] into the
5-amino compound. Coupling of this with 3-furfuryl propanoic acid and subsequent
phosphitylation leads to the desired phosphoramidite 2 in an overall yield of 53%.
SCHEME 1 Starting from deoxycytidine, the amino group was transaminated with furfuryl amine in a bisulphite-
catalysed reaction,[2] followed by standard dimethoxytrityl protection of the 5’ hydroxyl group and subsequent
phosphitylation. This series of reactions led to compound 1 in an overall yield of 40%. The coupling of this
phosphoramidite using standard cycles occurs as efficiently as standard monomers.

E. Anderson and D. Picken
763
SCHEME 2
SCHEME 3

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E. Anderson and D. Picken
This monomer was used for oligonucleotide synthesis and behaves in all respects as
a standard nucleoside phosphoramidite.
A further member of this series was prepared in order to provide a monomer
with a spacer portion where the reactive furan is further away from the oligo-
nucleotide, since it was felt that this would be a desirable feature for many ex-
periments. Starting with 5’-DMTr-5-iodo-deoxyuridine, propargylamine was
coupled using a Sonogoshira procedure[4] (see Scheme 3). Subsequent carbonyl
diimidazole-mediated coupling with furfuryl succinamidic acid and phosphitylation
yielded the required phosphoramidite 3 in 38% overall yield. This compound is not
soluble in acetonitrile at the concentrations used for synthesis, but can be
successfully incorporated into oligonucleotides by using THF as co-solvent.
CONJUGATIONS
Each of the monomers described above has been used in the synthesis of
oligonucleotides modified both at the 5’ end and at internal positions. Conjugation
experiments with commercially available fluorescent maleimides were then carried
out. In general, the conditions used have been to incubate a five-fold molar excess
of the maleimide with the oligonucleotide in 100 mM citrate/phosphate buffer pH
5.0 for several hours at 37°C. A typical example of a crude conjugation mixture is
shown in Figure 1, where Alexa 488 C5 maleimide is conjugated to the sequence
indicated, where X represents monomer 2. Experiments to optimize the con-
jugation reactions are ongoing.
FIGURE 1

E. Anderson and D. Picken
765
CONCLUSIONS
A variety of furan-bearing phosphoramidite monomers have been synthesized
and efficiently incorporated into oligonucleotides. Furan-modified oligonucleotides
enter into Diels-Alder reactions with maleimido-fluorophores to yield fluorescently
labelled products. This methodology is a promising route to multiply labeled
oligonucleotides and is orthogonal to amine-based strategies.