Synthesis of oligoribonucleic acid conjugates using a cyclooctyne phosphoramidite

Article abstract of DOI:10.1021/ol102357u

The conjugation of a ribonucleic acid 16-mer with the cationic amphiphilic peptide penetratin and an anionic hyaluronan tetrasaccharide by means of Cu-free "click" chemistry is reported. The alkyne-functionalized 16-mer was prepared by automated solid-pha

Full text of DOI:10.1021/ol102357u

ORGANIC
LETTERS
2010
Vol. 12, No. 23
5486-5489
Synthesis of Oligoribonucleic Acid
Conjugates Using a Cyclooctyne
Phosphoramidite
Pieter van Delft, Nico J. Meeuwenoord, Sascha Hoogendoorn, Jasper Dinkelaar,
Herman S. Overkleeft, Gijsbert A. van der Marel,* and Dmitri V. Filippov*
Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box 9502,
2300 RA Leiden, The Netherlands
filippoV@chem.leidenuniV.nl; marel_g@chem.leidenuniV.nl
Received September 30, 2010
ABSTRACT
The conjugation of a ribonucleic acid 16-mer with the cationic amphiphilic peptide penetratin and an anionic hyaluronan tetrasaccharide by
means of Cu-free “click” chemistry is reported. The alkyne-functionalized 16-mer was prepared by automated solid-phase synthesis, using a
newly developed strained cyclooctyne phosphoramidite in the final coupling. Cycloaddition of the alkyne functionalized RNA to the azide
containing biomolecules led to a clean conversion into the corresponding nucleic acid conjugates.
Oligonucleotides functionalized in such a manner that they
can be reliably connected to other entities (biomolecules,1,2
surfaces3,4 ) have attracted considerable interest in life
sciences, nanotechnology, and adjacent fields of research.
For instance, advances in the development of nucleic acid
based diagnostics and therapeutics rely on the availability
of well-defined oligonucleotides provided with reporter
molecules such as fluorescent labels5,6 or targeting devices.
In this respect, research toward inhibition of gene expression
via antisense, RNAi, or antigene approaches explores the
use of oligonucleotides conjugated to peptides,7,8 carbohy-
drates,9 or small molecules10 to acquire therapeutics with
improved properties in terms of stability to enzymatic
degradation, cellular uptake, and targeting.11,12 Over the past
decades, numerous conjugation methods, including amide,
disulfide, thioether, imine, and oxime formation, have been
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10.1021/ol102357u  2010 American Chemical Society
Published on Web 11/04/2010

Scheme 1. Synthesis of the Strained Cyclooctyne Phosphoramidite
reported.13-15 While most of these methods have their merits,
they are not generally applicable due to limitations such as
lack of selectivity and incomplete reactions.
The nucleic acid fragments, resulting from this approach,
are provided with dibenzocyclooctynol function at the 5′ end,
allowing cycloaddition with azides to give oligonucleotide
conjugates having intact primary and secondary structure.
Phosphoramidite 5 was designed as a suitable and easily
accessible phosphitylating reagent.
The advent of the Cu(I)-catalyzed16,17 Huisgen [3 + 2]
cycloaddition between an alkyne and an azide, commonly
referred to as the “click” reaction, enables the selective
formation of a specific triazole product in an aqueous and
otherwise complex chemical environment. In recent years,
DNA fragments have been conjugated to oligopeptides,
oligosaccharides, and fluorescent dyes in this fashion. In a
postsynthetic approach, oligonucleotides functionalized with
either an alkyne or an azide are conjugated with comple-
mentary functionalized molecules after standard automated
DNA synthesis and ensuing removal of the protecting
groups.18 In this approach, alkyne or azide functionalities
are appended to nucleobases, the ribose, or the terminal
phosphate of the synthetic DNA fragments.19 Alkyne func-
tions are more often incorporated in DNA fragments, as
azides are usually incompatible with the phosphoramidite
chemistry applied in the solid-phase synthesis of oligonucle-
otides. The necessity to use a Cu(I)-stabilizing ligand20 and
oxygen-free conditions to increase the cycloaddition rate and
to minimize the degradation of DNA has shifted the attention
to the development of Cu-free click reactions. This aim
corresponds with the new metal-free bioorthogonal reactions
developed in the field of chemical biology and involves the
reaction of strained cycloalkynes with azides,21-23 the
reaction of strained alkenes with tetrazines,24a,b the reaction
of oxabornadienes with alkynes,25 and a strategy based on a
photoreaction-mediated liberation of a strained alkyne for
ensuing click ligation.26 Recent methodologies to nucleic acid
conjugates apply the nitrile oxide-norbornene27 click chem-
istry and the Diels-Alder reaction.28a,b As part of a pro-
gram29 to design and evaluate nucleic acid conjugates, we
became interested in the bioconjugation procedure of the
group of Boons,23 in which 4-dibenzocyclooctynols are
applied for the visualization of metabolically labeled gly-
coconjugates. Dibenzocyclooctynols are easily synthesized
and react rapidly with azides. We envisaged that derivati-
zation of dibenzocyclooctynol and ensuing conversion into
a phosphoroamidite would afford a phosphitylation agent
applicable for automated solid-phase nucleic acid synthesis.
Previously described cyclooctynol 130 (Scheme 1) was
treated with p-nitrophenyl chloroformate. The resulting
carbonate 2 was used without further purification to react
with 2 equiv of TBDPS-protected aminohexanol,31 furnishing
3 in near quantitative yield. TBAF-mediated removal of the
TBDPS group of 3 and subsequent phosphitylation of the
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Org. Lett., Vol. 12, No. 23, 2010
5487

Scheme 2. Synthesis of the Alkyne-T5 Sequence (6) and Subsequent Conjugation with the Fluorescent Label BODIPY
obtained alcohol 4 with commercially available 2-cyanoet-
hoxy-N,N-diisopropylamino-chloro-phosphine and DIPEA in
DCM led to the isolation of new phosphitylation reagent 5
in 75% overall yield.
recently reported by us,33 as a reaction partner for the
cycloaddition with the alkyne-T5 oligonucleotide (6). The
progress of the conjugation of stoichiometric amounts of
azide label 7 and alkyne-funtionalized pentamer 6 was
monitored by LC-MS analysis. After stirring the reaction
overnight, we found the reaction to be complete as indicated
by a single peak in the LC trace, which was confirmed to be
the product 8 by mass spectrometry data.
Following our success in synthesizing a fluorescently
labeled DNA fragment, we shifted our attention to the
construction of conjugates of the less stable and therefore
more challenging RNA oligomers. We selected a 16-mer
RNA fragment, in which all common nucleosides are
incorporated, for functionalization with the dibenzocyclooc-
tynol moiety. The alkyne-functionalized RNA fragment 9
(Scheme 3) was constructed by a standard RNA automated
solid-phase synthesis protocol, again employing the powerful
BMT activating agent. At the end of the synthesis, depro-
tection of the phosphotriesters and the exocyclic amine
groups of the nucleobases with methylamine in methanol
was accompanied by cleavage from the solid support to yield
the 2′-OTBS-protected oligomer. Removal of the silyl groups
with TEA·HF and subsequent purification by anion exchange
chromatography yielded target RNA fragment 9.
The adequacy of reagent 1 for application in automated
DNA synthesis was explored by the construction of alkyne-
functionalized thymine pentamer 6 (Scheme 2). The penta-
nucleotide T5 was assembled by the use of a standard four-
step elongation cycle, comprising coupling with 5 equiv of
commercially available thymidine phosphoramidite under
influence of the activating agent 5-(benzylmercapto)-1H-
tetrazole (BMT), oxidation with I2/pyridine/H2O, capping
with acetic anhydride, and finally removal of the dimethoxy-
trityl group by dichloroacetic acid. In the ultimate and crucial
steps of the solid-phase protocol, the immobilized pentamer
was functionalized by coupling with 6 equiv of amidite 5
and ensuing oxidation32 of the intermediate phosphite using
the standard conditions. Deprotection and cleavage from the
CPG solid support using aqueous ammonia was followed
by purification with reversed-phase HPLC to afford the target
alkyne-functionalized pentamer 6 (Scheme 2). Notably, the
presence of the hydrophobic dibenzocyclooctyne moiety
increased the retention time of our target compound in
comparison with the unfunctionalized oligomer significantly,
thereby simplifying purification by RP-HPLC.
For conjugation with oligonucleotide 9, the cationic
amphiphilic peptide penetratin 11 (Scheme 3) and a tet-
rameric fragment of hyaluronan 10 were selected. Penetratin,
a 16-amino acid peptide having the sequence Arg-Gln-Ile-
Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-
NH2 is known for its ability to internalize covalently attached
For the first conjugation event, we selected the azide-
functionalized Azido-BODIPY derivative (7), obtained as
(29) (a) Khan, S.; Bijker, M. S.; Weterings, J. J.; Tanke, H. J.; Adema,
G. J.; van Hall, T.; Drijfhout, J. W.; Melief, C. J. M.; Overkleeft, H. S.;
van der Marel, G. A.; Filippov, D. V.; van der Burg, S. H.; Ossendorp, F.
J. Biol. Chem. 2007, 282, 21145–21159. (b) van der Heden van Noort,
G. J.; van der Horst, M. G.; Overkleeft, H. S.; van der Marel, G. A.; Filippov,
D. V. J. Am. Chem. Soc. 2010, 132, 5236–5240. (c) van der Heden van
Noort, G. J.; Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V. J.
Org. Chem. 2010, 75, 5733–5736.
(32) Solution-phase experiments revealed that the alkyne is also stable
towards t-BuOOH (100 equiv, 90 min).
(33) The final synthetic step and full compound characterization can be
found in the Supporting Information. For further synthetic information and
intermediates, see: Verdoes, M.; Florea, B. I.; Hillaert, U.; Willems, L. I.;
van der Linden, W. A.; Sae-Heng, M.; Filippov, D. V.; Kisselev, A. F.;
van der Marel, G. A.; Overkleeft, H. S. ChemBioChem 2008, 9, 1735–
1738.
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2547–2552.
(31) Bartoszewicz, A.; Kalek, M.; Stawinski, L. Tetrahedron 2008, 64,
8843–8850.
5488
Org. Lett., Vol. 12, No. 23, 2010

Scheme 3
.
Synthesis of 5′-Alkyne Functionalized Ribonucleic Acid 16-mer and Its Conjugation to Hyaluronic Acid Tetramer and
Penetratin
peptides and oligonucleotides into many cell types.7 Hyalu-
ronic acid (HA), a linear polysaccharide of the glycosamineg-
lycan family, is involved in a wide variety of biological
processes, such as cell migration and proliferation. HA and
fragments thereof are known to be recognized by the CD44
protein, and studies have revealed that conjugates of this
carbohydrate showed an increased uptake in cancer cells due
to overexpression of the CD44 protein.34 The preparation
of azido-functionalized penetratin 11 started with automated
solid-phase peptide synthesis (SPPS), using RAM Tentagel
and Fmoc chemistry, followed by manual coupling with
azidopropionic acid succinimidyl ester.35 Final deprotection
and cleavage from the solid support followed by HPLC
purification yielded the peptide penetratin equipped with an
azide moiety 11. Conjugation with the RNA oligomer (9)
was performed in a phosphate buffer saline pH 7.3. Although
the solubility of the reaction partners was initially incomplete,
the reaction proceeded toward a clear solution and after 5 h.
LC-MS analysis showed the formation of a single product.
Next the conjugation of the RNA fragment with a
hyaluronan tetramer was undertaken. The tetrasaccharide 10
containing an azide spacer on the reducing end was obtained
as previously reported by our group.36 Similar cycloaddition
of tetrasaccharide 10 with the RNA oligomer 9 in water as
the solvent and shaking for 4 h resulted in complete
disappearance of starting materials and formation of conju-
gate 12, as observed by LC-MS. HPLC purification afforded
the target conjugates 12 and 13. Both the DNA and the RNA
conjugates were purified for analytical purposes. Purification
on a semipreparative extended polar selectivity column
allowed only partial separation of the two putative regioi-
somers. Hence, the conjugates were isolated as mixtures.
The facile preparation of dibenzocyclooctynol containing
phosphoramidite 5, the seamless implementation of this new
reagent in standard automated nucleic acid synthesis, as well
as the efficiency of the ensuing cycloaddition reaction of
equimolar quantities of alkyne-functionalized RNA (DNA)
oligomers with azide-containing biomolecules make this
method a valuable alternative for existing procedures toward
RNA (DNA) conjugates. The existence of a variety of azide-
functionalized entities such as nanoparticles and surfaces
allows the application of the here presented cyclooctyne-
functionalized oligonucleotides to other fields of research.
Future work from our side includes the conjugation of
oligonucleotides to proteins and the application of other
nucleic acid derivatives such as thiophosphoryl DNA29a and
2′-OMe RNA.
Acknowledgment. This work was funded by the Dutch
Organization for Scientific Research (NWO).
(34) Luo, Y.; Ziebell, R.; Prestwich, G. D. Biomacromolecules 2000,
1, 208–218.
Supporting Information Available: Spectroscopic data
and experimental procedures. This material is available free
of charge via the Internet at http://pubs.acs.org.
(35) Grandjean, C.; Boutonnier, A.; Guerreiro, C.; Foernier, J-. M.;
Mulard, L. A. J. Org. Chem. 2005, 70, 7123–7132.
(36) Dinkelaar, J.; Code´e, J. D. C.; van den Bos, L. J.; Overkleeft, H. S.;
van der Marel, G. A. J. Org. Chem. 2007, 72, 5737–5742.
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