Versatile strategy for oligonucleotide derivatization. Introduction of lanthanide(III) chelates to oligonucleotides

Article abstract of DOI:10.1021/ol016093m

matrix presented R = a linker containg a protected functional or conjugate group Novel nucleosidic phosphoramidite blocks were synthesized by a Mitsunobu reaction between 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)uridine and a primary alcohol containing a conju

Full text of DOI:10.1021/ol016093m

ORGANIC
LETTERS
2001
Vol. 3, No. 16
2473-2476
Versatile Strategy for Oligonucleotide
Derivatization. Introduction of
Lanthanide(III) Chelates to
Oligonucleotides
Jari Hovinen* and Harri Hakala
PerkinElmer Life Sciences, Wallac Oy, P.O. Box 10, FIN-20101 Turku, Finland
jari.hoVinen@perkinelmer.com
Received May 10, 2001
ABSTRACT
Novel nucleosidic phosphoramidite blocks were synthesized by a Mitsunobu reaction between 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)uridine and
a primary alcohol containing a conjugate group in its structure (a protected functional group, an organic dye, or a precursor of a lanthanide-
(III) chelate) followed by phosphitylation. They were used in machine-assisted DNA synthesis in the standard manner. A slighty modified
deprotection procedure was used for the preparation of oligonucleotide conjugates tethered to lanthanide(III) chelates. For the latter application
one non-nucleosidic block was also synthesized.
Synthetic oligonucleotides tethered to various ligands have
been used as research tools in molecular biology.1 They have
been applied to genetic analysis and used to elucidate the
mechanism of gene function. Oligonucleotides carrying
reporter groups have had widespread use for automated DNA
sequencing, hybridization affinity chromatography, and
fluorescence microscopy. Oligonucleotide-biotin conjugates
are widely used as hybridization probes.
The fluorescent label monomers for solid phase oligo-
nucleotide chemistry are most commonly organic dyes, and
several of those blocks are even commercially available.
However, such labels and labeled biomolecules suffer from
many drawbacks such as Raman scattering, low water
solubility, and concentration quenching. Thus multilabeling
of oligonucleotides with organic fluorophores may not
enhance the detection sensitivity to the degree needed in
several applications. For these type of applications lanthani-
de(III) chelates are labels of choice because they do not suffer
from these phenomena.2
Multilabeling of oligonucleotides can be performed by
three alternative methods:3 (i) by coupling several base- or
carbohydrate-tethered nucleosidic building blocks to the
growing oligonucleotide chain, (ii) by functionalization of
the internucleosidic phosphodiester linkages, or (iii) by using
several multifunctional non-nucleosidic building blocks dur-
ing the oligonucleotide chain assembly. All of these methods
have their own drawbacks. Since the double helix formation
of DNA is based on hydrogen bonding between the comple-
mentary base residues, tethers attached to the base moieties
often weaken these interactions. This problem is easily
(1) For reviews, see: Iyer, R. P.; Roland, A.; Zhou, W.; Ghosh, K. Curr.
Opin. Mol. Ther. 1999, 1, 344. Uhlman, E.; Peyman, A. Chem. ReV. 1990,
90, 543. English, U.; Gauss, D. H. Angew. Chem., Int. Ed. Engl. 1991, 30,
613.
(2) Hemmila¨, I.; Dakubu, S.; Mukkala, V.-M.; Siitari, H.; Lo¨vgren, T.
Anal. Biochem. 1984, 137, 335.
(3) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123. Wojczewski,
C.; Stolze, K.; Engels, J. W. Synlett 1999, 1667.
10.1021/ol016093m CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/07/2001

overcome by using the tethered nucleosides at the 3′- or 5′-
terminus of the coding sequence or by using labels linked
to C5 of pyrimidine residues. Introduction of tethers to the
phosphate backbone often gives rise to new chiral centers
and makes the purification of these analogues difficult.
Introduction of the tether arm to the carbohydrate moiety,
in turn, often decreases the coupling efficiency of the
phosphoramidite. Although design of non-nucleosidic blocks
may look attractive on paper, very often their syntheses suffer
from complexity, low coupling yields, and problems associ-
ated with the storage and handling of the phosphoramidites.
For several applications design of base-tethered nucleosidic
building blocks is often the method of choice.
bis(methoxycarbonylmethyl)aminomethyl]pyridine (4)5 to
5-hexynol (Scheme 2). The dabsyl derivative, 7, was obtained
Scheme 2
The present synthetic strategy for the preparation of
nucleosidic phosphoramidite building blocks is shown in
Scheme 1. Accordingly, reaction of 2′-deoxy-5′-O-(4,4′-
Scheme 1. Present Strategy for the Preparation of Nucleosidic
Phosphoramidites
by allowing dabcyl chloride (6) to react with an excess of
6-aminohexanol in dichloromethane.
Synthesis of the terpyridine structure, 15, is outlined in
Scheme 3. In general, the synthesis was analogous to that
published for the corresponding 4-amino derivative.6 Ac-
cordingly, 4-bromobenzaldehyde was condensed with 2-acetyl-
pyridine by a Claisen-Schmidt reaction to give the (E)-prop-
2-enone, 8. Reaction of this with N-[2-(pyrid-2′-yl)-2-
oxoethyl]pyridinium iodide, 9, and ammonium acetate in
methanol yielded the terpyridine derivative, 10, in high yield.
The terminal pyridine moieties were then oxidized with
3-chloroperbenzoic acid to N,N′′-dioxides to give rise to 11.
The modified Reissert-Henze reaction yielded the 6,6′-
dicarbonitrile 12, which was reduced with borane to the
corresponding bis(aminomethyl) compound, 13. It was
carboxymethylated to the tetraester, 14, with methyl bro-
moacetate in the presence of diisopropylethylamine and
potassium iodide as a catalyst. Finally, reaction of the
bromide 14 with 5-hexynol in the presence of Pd(II) and
Cu(I) gave the ligand 15.
Synthesis of the Phosphoramidites and Solid Supports.
To demonstrate the versatility of the present method for
nucleoside derivatization 1 was allowed to react with N6-
trifluoroacetamidohexanol as well as the alcohols 5, 7, and
15 in the presence of triphenyl phosphine and DEAD.
The nucleoside derivatives 2a-d were obtained in high
yield. Their structures were confirmed on NMR analyses,
(4) Mitsunobu, O. Synthesis 1981, 1.
(5) This compound was synthesized as described for the corresponding
tert-butyl ester: Takalo, H.; Mukkala, V.-M.; Mikola, H.; Liitti, P.;
Hemmila¨, I. Bioconjugate Chem. 1994, 5, 278.
(6) Mukkala, V.-M.; Helenius, M.; Hemmila¨, I.; Kankare, J.; Takalo, H.
HelV. Chim. Acta 1993, 76, 1361.
(7) Mitsunobu reaction between of 3′,5′-O-protected dU and 5-trifluo-
roacetamidopentan-1-ol has been reported; see: Brossette, T.; Le Faou, A.;
Vaillex, A.; Criminon, C.; Grassi, J.; Mioskowski, C.; Lebeau, L. J. Org.
Chem. 1999, 64, 5083.
(8) Dahle´n, P.; Liukkonen, L.; Kwiatkowski, M.; Hurskainen, P.; Iitia¨,
A.; Siitari, H.; Ylikoski, J.; Mukkala, V.-M.; Lo¨vgren, T. Bioconjugate
Chem. 1994, 5, 268.
(9) Kwiatkowski, M.; Samiotaki, M.; Lamminma¨ki, U.; Mukkala, V.-
M.; Landegren, U. Nucleic Acids Res. 1994, 22, 2604.
(10) DELFIA and LANCE are trademarks of PerkinElmer Life Sciences.
dimethoxytrityl)uridine (1) with a primary alcohol under
Mitsunobu conditions4 gives the N3-derivatives, 2, phosphi-
tylation of which results in the desired phosphoramidites, 3.
The key steps are discussed below in detail.
Synthesis of the Alcohols. The ligand, 5, was synthesized
by palladium-catalyzed coupling of 4-bromo-2,6-bis[N,N-
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Org. Lett., Vol. 3, No. 16, 2001

Scheme 3
and the site of alkylation was in all cases undoubtedly N3
blocks were coupled to the 5′-terminus of the oligomers. No
differences in coupling efficiency between 3a-d and com-
mercial nucleoside monomers were detected. The oligo-
nucleotide conjugates synthesized in the aid of blocks 3a
and 3c were deprotected by conventional ammonialysis. An
oligonucleotide bearing 10 primary amino groups was further
labeled with a nonluminescent europium(III) chelate, 19,5
in solution8 according to Scheme 5.
(see Supporting Information). It is worth noting that under
the reaction conditions employed 3′-O-protection of the
nucleoside is not required.7 Finally, treatment of 2a-d with
2-cyanoethyl N,N,N’,N’-tetraisopropylphosphordiamidite in
the presence of 1H-tetrazole yielded the corresponding
phosphoramidites, 3a-d. Purification was performed on
silica gel or by precipitation from cold (-70 °C) hexanes.
To attach the labels to the 3′-terminus of oligonucleotides,
synthesis of the corresponding modified solid support is-
needed. As an illustrative example 2a was allowed to react
with succinic anhydride, giving the corresponding succinate,
17, which was immobilized on a long chain alkylamine CPG
polymer support with N,N′-diisopropylcarbodiimide (Scheme
4).
Direct introduction of lanthanide(III) chelates to oligo-
nucleotides required a modified deprotection strategy9 as
described in Scheme 6. Accordingly, after completion of the
oligonucleotide synthesis the fully protected oligomer was
initially treated with sodium hydroxide (0.1 M; 4 h at room
temperature) to ensure total hydrolysis of the ester protecting
groups followed by ammonialysis to complete the base
deprotection. Treatment of the deblocked oligomer with
europium(III) citrate converted the oligonucleotide con-
jugate to the corresponding europium(III) chelate.
Synthesis of the Oligonucleotides. To demonstrate the
applicability of the building blocks 3a-d for oligonucleotide
synthesis, several model sequences were synthesized using
standard machine-assisted phosphoramidite chemistry. These
Scheme 4. Synthesis of the Solid Support with the Aid of 2a
Org. Lett., Vol. 3, No. 16, 2001
2475

simplified by omitting the nucleobase, resulting in non-
nucleosidic phosphoramidite building blocks such as 16. This
block was successfully coupled to the 5′-terminus of a 45-
mer oligonucleotide. Deprotection strategy was similar to
that for nucleosidic lanthanide blocks 3b and 3d. The
oligonucleotides labeled with lanthanide chelates were used
in DELFIA10-based DNA hybridization assays as well as
LANCE fluorescence quenching assays (Helicase, RNase,
Molecular Beacons). These results will be published else-
where.
Scheme 5. Introduction of Europium(III) Chelates to
Oligonucletides in the Aid of Block 3a
The current method for oligonucleotide derivatization has
the following advantages: (i) Because the coupling reaction
between the nucleoside and the tether molecule is performed
under mild reaction conditions (at ambient temperature in
dry THF) a wide range of tethers and label molecules can
be introduced. The only requirement is that the tether
molecule has a primary hydroxyl group in its structure and
other functional groups are protected. (ii) The nucleosidic
blocks are solid materials, which makes their storage and
handling convenient (by contrast, the non-nucleosidic block
16 is an oil). (iii) The blocks can be incorporated into the
oligonucleotide structure in high efficiency using standard
protocols of machine-assisted DNA chemistry. In is worth
noting that since the labels attached to the N3 positon of
uracil residues naturally weaken hydrogen bonds of in the
dublex, these labels should be used only up- or downstream
of the coding sequence.
Acknowledgment. We thank Mrs. Mirja Koski and Ms.
Jenni Degerholm for skillful technical assistance.
Supporting Information Available: Experimental pro-
cedures and characterization data for the compounds pre-
pared. This material is available free of charge via the Internet
at http://pubs.acs.org.
For several applications, introduction of only a single label
molecule to the 5′-terminus the oligonucleotide structure is
needed. For these applications the ligand structure can be
OL016093M
Scheme 6. Introduction of Lanthanide(III) Chelates to Oligonucletides in the Aid of Block 3b
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Org. Lett., Vol. 3, No. 16, 2001