Synthesis of anthraquinone oligonucleotides for triplex stabilization

Article abstract of DOI:10.1080/15257770701506491

The synthesis of two anthraquinone phosphoramidites is described. In both cases the anthraquinone moiety is attached via a linker to the 5-position of a uracil base, allowing incorporation at any thymidine position in an oligonucleotide sequence. Anthraqu

Full text of DOI:10.1080/15257770701506491

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Synthesis of Anthraquinone
Oligonucleotides for Triplex Stabilization
Zhengyun Zhao a , Guomei Peng a , Jan Michels b , Keith R. Fox c &
Tom Brown a
a School of Chemistry, University of Southampton, Highfield,
Southampton, United Kingdom
b Department of Chemistry, University of Hamburg, Hamburg,
Germany
c School of Biological Sciences, University of Southampton,
Southampton, United Kingdom
Published online: 10 Dec 2007.
To cite this article: Zhengyun Zhao , Guomei Peng , Jan Michels , Keith R. Fox & Tom Brown (2007):
Synthesis of Anthraquinone Oligonucleotides for Triplex Stabilization, Nucleosides, Nucleotides and
Nucleic Acids, 26:8-9, 921-925
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Nucleosides, Nucleotides, and Nucleic Acids, 26:921–925, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701506491
SYNTHESIS OF ANTHRAQUINONE OLIGONUCLEOTIDES FOR
TRIPLEX STABILIZATION
Zhengyun Zhao and Guomei Peng
School of Chemistry, University of
2
Southampton, Highfield, Southampton, United Kingdom
Jan Michels
Department of Chemistry, University of Hamburg, Hamburg, Germany
2
2
Keith R. Fox
School of Biological Sciences, University of Southampton, Southampton,
United Kingdom
To m B rown
School of Chemistry, University of Southampton, Highfield,
2
Southampton, United Kingdom
The synthesis of two anthraquinone phosphoramidites is described. In both cases the an-
2
thraquinone moiety is attached via a linker to the 5-position of a uracil base, allowing incorporation
at any thymidine position in an oligonucleotide sequence. Anthraquinone-modified oligonucleotides
have potential applications as triplex stabilizers and fluorescence quenchers.
Keywords Anthraquinone; triplex stabilizers; fluorescence quenchers
INTRODUCTION
Triplex-forming oligonucleotides (TFOs),[1] have potential uses as ther-
apeutic agents, since they could regulate gene expression by direct inter-
action with genomic dsDNA. However, the resulting triplex is usually less
stable than the corresponding duplex. In order to improve triplex stability,
we have attached substituted anthraquinones to uracil bases in the expecta-
tion that intercalation of the anthraquinone moiety will stabilize the triple
helix.[2] It also was anticipated that an attached tetramine[3] would further
increase triplex stability by interacting with the phosphodiester groups of
the DNA. We now report the synthesis of oligonucleotides containing the
anthraquinone nucleosides in Figure 1.
This research is funded by BBSRC. All oligonucleotide synthesis and purification was carried out by
ATDBio Ltd., UK (www.atdbio.com).
Address correspondence to Tom Brown, School of Chemistry, University of Southampton, High-
field, Southampton, SO17 1BJ, UK. E-mail: tb2@soton.ac.uk
921

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Z. Zhao et al.
FIGURE 1 Anthraquinone moiety linked to the 5-position of 2ꢁ-deoxyuridine.
SYNTHESIS
The tetramine, tris(2-aminoethyl)amine and 1,6-diaminohexane were
introduced onto the anthraquinone nucleus B by sequential nucleophilic
substitution reactions (Scheme 1). Firstly the tetramine was added and pro-
tected with trifluoroacetyl to give C. The trifluoroacetyl protecting groups
are compatible with standard DNA synthesis deprotection conditions. Af-
ter this the diaminohexane was coupled to the remaining nucleophilic
site.
Reaction of 5ꢁ-O-DMTr-5-iodo-2ꢁ-deoxyuridine E with heptynoic acid
gave the 5-substituted nucleoside F (Scheme 2). The anthraquinone-bearing
tetramine D was coupled to the carboxylic acid group of the substituted de-
oxyuridine F using EDC and HOBT, then converted into phosphoramidite
G for incorporation into oligonucleotides.
SCHEME 1 Preparation of anthraquinone-tetramine derivatives. (i) tris(2-aminoethyl)amine (3 eq),
toluene, reflux 3 hours; (ii) ethyl trifluoroacetate (10 eq), triethylamine (10 eq), rt 5 hours, combined
yield 31%, (iii) 1,6-diaminohexane, xylene, reflux overnight, 40%.

Anthraquinone Oligonucleotides
923
SCHEME 2 Synthesis of uridine bearing anthraquinone-tetramine phosphoramidite. (i) heptynoic acid,
triethylamine, CuI, (Ph3P)4Pd, DMF, 85%; (ii) D, EDC, HOBT, DIPEA, DMF, overnight, 65%; (iii) 2-
cyanoethyl bis-diisopropylphosphoramidite, DIHT, acetonitrile, 68%.
SCHEME 3 Synthesis of chloroanthraquinone bearing 2ꢁ-deoxyuridine phosphoramidite. (i) 1,6-
diaminohexane (10 eq), xylene, reflux, 63%; (ii) F, EDC, HOBT, DIPEA, DMF, 86%; (iii) 2-cyanoethyl
bis-diisopropylphosphoramidite (1.1 eq), DIHT (0.5 eq), acetonitrile, 88%.

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Z. Zhao et al.
FIGURE 2 Sequences used for fluorescence quencher and fluorescence melting experiments. (a) X =
FAM Y = tetramine-anthraquinone dU (A2); (b) 5 = 5-methyl dC, 6 = propargylamino-dU, 7 = HEG, t
= Fluorescein dT (Glen Research), Y = tetramine-anthraquinone dU (A2).
The synthesis of chloroanthraquinone 2ꢁ-deoxyuridine (Figure 1, A1),
is shown in Scheme 3. Applying a similar strategy, 1,6-diaminohexane was
introduced onto the anthraquinone B, followed by coupling with the car-
boxylic function of nucleoside F using EDC and HOBT, and was finally
converted into phosphoramidite J. The coupling and phosphitylation yields
were higher than the corresponding tetramine bearing compound G.
Oligonucleotides were prepared using phosphoramidite monomers G
and J, and standard phosphoramidite DNA synthesis reagents on an ABI
394 DNA/RNA synthesizer. The coupling time for G and J was extended
from 25 seconds to 6 minutes and deprotection was carried out with con-
centrated aqueous ammonia at 55◦C overnight. Oligonucleotide products
were analyzed and purified by reversed-phase HPLC and gel electrophore-
sis, and characterized by MALDI-TOF MS.
The oligonucleotide bearing anthraquinone-tetramine A2 was evaluated
as a fluorescence quencher.[4] It was labelled at the 3ꢁ-end of the Taqman
sequences in Figure 2a and used in real-time PCR to monitor the fluores-
cence changes. The short Taqman sequence was found to be more efficient
in terms of cleavage and fluorescence change.
Preliminary fluorescence melting experiments,[5] on TFOs formed
by the sequences in Figure 2b, indicate that the anthraquinone-bearing
tetramine (A2) significantly stabilizes triplexes especially when the modi-
fication is at the 5ꢁ-end of the TFO. It is significantly more effective than the
chloro-version (A1).
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1. Felsenfeld, G.; Davies, D.R.; Rich, A. Formation of a three-stranded polynucleotide molecule. J. Am
Chem. Soc. 1957, 2023–2024.

Anthraquinone Oligonucleotides
925
2. Huang, H.-S.; Chiu, H.-F.; Yeh, P.-F.; Yuan, C.-L. Structure-based design and synthesis of regioisomeric
disubstituted aminoanthraquinone derivatives as potential anticancer agents. Helv. Chim. Acta 2004,
87, 999–1006.
3. Zafrul Azam, A.; Moriguchi, T.; Shinozuka, K. Modified α–β chimeric oligoDNA bearing a multi-
conjugate of 2,2-bis(hydroxymethyl) propionic acid-anthraquinone-polyamine exhibited improved
and stereo-non-specific triplex-forming ability. Chem. Commun. 2006, 335–337.
4. May, J.P.; Brown, L.J.; van Delft, I.; Thelwell, N.; Harley, K.; Brown, T. Synthesis and evaluation of a
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