2′-O-dimethylaminoethoxyuridine and 5-dimethylaminopropargyl deoxyuridine for at base pair recognition in triple helices

Article abstract of DOI:10.1080/15257770701530525

The nucleoside analogues 2′-O-dimethylaminoethoxy uridine and 5-dimethylaminopropargyl deoxyuridine have been synthesised and incorporated into oligonucleotides. Their triplex-stabilising properties have been determined in fluorescence melting experiments

Full text of DOI:10.1080/15257770701530525

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2′-O-Dimethylaminoethoxyuridine and
5-Dimethylaminopropargyl Deoxyuridine
for at Base Pair Recognition in Triple
Helices
Lavinia Brennan a , Guomei Peng a , Natarajan Srinivasan a , Keith R.
Fox b & Tom Brown a
a School of Chemistry , University of Southampton , Highfield,
Southampton, United Kingdom
b School of Biological Sciences , Basset Crescent East, University of
Southampton , Southampton, United Kingdom
Published online: 05 Dec 2007.
To cite this article: Lavinia Brennan , Guomei Peng , Natarajan Srinivasan , Keith R. Fox & Tom Brown
(2007) 2′-O-Dimethylaminoethoxyuridine and 5-Dimethylaminopropargyl Deoxyuridine for at Base Pair
Recognition in Triple Helices, Nucleosides, Nucleotides and Nucleic Acids, 26:10-12, 1283-1286, DOI:
10.1080/15257770701530525
To link to this article: http://dx.doi.org/10.1080/15257770701530525
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1283–1286, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701530525
2ꢀ-O-DIMETHYLAMINOETHOXYURIDINE
AND 5-DIMETHYLAMINOPROPARGYL DEOXYURIDINE
FOR AT BASE PAIR RECOGNITION IN TRIPLE HELICES
Lavinia Brennan, Guomei Peng, and Natarajan Srinivasan
School
2
of Chemistry, University of Southampton, Highfield, Southampton, United Kingdom
Keith R. Fox School of Biological Sciences, Basset Crescent East, University
2
of Southampton, Southampton, United Kingdom
To m B rown School of Chemistry, University of Southampton, Highfield,
2
Southampton, United Kingdom
The nucleoside analogues 2ꢁ-O-dimethylaminoethoxy uridine and 5-dimethylaminopropargyl
2
deoxyuridine have been synthesised and incorporated into oligonucleotides. Their triplex-stabilising
properties have been determined in fluorescence melting experiments.
Keywords Antigene; Triple helices; Triplexes; AT recognition
INTRODUCTION
Triplex-forming oligonucleotides (TFOs) containing 2ꢁ-O-aminoethoxy-
5-aminopropargyluridine (bis-amino U) form very stable triplets with AT
base pairs in double stranded DNA, making these modified TFOs poten-
tially useful in antigene therapy.[1] The stabilising effect is due to specific in-
teractions between the charged primary amino groups attached to the mod-
ified nucleoside and the phosphodiester backbone of the complementary
duplex.[2] (The pro-R phosphate group in the case of the 2ꢁ-aminoethoxy
side-chain.)
One of the drawbacks of the bis-amino U modified nucleoside is
the requirement for amino protecting groups during solid-phase oligonu-
cleotide synthesis. In certain cases the conditions required to remove these
This research was funded by grants from BBSRC and EU. Oligonucleotide synthesis was carried out
by ATDBio Ltd. (www.atdbio.com).
Address correspondence to L. Brennan, School of Chemistry, University of Southampton,
Highfield, Southampton SO17 1BJ, UK. E-mail: L. Brennan@soton.ac.uk
1283

1284
L. Brennan et al.
SCHEME 1 Synthesis of 5-dimethylaminopropargyl deoxyuridine. (i) DMTCl, pyridine, rt, 3 hours,
85%; (ii) (Me)2NCH2CCH, Et3N, CuI, Pd0(PPh3)4, DMF, rt, 16 hours, 84%; (iii) (i Pr)2NPCl
(OCH2CH2CN), THF, 2 hr, rt, 72%.
protecting groups (e.g., aqueous methylamine) are too harsh for certain
other modifications in the DNA strand (e.g., the cross-linker Psoralen).
In order to circumvent this problem we have prepared phos-
phoramidites of 5-dimethylaminopropargyl deoxyuridine 3 and 2ꢁ-O-
dimethylaminoethoxyuridine 7, incorporated them into oligonucleotides
and determined their triplex-stabilising properties in fluorescence melting
experiments. The synthesis of the two phosphoramidite monomers 3 and 7
is described and results of fluorescence melting experiments are presented.
RESULTS AND DISCUSSION
Synthesis of 5-Dimethylaminopropargyl Deoxyuridine
The synthesis of 5-aminopropargyl deoxyuridine has been described
previously.[3] A similar strategy was employed for 5-dimethylaminopropargyl
deoxyuridine (Scheme 1). Starting from 5-iodo-2ꢁ-deoxyuridine, protection
of the 5ꢁ-hydroxyl group was achieved using dimethoxytrityl chloride in pyri-
dine to give the corresponding 5ꢁ-O-dimethoxytrityl ether 1. Compound 1
was then coupled with 3-dimethylaminopropyne under Pd-catalysed cross-
coupling conditions[4] to give compound 2, which was then converted to
the corresponding phosphoramidite 3.
Synthesis of 2ꢁ-O-Dimethylaminoethoxyuridine
This synthesis was achieved using published procedure[5,6] (Scheme 2).
Uridine was converted into 2,2ꢁ-anhydro-1-β-D-arabinofuranosyluracil 4 us-
ing phenyl carbonate in the presence of sodium hydrogen carbonate

2ꢁ-O-Dimethylaminoethoxyuridine and 5-Dimethylaminopropargyl
1285
SCHEME 2 Synthesis of 2ꢁ-O-dimethylaminoethoxyuridine. (i) (PhCO)2O, NaHCO3, DMF, 100◦C,
4 hours, 100%; (ii) Al foil, dimethylaminoethanol, 140◦C, 24 hours; (iii) DMTCl, pyridine, rt, 3 hours,
50%; (iv) [(iPr)2N]2P(OCH2CH2CN), DIHT, DCM, 16 hours, rt, 80%.
in DMF before reaction with the aluminium alkoxide of dimethy-
laminoethanol to give 2ꢁ-dimethylaminoethoxyuridine 5. Compound
5 was subsequently converted into its 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-3ꢁ-O-
phosphoramidite 7.
Compounds 3 and 7 were incorporated into two different 17-mer TFOs
by automated solid-phase DNA synthesis (phosphoramidite method) to give
oligonucleotides TFO1 and TFO2 respectively. TFOs 3 and 4 were synthe-
sised for comparative fluorescence melting studies, as well as the unmodi-
fied control oligomer. All oligomers were purified by reverse-phase HPLC
and characterised by MALDI-TOF mass spectrometry.
5ꢁ-Q-XCCTXCTCXTXTXTCXT-3ꢁ (TFO)
5ꢁ-F-GTGTTAGGAAGAGAAAAAAGAACTGGT-HEG
|
CACAATCCTTCTCTTTTTTCTTGACCA-HEG
In the TFOs, C = 5-methyldeoxycytidine and Q = amidohexyldabcyl. In
the hairpin duplex F = 6-amidohexylfluorescein, HEG = (hexaethylene)
glycol.
Dabcyl-labelled TFOs were hybridized to a complementary hairpin du-
plex labelled with fluorescein. Results from fluorescence melting experi-
ments are summarised in Table 1.
It has been shown previously that uridine based derivatives such as bis-
amino U show selective binding affinity towards AT base pairs,[7] and it is en-
visaged that other uridine based derivatives such as used in this study would
also will show similar selectivity towards AT base pairs.

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L. Brennan et al.
TABLE 1 Tm values determined at pH 6.0 and pH 6.5. Concentration 2.5 µM:0.25 µM (TFO:duplex)
in 20 mM sodium acetate with 200 mM NaCl. Modifications in TFOs 3 and 4 have been described
previously.[3,8]
TFO
Modification (x)
Tm (pH 6.5) ꢀTm (pH 6.5) Tm(pH 6.0) ꢀTm (pH 6.0)
Unmod thymidine
38.1
48.5
—
10.4
45.0
56.5
—
11.5
1
2
3
4
5-dimethylaminopropargyl
dU (DMAPdU)
2ꢁ-O-dimethylaminoethoxy
U (DMAEU)
5-aminopropargyl dU
(APdU)
41.2
56.2
46.6
3.1
18.1
8.5
49.2
65.1
54.4
4.2
20.1
9.4
5-propynyl dU (PdU)
It has been suggested that modification with a propynyl group bearing
a charged or an electronegative group at the 5-position of the nucleobase,
may lead to an X.AT triplet that is more stable than T.AT.[8] Our results
confirm this and show that TFOs with a positive charge attached to a
propynyl group in the 5-position of thymine analogues are more stable
than those with uncharged propynyl dU, even in the case of the relatively
bulky dimethylamino group. It appears that hydrogen bonding is not the
sole determinant of stability as the dimethylaminopropargyl substituent en-
hances triplex stability (although to a lesser extent than aminopropargyl).
In future it will be informative to make a comparison between the 2ꢁ-O-
dimethylaminoethoxy uridine analogue in this study and 2ꢁ-O-aminoethoxy
uridine.[9]
In conclusion, two novel nucleoside analogues, each containing a
dimethylamino group, have been synthesised and incorporated into triplex-
forming oligonucleotides. Fluorescence melting experiments with a comple-
mentary hairpin duplex have been carried out. Results show that although
the 5-dimethylaminopropargyl group is not as stabilising as the analogous
5-aminopropargyl modification, the charge seems to impart more stability
than the uncharged 5-propynyl-derivative.
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Angew. Chem. Int. Ed. 1998, 37, 1288.
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