On the stability of furanopyrimidin-2-one bases in oligonucleotides

Article abstract of DOI:10.1039/b402559h

The fluoresceinated furanopyrimidin-2-one nucleobase 4 incorporated into an oligonucleotide undergoes unexpectedly facile hydrolytic ring-opening in aqueous buffer at slightly elevated temperatures.

Full text of DOI:10.1039/b402559h

On the stability of furanopyrimidin-2-one bases in oligonucleotides†
Guan-Sheng Jiao and Kevin Burgess*
Department of Chemistry, PO Box 30012, Texas A & M University, College Station, TX 77842-3012.
E-mail: burgess@tamu.edu; Fax: (+1)979 845 8839; Tel: (+1)979 845 4345
Received (in Cambridge, UK) 19th February 2004, Accepted 8th April 2004
First published as an Advance Article on the web 7th May 2004
The fluoresceinated furanopyrimidin-2-one nucleobase 4 incor-
porated into an oligonucleotide undergoes unexpectedly facile
hydrolytic ring-opening in aqueous buffer at slightly elevated
temperatures.
imply the modified base in oligonucleotide 5 resembled a T residue
more than a C.
A standard procedure for measuring thermal denaturation was
used to obtain the data shown in Fig. 1.10 This involved first heating
the sample in phosphate buffer (pH 7.2) to 90 °C for 5 min, then
2A-Deoxy-5-alkynyluracil bases A are useful analogs of uracil and
thymidine which are conveniently prepared via Sonogashira
reactions1 of 5-iodouracil.2 Their alkyne substituents tend to be
directed away from the helical structure of double strand (ds)-DNA
hence decreasing the likelihood that they will perturb its structure
and function.3 However, 5-alkynyluracil bases tend to cyclize onto
the alkyne substituent giving the corresponding furanopyrimidin-
2-ones B.4 These derivatives are also interesting, particularly for
their applications in medicinal chemistry.5–7 The most recent phase
of research in this area was initiated by Yu et al. who incorporated
furanopyrimidin-2-ones into oligonucleotides and studied the
stabilities of modified ds-DNA produced in this way via thermal
denaturation experiments.8 Reported here is a potential side
reaction that can compromise data obtained from that kind of
approach.
Our interest in this area began when attempting to couple
5-iodofluorescein 19 with the 5-alkynyluracil derivative 2.10 The
non-cyclized Sonogashira products were formed when the reaction
was performed at near ambient temperature for relatively short
times (cat. Pd(PPh3)4, CuI, NEt3, THF, 40 °C, 0.5 h; 83%; not
shown),11 but longer reaction times at higher temperatures favoured
formation of the cyclic product 3 ( Scheme 1).
The next step was to investigate the properties of the fluorescei-
nated furanopyrimidin-2-one in DNA. Consequently, compound 3
was converted to the corresponding phosphoramidite 4 via standard
methods, then incorporated into the oligonucleotide 5 via solid
phase syntheses.12
In their work on oligonucleotides modified with a furanopyr-
imidin-2-one base, Yu et al. reported thermal denaturation data that
implied this type of analog anneals with highest affinity to a G
complement, i.e. the base behaves as a C analog. However, the data
in Fig. 1 indicate the ds-DNA sequence 6 dissociates at 11 °C less
than 7; this melting temperature is comparable to the T:G
mismatched system 8. Conversely, when the modified base was set
opposite an A residue in the duplex 9 then the melting temperature
was within 0.8 °C of the T:A matched situation 10 and 11.8 °C
greater than the A:A mismatched one in 11. These results strongly
Scheme 1 Preparation of 3 and 4, Reagents and conditions: (i) cat.
Pd(PPh3)4, CuI, NEt3, THF, 55 °C, 12 h; (ii) ClP(NiPr2)(OCH2CH2CN), Et
NiPr2, CH2Cl2, 25 °C, 4 h.
† Electronic supplementary information (ESI) available: experimental
procedures for the preparation of the new compounds, protocols for the
thermal denaturation and enzyme digestion experiments. See http://
www.rsc.org/suppdata/cc/b4/b402559h/
Fig. 1 Sequence of 5 where Z is the position at which 4 was incorporated,
the ds-DNA oligomers that were made from it, and their melting
temperatures.
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C h e m . C o m m u n . , 2 0 0 4 , 1 3 0 4 – 1 3 0 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

cooling it rapidly to 0 °C; it was then heated from 0 °C to 90 °C in
small temperature increments over 6 h while the absorbance was
recorded. To test if this protocol caused a chemical change in 5, it
was repeated on this oligonucleotide alone. The molecular ion
detected before this treatment was 4296 Da (MALDI), and the
calculated mass was 4297 Da, these readings are within experi-
mental error. However, after the thermal denaturation cycle the
observed mass was 4315 Da, representing an increase of
approximately 18 units.
A literature search revealed that a nucleoside containing a
furanopyrimidin-2-one base had been treated with 2 M NaOH and
was found to open at the furan ring.13 When the fluorescein-
labelled furanopyrimidin-2-one 3 was subjected to the same
conditions (2 M NaOH, THF, 25 °C, 6 h), it was found to ring-open
in a similar way giving the ketone 12.
syntheses of 2A-deoxy-5-alkynyluracil derivatives in the literature
where this cyclization has not been observed. However, work by
Yu et al.8 and others14 indicate that the ferrocene-substituted
compounds B, R = Fc, seem to undergo this cyclization readily.
Conversely, that same ferrocenyl derivative seems to be stable to
thermal denaturation conditions (though Yu et al. did note that
some unidentified materials were observed on enzyme digestion of
their modified oligonucleotides) while the fluorescein-substituted
compounds featured here appear to be more vulnerable to this ring-
opening process. We conclude that the substituent R may have a
relatively strong influence on the ease with which structures A
convert to the cyclized forms B, and on subsequent ring-opening
reactions. Overall, these studies indicate that thermal denaturation
data of modified oligonucleotides containing furanopyrimidin-
2-ones should be interpreted with care, and medicinal chemists
working with these types of nucleosides as pharmaceutical leads
might consider hydrolytic ring-opening of the nucleobase as a
possible metabolic pathway in vivo.
Support for this work was provided by The National Institutes of
Health (HG 01745) and by The Robert A. Welch Foundation. The
TAMU/LBMS-Applications Laboratory, and useful discussions
with Dr Shane Tichy and with Dr Lars H. Thoresen are
acknowledged.
Notes and references
1 K. Sonogashira, Y. Tohda and N. Hagihara, Tetrahedron Lett., 1975, 16,
4467.
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4 M. J. Robins and P. J. Barr, J. Org. Chem., 1983, 48, 1854.
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Andrei, R. Snoeck, E. De Clercq and J. Balzarini, J. Med. Chem., 2000,
43, 4993.
6 S. Blewett, C. McGuigan, H. Barucki, G. Andrei, R. Snoeck, E. D.
Clercq and J. Balzarini, Nucleosides, Nucleotides Nucleic Acids, 2001,
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7 S. Srinivasan, C. McGuigan, G. Andrei, R. Snoeck, E. D. Clercq and J.
Balzarini, Nucleosides, Nucleotides Nucleic Acids, 2001, 20, 763.
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Donilon, J. F. Kayyem, M. Gozin and G. F. Blackburn, J. Am. Chem.
Soc., 2000, 122, 6767.
Finally, in a key experiment, the ss-oligonucleotide that had been
subjected to the thermal denaturation sequence described above,
was then digested with a combination of enzymes to cleave all the
phosphodiester bonds. HPLC analyses of the digest indicated it
contained dA, dT, dC, dG, and one extra peak. Co-injection of the
digest with standard samples indicated this extra peak did not
correspond to the furanopyrimidin-2-one (i.e. compound 3 without
DMT protection), but it did co-elute with 12 (Scheme 2).
It is clear that experiments with modified nucleosides can be
complicated by the formation of furanopyrimidin-2-ones and their
hydrolytic ring-opening reactions. Curiously, there are many
9 G.-S. Jiao, J. W. Han and K. Burgess, J. Org. Chem., 2003, 68, 8264.
10 D. J. Hurley and Y. Tor, J. Am. Chem. Soc., 1998, 120, 2194.
11 G.-S. Jiao and K. Burgess, Bioorg. Med. Chem. Lett., 2003, 13, 2785.
12 M. H. Caruthers, Acc. Chem. Res., 1991, 24, 278.
13 D. Loakes, D. M. Brown, S. A. Salisbury, M. G. McDougall, C. Neagu,
S. Nampalli and S. Kumar, Helv. Chim. Acta, 2003, 86, 1193.
14 A. R. Pike, L. C. Ryder, B. R. Horrocks, W. Clegg, M. R. J. Elsegood,
B. A. Connolly and A. Houlton, Chem. Eur. J., 2002, 8, 2891.
Scheme 2 Thermal denaturation and enzymatic degradation of 5. Reagents
and conditions: (i) 90 °C, phosphate buffer pH 7.2, 5 min then 0 °C to 90 °C
over 6 h; (ii) phosphodiesterase 1, alkaline phosphatase, and nuclease,
MgCl2, Tris HCl pH 7.5, 37 °C, 24 h.
C h e m . C o m m u n . , 2 0 0 4 , 1 3 0 4 – 1 3 0 5
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