Synthesis of a fluorescent PNA monomer containing 5-((9H-fluoren-2-yl) ethynyl)uracil

Article abstract of DOI:10.1080/15257770701527802

Pyrimidine nucleobases bearing 5-phenylethynyl substitution represent compact and intrinsically fluorescent nucleobases. Such nucleobases are capable of selective recognition of a complementary base and may fluorimetrically report on hybridization events. Our past work has demonstrated that the fluorescence of 5-phenylethynyluracils is sensitive to substitution on the phenyl ring, however these are relatively weak fluorophores. We currently are pursuing the functionalization of the phenyl group of these modified nucleobases in order to further improve their fluorescence response, increase their aqueous solubility and stabilize hybrids formed with complementary nucleic acids. As an example of this work, we have synthesized the 5-((9 H-fluoren-2-yl)ethynyl)uracil PNA monomer that will be incorporated into oligomers using Fmoc-based chemistry. Initial evaluation of the fluorescence of the 5-((9 H-fluoren-2-yl)ethynyl) uracil derivative shows that the fluorescence intensity is approximately 50 times greater than a similar 5-phenylethynyluracil derivative when under identical conditions. Copyright Taylor & Francis Group, LLC.

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Synthesis of A Fluorescent PNA
Monomer Containing 5-((9 H -Fluoren-2-
YL)Ethynyl)Uracil
Filip Wojciechowski a & Robert H. E. Hudson a
a Department of Chemistry , The University of Western Ontario ,
London, Ontario, Canada
Published online: 10 Dec 2007.
To cite this article: Filip Wojciechowski & Robert H. E. Hudson (2007) Synthesis of A Fluorescent PNA
Monomer Containing 5-((9 H -Fluoren-2-YL)Ethynyl)Uracil, Nucleosides, Nucleotides and Nucleic Acids,
26:8-9, 1199-1202, DOI: 10.1080/15257770701527802
To link to this article: http://dx.doi.org/10.1080/15257770701527802
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1199–1202, 2007
Copyright ꢀ Taylor & Francis Group, LLC
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ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701527802
SYNTHESIS OF A FLUORESCENT PNA MONOMER CONTAINING
5-((9H-FLUOREN-2-YL)ETHYNYL)URACIL
Filip Wojciechowski and Robert H. E. Hudson
University of Western Ontario, London, Ontario, Canada
Department of Chemistry, The
2
Pyrimidine nucleobases bearing 5-phenylethynyl substitution represent compact and intrinsi-
2
cally fluorescent nucleobases. Such nucleobases are capable of selective recognition of a complemen-
tary base and may fluorimetrically report on hybridization events. Our past work has demonstrated
that the fluorescence of 5-phenylethynyluracils is sensitive to substitution on the phenyl ring, how-
ever these are relatively weak fluorophores. We currently are pursuing the functionalization of the
phenyl group of these modified nucleobases in order to further improve their fluorescence response,
increase their aqueous solubility and stabilize hybrids formed with complementary nucleic acids. As
an example of this work, we have synthesized the 5-((9 H-fluoren-2-yl)ethynyl)uracil PNA monomer
that will be incorporated into oligomers using Fmoc-based chemistry. Initial evaluation of the fluo-
rescence of the 5-((9 H-fluoren-2-yl)ethynyl)uracil derivative shows that the fluorescence intensity is
approximately 50 times greater than a similar 5-phenylethynyluracil derivative when under identi-
cal conditions.
Keywords Fluorescent nucleobases; 5-(9H-fluoren-2-yl)ethynyl)uracil; PNA
INTRODUCTION
Recently we have described the synthesis, properties and incorpora-
tion of fluorescent nucleobases derived from 5-phenylethynylpyrimidines
into PNA.[1,2] The 5-alkynyl-substituted nucleobases show weaker fluores-
cence than the cyclized pyrrolocytosines or furanouracils.[1] Since the
cross-coupled phenylethynylpyrimidines show substituent-dependent fluo-
rescence we have decided to introduce a larger aromatic group, such as
fluorene, which is expected to be more emissive and corroborated by
the work of others. For example, Kim et al. have synthesized a fluorene-
labeled phosporamidite followed by incorporation into a central posi-
tion of an oligodeoxyribonucleotide. Their results indicate that a fully
matched duplex exhibits a 3.4-fold enhancement in fluorescence intensity
The authors wish to thank NSERC (Canada) for support of this work.
Address correspondence to Robert H. E. Hudson, Department of Chemistry, The University of
Western Ontario, London, Ontario N6A 5B7, Canada. E-mail: rhhudson@uwo.ca
1199

1200
F. Wojciechowski and R. H. E. Hudson
SCHEME 1
(λmax = 425 nm) relative to the single strand.[3] The attachment of fluo-
rene to a PNA backbone using a novel lysine PNA monomer was recently
accomplished by Appella and coworkers. The modified oligomers showed
comparable thermal stability to the unmodified aegPNA oligomer. A 4-fold
increase in fluorescence intensity was observed when hybridized to the com-
plementary strand compared to the unbound PNA.[4] Our aim is to intro-
duce fluorene-containing pyrimidines into PNA in order to avoid introduc-
ing chirality into the backbone, increase the fluorescence response of the
oligomer, stabilize the hybrids formed and study the fluorescence response
of fluorene incorporated into oligonucleotides and PNAs.
RESULTS AND DISCUSSION
The synthesis of 2-ethynylfluorene began with commercially available
fluorene which was monoiodinated at the 2 position with periodic acid dihy-
drate and iodine according to a literature procedure.[5] Next, Sonogashira
coupling between TMS-acetylene and 2-iodofluorene followed by removal
of the TMS group (K2CO3/MeOH) gave 2-ethynylfluorene in 82% over two
steps, as shown in Scheme 1.
The synthesis of the 5-((9H-fluoren-2-yl)ethynyl)uracil PNA monomer
was accomplished by palladium catalyzed cross-coupling of 2-ethynylfl-
uorene with tert-butyl (5-iodouracil-1-yl)acetate to give tert-butyl (5-((9H-
fluoren-2-yl)ethynyl)uracil-1-yl)acetate in 84% yield, after chromatographic
purification. The tert-butyl group was conveniently removed with tri-
fluoroacetic acid in DCM containing triethylsilane to yield (5-((9H-
fluoren-2-yl)ethynyl)uracil-1-yl)acetic acid in 93%, Scheme 2. Finally, the
SCHEME 2

Fluorescent PNA Monomer
1201
5-((9H-fluoren-2-yl)ethynyl)uracil PNA monomer was achieved by 1-(3-
(dimethylamino)propyl)-3-ethyl-carbodiimide hydrochloride (EDC) medi-
ated coupling of the base acetic acid derivative to the Fmoc-protected back-
bone and removal of the tert-butyl ester, Scheme 2.
In order to judge our success at creating a more emissive fluorophore,
we have chosen a practical approach. Probe strands are commonly used in
the micromolar concentration range concentration when observing by UV-
vis spectrophotometry. Since bimolecular duplex stability is concentration
dependent, we have often characterized thermal denaturation by observ-
ing changes in fluorescence with identical solutions (µM concentration) to
allow easy comparison of results between the two techniques. In using this
approach, we get a qualitative, operational ranking of the sensitivity of a par-
ticular fluorophore. By defining “brightness” as the fluorescence emission
under identical instrumental conditions and concentration we can qualita-
tively rank similar fluorophores. Applying this approach to the present case,
we studied tert-butyl (5-((9H-fluoren-2-yl)ethynyl)uracil-1-yl)acetate (1) and
ethyl (5-phenylethynyl)uracil-1-yl acetate (2) at 2.5 µM in dichloromethane.
FIGURE 1 Comparison of the fluorescence of tert-butyl (5-((9H-fluoren-2-yl)ethynyl)uracil-1-yl)acetate
1 and ethyl (5-phenethynyl)uracil-1-yl acetate 2.

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F. Wojciechowski and R. H. E. Hudson
Each fluorophore has a peak absorption band near 350 nm which when ir-
radiated leads to blue fluorescence (λmax = 412 nm for 1; λmax = 420 nm
for 2). As illustrated in Figure 1, the peak emission intensity of 1 is approxi-
mately 2.5 × 106 while that of 2 is 5 × 104 (inset).
The above monomer will be incorporated into a PNA oligomer and its
hybridization properties and fluorescence response evaluated. We also are
currently pursuing the synthesis of pyrrolocytosine containing fluorene via
cyclization of 5-((9H-fluoren-2-yl)ethynyl)uracil. Since PNA suffers from low
aqueous solubility introduction of the nonpolar fluorene ring will further
decrease the solubility. Therefore, derivatization of fluorene with cationic
groups is underway with the expectation that this will increase aqueous sol-
ubility and favourably affect the hybridization properties.
REFERENCES
1. Hudson, R.H.E.; Dambenieks, A.K.; Moszynski, J.M. New fluorescent nucleobases derived from 5-
alkynylpyrimidines. Proc. of SPIE 2005, 5969, 59690J1–J10.
2. Hudson, R.H.E.; Moszynski, J.M. A facile synthesis of fluorophores based on 5-phenylethynyluracils.
Synlett 2006, 18, 2997–3000.
3. Kim, B.H.; Seo, Y.J.; Hwang, G.T. A highly discriminating quencher-free molecular beacon for prob-
ing DNA. J. Am. Chem. Soc. 2004, 126, 6528–6529.
4. Appella, D.H.; Englund, E.A. Synthesis of γ -substituted peptide nucleic acids: A new place to attach
fluorophores without affecting DNA binding. Org. Lett. 2005, 7, 3465–3467.
5. Lee, S.H.; Nakamura, T.; Tsutsui, T. Synthesis and characterization of oligo(9,9-dihexyl-2,7-fluorene
ethynylene)s: For application as blue light-emitting diode. Org. Lett. 2001, 3, 2005–2007.