8-vinylguanine nucleo amino acid: A fluorescent PNA building block

Article abstract of DOI:10.1021/ol3000603

Attachment of a vinyl group at guanine position 8 provides fluorescent properties of the nucleobase. Therefore, 8-vinylguanine was introduced as a 2-aminoethylglycine peptide nucleic acid (PNA) building block. Incorporation of the guanine analog in short

Full text of DOI:10.1021/ol3000603

ORGANIC
LETTERS
2012
Vol. 14, No. 6
1382–1385
8-Vinylguanine Nucleo Amino Acid: A
Fluorescent PNA Building Block
€
Stefan Mullar, Julian Strohmeier, and Ulf Diederichsen*
€
Institut fu€r Organische und Biomolekulare Chemie, Georg-August-Universitat
€
Gottingen, Tammannstrasse 2, 37077 Gottingen, Germany
€
udieder@gwdg.de
Received January 11, 2012
ABSTRACT
Attachment of a vinyl group at guanine position 8 provides fluorescent properties of the nucleobase. Therefore, 8-vinylguanine was introduced as
a 2-aminoethylglycine peptide nucleic acid (PNA) building block. Incorporation of the guanine analog in short PNA sequences by Fmoc solid
phase peptide synthesis allowed the differentiation between hybridization states of specific double strands with DNA, RNA, and PNA as well as
quadruplex forming RNA/PNA oligomers based on fluorescence intensity.
Oligonucleotides use base pair recognition for the for-
mation of various double strands, tetrades and other folds
in multiple topologies. The incorporation of fluorescent
nucleobase analogs allows the detection of these kinds
of hybridization states and oligonucleotide topologies.1
Typical approaches are the preparation of fluorophore-
nucleobase conjugates, expanded nucleobases or nucleo-
tides bearing a fluorescent aromatic moiety instead of a
purine or pyrimidine.2 Fluorescent analogs should closely
resemble their parent nucleobases to minimize the influ-
ence of the modification with respect to recognition, con-
formation, and the micro environment.
Recently, we reported on 8-vinyl-20-deoxyguanosine asa
fluorescent 20-deoxyguanosine mimic for the investigation
of DNA hybridization and topology.3 This only slightly
modified guanosine analog maintains the nucleobase
core, hydrogen bonding options, and resembles the torsion
angles of DNA complexes while being fluorescent. 2-
Aminoethylglycine peptide nucleic acids (PNA) attend
broadinterest asuncharged and achiral DNA/RNA mimic
manifolds applied in diagnostics and as oligonucleotide
analogs.4 High binding affinity to complementary DNA
and RNA as well as resistance to nucleases and proteases
makes PNA an ideal tool for clinical diagnostics or biomo-
lecular probes.5,6 To take advantage of the fluorescent
properties of 8-vinylguanine also in PNA oligomers,
an 8-vinylguanine PNA building block was synthe-
sized, incorporated into PNA oligomers, and investigated
in PNA/PNA, PNA/oligonucleotide duplex formation and
within a RNA/PNA quadruplex fold.
Synthesis of the 8-vinylguanine PNA building block 1
was provided by bromination at C8 of the guaninyl acetic
acid 2, attachment to the aminoethylglycine backbone
followed by palladium catalyzed vinylation. Starting from
2-amino-6-chloro-purine 3, the guaninyl acetic acid 2
was accessible following literature protocols (Scheme 1).7
(1) (a) Wilson, J. N.; Kool, E. T. Org. Biomol. Chem. 2006, 4, 4265–
4274. (b) Okamoto, A.; Saito, Y.; Saito, I. Photochem. Photobiol. C 2005,
6, 108–122. (c) Rist, M. J.; Marino, J. P. Curr. Org. Chem. 2002, 6, 775–
793.
(4) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 245, 1497–1500.
(5) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier,
S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E.
Nature 1993, 365, 566–568.
€
€
(2) (a) Forster, U.; Gildenhoff, N.; Grunewald, C.; Engels, J. W.;
Wachtveitl, J. J. Lumin. 2009, 129, 1454–1458. (b) Krueger, A. T.; Kool,
E. T. J. Am. Chem. Soc. 2008, 130, 3989–3999. (c) Hainke, S.; Seitz, O.
Angew. Chem., Int. Ed. 2009, 48, 8250–8253.
(3) Nadler, A.; Strohmeier, J.; Diederichsen, U. Angew. Chem., Int.
Ed. 2011, 50, 5392–5396.
(6) Uhlmann, E.; Peyman, A.; Breiphol, G.; Will, D. Angew. Chem.,
Int. Ed. 1998, 37, 2796–2823.
r
10.1021/ol3000603
Published on Web 02/27/2012
2012 American Chemical Society

The acetic acid was regioselectively introduced at position
N9 using benzyl 2-bromoacetate followed by saponifica-
tion.7a An O6-benzyl-group was advantageous regarding
solubility, purification, and coupling yields.8 Bromination
at C8 was performed in EtOAc with bromine to give 4
which was coupled to the backbone of Fmoc protected
tert-butylester ofN-(2-aminoethyl)glycine usingPyBOB as
activation reagent. Nucleo amino acid 5 was subjected to
Stille coupling with tributylvinyltin to provide the vinyl-
guanine derivative 6 in good yields. After treatment with
trifluoroacetic acid in DCM the building block 1 (Vg) was
obtained by precipitation from diethyl ether ready to be
used in SPPS. To determine the extinction coefficient and
the quantum yield of building block 1 (Vg) (Supporting
Information), N-terminal deprotection was performed
with 20% piperidine in DMF providing amino acid 7.
PNA oligomers (Table 1) were synthesized via manual
Fmoc SPPS considering reduced deprotection periods and
capping cycles9 as well as double couplings with shorter
coupling times for the guaninyl nucleo amino acid10 and
the incorporation of the 8-vinlyguanine building block 1
(Vg). Furthermore, self-capping by acetylated nucleobase
species during the neutralization and deprotection was
avoided by an NMP/DIEA washing step after coupling
and capping cycles.11 Due to potential acetylation of the
exocyclic amine during SPPS, no capping was applied after
incorporation of 1 (Vg).8 However, byproducts were not
detected apart from oligomers lacking Vg in cases where
single coupling was applied for the fluorescence building
block. To enhance water solubility of PNA oligomers a lysine
residue was attached to the C-terminus.4 After cleavage from
the resin, the PNA oligomers were purified using reversed
phase HPLC and characterized by ESIꢀMS (Supporting
Information).
Table 1. Hybridization Stabilities of PNA/PNA, PNA/DNA,
and PNA/RNA Hybrids based on UV Melting Curves
complementary
strand
Tm
sequence
ratio (°C)
H-(gc[Vg]tgg)Lys-NH2 (PNA1)
H-(gcgtgg)Lys-NH2 (PNA2)
50-CCACGC-30 (DNA1)
PNA3
PNA3
PNA1
RQ
1:1 36.0
1:1 37.2
1:1 36.8
2:1 71.6
2:1 74.0
1:1 59.4
1:1 62.6
H-(g[Vg]gtcgg)Lys-NH2 (PVgH)
H-(gggtcgg)Lys-NH2 (PgH)
H-(ccca[Vg]cc)Lys-NH2 (PVgC)
H-(cccagcc)Lys-NH2 (PgC)
RQ
RQ
RQ
Hybridization properties of Vg containing PNAs with
complementary PNA and DNA oligomers were investi-
gated by fluorescence spectroscopy and thermal denatur-
ation studies. The stability of the double strand formed by
PNA1 (H-(gc[Vg]tgg)Lys-NH2) containing the Vg moiety
as guanine nucleo amino acid analog with the complemen-
tary sequence H-(cgcacc)Lys-NH2 (PNA3) provided a
comparable stability (ΔTm = 1.2 °C) as the guanine
analogous PNA2 (H-(gcgtgg)Lys-NH2)/PNA3 complex
(Supporting Information). Thermal denaturation studies
Scheme 1. Syntheses of 8-Vinylguanine PNA Amino Acid Vga
0
0
of the respective PNA1/DNA1 (5 CCACGC3 ) duplex in-
dicated a stability (Tm = 36.8 °C) in the extent of the PNA
double strand. PNA1/PNA3 duplex formation was also
clearly indicated by fluorescence spectroscopy. Excitation
of Vg at 277 nm provided fluorescence response for the
PNA1 single strand and the respective PNA1/PNA3 du-
plex (Figure 1). Hybrid formation at 5 °C led to a 72%
decrease in emission intensity. Heating the PNA1/PNA3
duplex above the melting temperature to 70 °C provided
a fluorescence spectrum that is almost identical to
the PNA1 single strand. The fluorescence curves ob-
tained for the PNA1/DNA1 duplex were similar to the
spectra recorded for the respective PNA double strand.
Therefore, incorporation of Vg in PNA oligomers allows
the detection of PNA/PNA and PNA/DNA duplex
formation.
(7) (a) Ura, Y.; Beierle, J. M.; Leman, L. J.; Orgel, L. E.; Ghadiri,
M. R. Science 2009, 325, 73–77. (b) Schwergold, C.; Depecker, J.; Di Giorgio,
C.; Patino, N.; Jossinet, F.; Ehresmann, B.; Terreux, R.; Cabrol-Bass,
Condom, R. Tetrahedron 2002, 58, 5675–5687.
(8) Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.;
Hansen, H. F.; Vulpius, T.; Petersen, K. H.; Berg, R. H.; Nielsen,
P. E.; Buchardt, O. J. Org. Chem. 1994, 59, 5767–5773.
a Abbreviations: OBn, O-benzyl; DIEA, diisopropylamine; PyBOB,
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate;
Fmoc, 9-fluoroenylmethoxycarbonyl; aeg, aminoethylglycine; TFA, tri-
fluoracetic acid.
€
(9) Schneggenburger, P. E.; Mullar, S.; Worbs, B.; Steinem, C.;
Diederichsen, U. J. Am. Chem. Soc. 2010, 132, 8020–8028.
(10) Chen, Y.; Wallace, B. A. Eur. Biophys. J. 1997, 26, 299–306.
(11) Koch, T.; Hansen, H. F.; Andersen, P.; Larsen, T.; Batz, H. G.;
Otteson, K.; Økrum, H. J. Pept. Res. 1997, 80–88.
Org. Lett., Vol. 14, No. 6, 2012
1383

In analogy to the PNA hybridization experiments with
RQ done by Armitage et al. PNA sequences were intro-
duced containing Vg (Table 1) investigating the fluores-
cence readout for the various topologies.12 UV melting
curves of the tetrade complexes RQ þ PgH and RQ þ
PVgH as well as the duplexes RQ þ PgC and RQ þ PVgC
(ΔTm = 3 °C) both turned out quite similar (Figure 3). The
introduction of the guaninylnucleoamino acidvinyl group
has negligible influence on the complex stabilities.
Figure 1. Fluorescence quenching upon double helix formation,
shown for 1:1 mixture of PNA1 in the presence and absence of
complementary PNA3 or DNA1 (2.5 μM, 10 mM phosphate
buffer pH 7, 100 mM NaCl, excitation wavelength: 277 nm).
Figure 3. UV melting curves for (left) RQ duplex forming PVgC
and PgC (275 nm) and (right) quadruplex inducing PVgH and
PgH (295 nm, 275 nm, 2.0 μM, 10 mM Tris-HCl buffer pH 7,
100 mM KCl, 1 cm cuvette).
For investigating the hybridization properties of RNA/
PNA interacting oligomers by fluorescence spectroscopy,
a quadruplex forming RNA (RQ) was prepared as intro-
duced by Armitage et al.12 RQ is a truncated version of RDQ,
an RNA aptamer originally selected for binding to the
Fragile X mental retardation protein.13 With complementary
and homologous PNA probes Armitage et al. were able to
invade the RNA guanine quadruplex and form stable 1:1
duplexes (complementary probe) or 1:2 quadruplex (homol-
ogous probe) PNA-RNA hybrids (Figure 2).12,14
The guaninyl vinyl group also does not affect the duplex
or quadruplex secondary structures as indicated by CD
spectroscopy (Figure 4). In complex with RQ the PNA
sequences PVgH and PVgC provided similar CD spectra
as the complexes induced by PgH and PgC, respectively.
Figure 4. CD spectra of RQ with 1 equiv PVgC (black)/PgC
(red) or 2 equiv PVgH (black)/PgH (red) (1:1 and 1:2 mixture,
1.0 μM RQ, 10 mM Tris-HCl buffer pH 7, 100 mM KCl, 22 °C).
The fluorescence response of the modified PNA oligomers
PVgH and PVgC was evaluated upon hybridization to RQ.
Duplex formation between RNA RQ and PNA PVgC was
indicated by fluorescence response after excitation at 277 nm.
The fluorescence intensity decreased by 70% upon duplex
formation of PVgC with RQ at 22 °C (Figure 5). As a control
experiment, the fluorescence spectrum of the equimolar
mixture of RQ and PVgC was measured above the melting
temperature (95 °C) and was found to be almost identical to
the spectrum of single-stranded PVgC.
Figure 2. RNA quadruplex RQ opens to an RNA/PNA duplex
adding PNA oligomer PVgC and leads to a dimer of qua-
druplexes adding PNA PVgH (only one of four possible qua-
druplexes shown).12
(13) Darnell, J. C.; Jensen, K. B.; Jin, P.; Brown, V.; Warren, S. T.;
Darnell, R. B. Cell 2001, 107, 489–499.
(14) Marin, V. L.; Armitage, B. A. J. Am. Chem. Soc. 2005, 127,
8032–8033.
(12) Marin, V. L.; Armitage, B. A. Biochemistry 2006, 45, 1745–1754.
1384
Org. Lett., Vol. 14, No. 6, 2012

PNA PVgH was expected to induce a quadruplex topology
by interaction with RNA RQ. Therefore, the change in
hydrogen bonding interaction and surrounding of the
vinylguanine should be reflected in the fluorescence
response. In absence of RQ quite a low fluorescence
intensity of PNA PVgH was detected, that seems to
result from quenching by neighboring guanine nucleo-
bases (Figure 6). In the presence of RQ, the fluorescence
intensity increased by about 93%, indicating the forma-
tion of the hybrid quadruplex at 22 °C. At 95 °C, the
intensity was decreased due to melting of the quadruplex
(Figure 6).
In conclusion, the synthesis of the fluorescent 8-vinyl-
guaninyl peptide nucleic acid building block Vg was
reported. Incorporation into PNA sequences by Fmoc-
SPPS chemistry was provided under standard coupling
and cleavage conditions. The fluorescent Vg label was used
to probe the hybridization of PNA/PNA, PNA/DNA,
PNA/RNA doublestrands and an RNA/PNAquadruplex.
A clear differentiation between the PNA single strands and
the pairing complexes with their respective topology was
indicated by fluorescence intensity. The vinyl modification
of the guaninyl nucleo amino acid was proven to have
minimal influence on the complex stabilities and secondary
structures. Vinylguanine does not interfere with a guanine-
like hydrogen bonding pattern, adapts structurally well to
the respective pairing topology, and allows a sensitive
fluorescent readout of the topology and the direct environ-
ment of the nucleo amino acid Vg.
Figure 5. Fluorescence quenching upon double helix formation,
shown for 1:1 mixture of PVgC in presence (red) and absence
(black) of RQ (2.0 μM, 10 mM Tris-HCl buffer pH 7, 100 mM
KCl, excitation wavelength: 277 nm).
Acknowledgment. Financial support from the Deutsche
Forschungsgemeinschaft (SFB 803) is gratefully acknowl-
ꢀ
edged. We are thankful to Dr. Andre Nadler for extensive
discussions.
Supporting Information Available. General methods,
characterization data and experimental procedures for all
new compounds as well as synthesized oligoamides. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 6. Fluorescence increase upon quadruplex formation,
shown for 1:2 mixture PVgH in presence (red) and absence
(black) of RQ (1.0 μM, 10 mM Tris-HCl buffer pH 7, 100 mM
KCl, excitation wavelength: 277 nm).
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 6, 2012
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