Peptide nucleic acid containing a meta-substituted phenylpyrrolocytosine exhibits a fluorescence response and increased binding affinity toward RNA

Article abstract of DOI:10.1021/ol9019474

Peptide nucleic acids (PNA) containing meta-substituted 6-phenylpyrrolocytosine (PhpC), [mono-m-(aminoethoxy)phenyl]pyrrolocytosine (mmePhpC), [mono-m-(aminopropoxy)phenyl]pyrrolocytosine (mmpPhpC), and [mono-m-(guanidinoethoxy)phenyl]pyrrolocytosine (mmg

Full text of DOI:10.1021/ol9019474

ORGANIC
LETTERS
2009
Vol. 11, No. 21
4878-4881
Peptide Nucleic Acid Containing a
Meta-Substituted Phenylpyrrolocytosine
Exhibits a Fluorescence Response and
Increased Binding Affinity toward RNA
Filip Wojciechowski and Robert H. E. Hudson*
Department of Chemistry, The UniVersity of Western Ontario,
London, Ontario, Canada N6A 5B7
robert.hudson@uwo.ca
Received August 21, 2009
ABSTRACT
Peptide nucleic acids (PNA) containing meta-substituted 6-phenylpyrrolocytosine (PhpC), [mono-m-(aminoethoxy)phenyl]pyrrolocytosine
(mmePhpC), [mono-m-(aminopropoxy)phenyl]pyrrolocytosine (mmpPhpC), and [mono-m-(guanidinoethoxy)phenyl]pyrrolocytosine (mmguaPhpC),
have been synthesized. Meta-substituted PhpCs have been hybridized with overall higher binding affinity toward DNA and RNA than previously
synthesized moePhpC or newly synthesized mopPhpC. The guanidinium-containing nucleobase, mmguaPhpC, exhibited the highest increase
in binding affinity toward RNA while fluorometrically responding on the state of hybridization.
The design and study of fluorescent nucleobase analogues
continues to be of interest as they find use in numerous
biotechnological and biomedical applications.1 Many of these
applications would also benefit from increased binding
affinity between the oligonucleotide anologue and the target
nucleic acid.2 The nucleic acid complex is governed by
Watson-Crick hydrogen bonding as a source of comple-
mentary recognition and is further stabilized by π-π
stacking. Introducing modified nucleobases with improved
stacking interactions and hydrogen bonding is thereby a
viable approach to increasing binding affinity in a sequence-
discriminating manner. For some applications, it is desirable
not only to increase the binding affinity but also to construct
a nucleobase that concurrently undergoes a fluorescent
response upon hybridization or change in environment.
Peptide nucleic acid (PNA) is an oligonucleotide analogue
capable of forming highly stable complexes with its target
nucleic acid.3 Numerous modified nucleobases have been
incorporated into PNA.4 Two notable fluorescent cytosine
analogues are phenoxazine5 and phenylpyrrolocytosine (PhpC).6
When containing an aminoethoxy moiety, the phenoxazine
nucleobase forms a fourth hydrogen bond to guanine and has
been termed the G-clamp7 (Figure 1a). Both the phenoxazine
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10.1021/ol9019474 CCC: $40.75
Published on Web 09/29/2009
 2009 American Chemical Society

investigate second generation PhpCs by varying the position
and nature of substitution on the phenyl ring. It was our
intention in the design that the bases remain capable of
forming an additional hydrogen bond to guanine while
fluorometrically responding on the state of hybridization
(Figure 2).
We first decided to synthesize the ortho-substituted
analogue with a three-carbon chain, mopPhpC (Figure 2a),
in order to establish if the ethyl linker (moePhpC) was too
short. Further, we reasoned that ortho-substituted PhpCs may
form an undesirable secondary repulsive interaction between
the oxygen on the phenyl ring and the O6 of guanine. The
meta-substituted PhpCs (mmePhpC and mmpPhpC, Figure
2b) were synthesized in order to determine if this substitution
was more favorable, potentially by relief of secondary
repulsive interactions. Finally, we chose the nucleobase with
the highest affinity toward both DNA and RNA as a good
candidate for derivatiztion to the guanidinium group. Instal-
lation of the guanidinium group on the mmePhpC scaffold
holds the potenial to further increase the binding affinity
through dual O6- and N7-Hoogsteen hydrogen bonds (Figure
2c).
Figure 1. Previously studied cytosine analogues, based on the
phenoxazine and PhpC nucleobases, capable of forming a fourth
hydrogen bond to guanine: (a) phenoxazine-based G-clamp and (b)
phenylpyrrolocytosine-based boPhpC.
and G-clamp nucleobases continue to be modified and exploited
in various applications.8
We have recently designed a novel intrinsically fluorescent
cytosine analogue based on PhpC, termed boPhpC9 (Figure
1b).
Our previous work indicated that the boPhpC nucleobase
also formed a fourth hydrogen bond to guanine, thereby
increasing binding affinity, but also fluorimetrically reported
on PNA/DNA hybridization.9 This dual behavior places
boPhpC and its analogues in a unique class of molecules.
While studying the hybridization properties of moePhpC
(Figure 2a), we noticed significantly greater stabilization of
The required alkyne, 2, for the synthesis of mopPhpC was
prepared by treating 2-iodophenol with tert-butyl 2-bro-
mopropylcarbamate to give 1 which was subjected to
Sonogashira reaction conditions and TMS removal to give
the terminal alkyne 2 (Scheme 1a). Similarly, the synthesis
Scheme 1. Synthesis of Alkynes 2, 5, and 6
Figure 2. Structures of modified PhpCs designed to engage guanine
with an additional hydrogen bond. (a) Previously studied moePhpC
and newly synthesized mopPhpC; (b) meta-substituted PhpCs
mmePhpC (n ) 1) and mmpPhpC (n ) 2); (c) proposed interaction
of guanidino-PhpC (mmguaPhpC) with guanine.
of alkynes 5 and 6 was accomplished by treating 3-bro-
mophenol with tert-butyl 2-bromoethylcarbamate or tert-
butyl 2-bromopropylcarbamate to give 3 or4, which were
subjected to Sonogashira reaction conditions and TMS
removal to give alkynes 5 and 6 (Scheme 1b).
a PNA/DNA duplex over a PNA/RNA duplex of the same
sequence. Furthermore, the flanking nucleobases in PNAs
containing moePhpC would affect the overall duplex stability.
In a recent study, an unusual sequence-dependent behavior
has also been observed with the G-clamp in both DNA and
PNA.10
Alkynes 2, 5, or 6 and ethyl (N4-benzoyl-5-iodocytosin-
1-yl)acetate6a were subjected to a one-pot Sonogashira cross-
In order to address the lower binding affinity toward RNA
and the sequence-dependent behavior, we have decided to
(8) (a) Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y;
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Org. Lett., Vol. 11, No. 21, 2009
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coupling/annulation reaction to give 7, 8, or 9 in good yield
(Scheme 2).
Scheme 3. Synthesis of mmguaPhpC Monomer
Scheme 2. Synthesis of Substituted PhpCs 16-18
DNA and +3.0 °C with RNA, seq 10). Therefore increasing
the tether length in ortho-substituted PhpC does not eliminate
Hydrolysis of the ester group followed by carbodiimide-
mediated condensation with N-[2-(Fmoc)aminoethyl]glycine
benzyl ester11 gave the monomer ester 13, 14, or 15. The
benzyl ester was hydrogenated to give the final monomers
16 (mopPhpC), 17 (mmePhpC), or 18 (mmpPhpC).
An orthogonally protected PNA monomer containing a
protected guanadinium group (22) was synthesized by
treating 8 with TFA, followed by guanylation of the
intermediate amine with N,N′-di-Boc-1H-pyrazole-1-car-
boxamidine in the presence of DIPEA. The monomer,
mmguaPhpC 22, was synthesized similarly to the above
synthesized monomers (Scheme 3).
Monomers 16, 17, 18, and 22 were incorporated into two
PNA oligomers; GTA GAT XAC T-Lys and GTA GAT
CYC T-Lys. The oligomers were cleaved from the solid
support by standard conditions, purified by RP-HPLC and
characterized by ESI-MS (see the Supporting Information).
The hybridization properties of previously synthesized
sequences (1-3 and 8) containing cytosine,9a PhpC,9a and
moePhpC9a were compared to newly synthesized sequen-
ces (4-7 and 11-14) containing mopPhpC, mmePhpC,
mmpPhpC, and mmguaPhpC (Table 1). A decrease in
binding affinity was observed when mopPhpC was incor-
porated into the sequence 4 (+2.5 °C with DNA and +1.0
°C with RNA, seq 4) compared to moePhpC (+10.5 °C with
DNA and +4.0 °C with RNA, seq 3). However the propyl
linker performed better in seq 11, which resulted in an
increase in binding affinity (+11.0 °C with DNA and +7.0
°C with RNA, seq 11) compared to moePhpC (+7.0 °C with
Table 1. Tm Data (°C) for PNA:DNA and PNA:RNA Duplexesa
DNA
∆Tm
RNA
∆Tm
seq
nucleobase
Tm
Tm
19a
29a
39a
4
5
6
X ) cytosine
X ) PhpC
49.5
52.0
60.0
52.0
63.0
60.0
60.0
51.0
53.0
58.0
62.0
61.5
64.5
63.0
57.0
55.0
61.0
58.0
63.0
61.0
63.5
51.0
52.0
54.0
58.0
59.0
54.0
63.0
+2.5
+10.5
+2.5
+13.5
+10.5
+10.5
-2.0
+4.0
+1.0
+6.0
+4.0
+6.5
X ) moePhpC
X ) mopPhpC
X ) mmePhpC
X ) mmpPhpC
X ) mmguaPhpC
Y ) cytosine
7
89a
99a
109a
11
12
13
14
Y ) PhpC
+2.0
+7.0
+11.0
+10.5
+13.5
+12.0
+1.0
+3.0
+7.0
+8.0
+3.0
+12.0
Y ) moePhpC
Y ) mopPhpC
Y ) mmePhpC
Y ) mmpPhpC
Y ) mmguaPhpC
a Sequences: GTA GAT XAC T-Lys and GTA GAT CYC T-Lys.
sequence dependent binding affinity nor does it increase the
overall binding affinity toward RNA.
The introduction of meta-substituted PhpCs, mmePhpC
and mmpPhpC revealed very promising results. In both
oligomers an increase in binding affinity toward RNA was
observed. Likewise, a ∆Tm > 10.0 °C was maintained for
DNA in both sequences.
An average melting temperature, ∆Tm(avg), for the modified
nucleobases is shown in Table 2. This represents a measure
of the consistency of performance of the base modifica-
(11) Wojciechowski, F.; Hudson, R. H. E. J. Org. Chem. 2008, 73, 3807–
3816.
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Org. Lett., Vol. 11, No. 21, 2009

Table 2. Average Melting Temperature ∆Tm(avg) (°C) for
PNA:DNA and PNA:RNA Containing the Modified
Nucleobasesa
nucleobase
DNA (∆Tm(avg)
)
RNA (∆Tm(avg))
cytosine
PhpC
+2.25
+8.8
+6.75
+12.0
+12.0
+11.25
-0.5
+3.5
+4.0
+7.0
+5.5
+9.25
moePhpC
mopPhpC
mmePhpC
mmpPhpC
mmguaPhpC
Figure 3. Fluorescence emission spectra of sequences 7 and 14
containing mmguaPhpC when hybridized to DNA and RNA.
a Average ∆Tm values are the sum of the ∆Tm values from both
In summary, we have shown that rational modification of
PhpC produces high-affinity cytosine analogues potentially
capable of forming a fourth and fifth hydrogen bond to
guanine while fluorometrically reporting on the state of
hybridization. We have also shown that switching the
aminoethoxy or aminopropoxy tether from the ortho position
to the meta position in PhpC maintains high affinity binding
to DNA but, more importantly, significantly increases the
binding affinity toward RNA. Preliminary data also show
that meta-substituted PhpC may alleviate unusual sequence-
dependent binding affinity such as that shown with the
G-clamp.
sequences in Table 1 divided by two.
tion in different sequence contexts. The meta-substituted
mmePhpC hybridized with a ∆Tm(avg) of +12.0 with DNA
and +7.0 with RNA. Likewise the propyl tether in mmpPhpC
hybridized with high affinity (∆Tm(avg) +12.0 °C with DNA)
but slightly lower toward RNA (∆Tm(avg) +5.5 °C). Therefore,
mmePhpC was chosen for derivatization to the guanidinium-
containing nucleobase.
The introduction of mmeguaPhpC into sequence 7 and 14
resulted in high affinity binding toward DNA (+10.5 °C and
+12.0 °C, ∆Tm(avg) +11.25) but also exhibited a significant
increase in binding affinity toward RNA (+6.5 °C and +12.0
°C, ∆Tm(avg) +9.25).
The fluorescence responses of sequences 4-7 and 11-14
were evaluated against DNA and RNA (Supporting Informa-
tion). The fluorescence response of sequences containing
mmguaPhpC in the single stranded state and hybridized to
DNA or RNA is shown in Figure 3. A 60% decrease in
emission intensity is observed in sequence 7,but only a 35%
decrease in intensity is observed in sequence 14 when
hybridized to DNA.
The fluorescence response of oligomers containing mmgua-
PhpC is better with RNA; upon duplex formation, a 50%
decrease in emission intensity is observed in sequence 7 and
a 70% in sequence 14 (Figure 3). The quenching of
fluorescence upon duplex formation is consistent with MepC
and PhpC in DNA and RNA and is likely due to both
hydrogen-bonding and base-stacking interactions.6b,12
Future work will be aimed at evaluating the binding affinty
of oligonucleotide analogues containing meta-substituted
PhpC in various sequence contexts. These nucleobases will
also be evaluated in antisense or antigomer applications or
as fluorescent-based hybridization probes.
Acknowledgment. We thank the Natural Science and
Engineering Research Council of Canada for support of this
work including a PGS-D scholarship (F.W.).
Supporting Information Available: Characterization data
and experimental procedures for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL9019474
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Org. Lett., Vol. 11, No. 21, 2009
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