3-Aminopyrrolidine-4-carboxylic acid as versatile handle for internal labeling of pyrrolidinyl PNA

Article abstract of DOI:10.1016/j.bmcl.2011.08.079

Conformationally restricted pyrrolidinyl PNAs with an a/b-dipeptide backbone consisting of a nucleobase- modified proline and a cyclic five-membered amino acid spacer such as (1S,2S)-2-aminocyclopentanecarboxylic acid (ACPC) (acpcPNA) can form very stable hybrids with DNA with high Watson-Crick base pairing specificity. This work aims to explore the effect of incorporating 3-aminopyrrolidine-4-carboxylic acid (APC), whi h is isosteric to the ACPC spacer, into the acpcPNA. It is expected that the modification should not negatively affect the DNA binding properties, and that the additional nitrogen atom in the APC should provide a handle for internal modification. Orthogonally-protected (N3-Fmoc/N1-Boc and N3-Fmoc/N1-Tfa) APC monomers have been successfully synthesized and incorporated into the acpcPNA by Fmoc-solid-phase peptide synthesis. Tm, UV and CD spectroscopy confirmed that the (3R,4S)-APC could substitute the (1S,2S)-ACPC spacer in the acpcPNA with only slightly decreasing the stability of the hybrids formed between the modified acpc/apcPNA and DNA. In contrast, the (3S,4R) enantiomer of APC caused substantial destabilization of the hybrids. Furthermore, a successful on-solid-support internal labeling of the acpc/apcPNA via amide bond formation between pyrene-1-carboxylic acid or 4-(pyrene-1-yl) butyric acid and the pyrrolidine nitrogen atom of the APC spacer has been demonstrated. Fluorescence properties of the pyrene-labeled acpc/apcPNAs are sensitive to their hybridization states and can readily distinguish between complementary and single-mismatched DNA targets.

Full text of DOI:10.1016/j.bmcl.2011.08.079

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6465–6469
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Aminopyrrolidine-4-carboxylic acid as versatile handle for internal labeling
of pyrrolidinyl PNA
Nisanath Reenabthue a, Chalothorn Boonlua b, Chotima Vilaivan b, Tirayut Vilaivan b,
Chaturong Suparpprom a,
⇑
a Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Naresuan University, Ta-Po District, Muang, Phitsanulok 65000, Thailand
b Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Conformationally restricted pyrrolidinyl PNAs with an
a/b-dipeptide backbone consisting of a nucleo-
Received 3 June 2011
Revised 15 August 2011
Accepted 17 August 2011
Available online 23 August 2011
base-modified proline and a cyclic five-membered amino acid spacer such as (1S,2S)-2-aminocyclopen-
tanecarboxylic acid (ACPC) (acpcPNA) can form very stable hybrids with DNA with high Watson–Crick
base pairing specificity. This work aims to explore the effect of incorporating 3-aminopyrrolidine-4-car-
boxylic acid (APC), which is isosteric to the ACPC spacer, into the acpcPNA. It is expected that the mod-
ification should not negatively affect the DNA binding properties, and that the additional nitrogen atom
in the APC should provide a handle for internal modification. Orthogonally-protected (N3-Fmoc/N1-Boc
and N3-Fmoc/N1-Tfa) APC monomers have been successfully synthesized and incorporated into the
Keywords:
Peptide nucleic acid (PNA)
b-Amino acid
acpcPNA by Fmoc-solid-phase peptide synthesis. Tm, UV and CD spectroscopy confirmed that
Foldamer
the (3R,4S)-APC could substitute the (1S,2S)-ACPC spacer in the acpcPNA with only slightly decreasing
the stability of the hybrids formed between the modified acpc/apcPNA and DNA. In contrast, the
(3S,4R) enantiomer of APC caused substantial destabilization of the hybrids. Furthermore, a successful
on-solid-support internal labeling of the acpc/apcPNA via amide bond formation between pyrene-1-car-
boxylic acid or 4-(pyrene-1-yl) butyric acid and the pyrrolidine nitrogen atom of the APC spacer has been
demonstrated. Fluorescence properties of the pyrene-labeled acpc/apcPNAs are sensitive to their hybrid-
ization states and can readily distinguish between complementary and single-mismatched DNA targets.
Ó 2011 Elsevier Ltd. All rights reserved.
Molecular beacon
Fluorescence
Peptide nucleic acid or PNA1,2—an analogue of DNA with achiral,
electrostatically neutral, peptide-like backbone—is an important
tool in nucleic acid-related applications ranging from diagnostic,
therapeutic, materials science and nanotechnology. Despite the
completely different core structure of Nielsen’s PNA from native
nucleic acids, PNA can form a very stable hybrid with DNA/RNA
in a highly sequence specific manner according to the Watson–
Crick base pairing rules.3 An additional advantage of the unnatural
backbone of PNA is its high chemical/physiological stability.4 The
ability to functionalize PNA, for example, by biotin, a fluorophore
or other reporter groups, is very important for many of its applica-
tions. In principle, the peptide backbone of PNA allows facile mod-
ification by standard amide bond formation. However, in practice
this is generally true only for terminal modifications, whereby
the amino group is already present (N-terminus) or can be incorpo-
rated without disrupting the overall structure of the PNA (e.g., as a
lysine residue at the C-terminus).5 Backbone modification at non-
terminal positions requires incorporation of specialized monomers
during the PNA synthesis on the solid support.6–8 These modified
PNA monomers often cause disturbance to the PNA structures
and their DNA binding properties.9 Recently, Appella et al. has
developed Nielsen’s type aegPNA monomers with a lysine side-
chain attaching at the gamma-position with the aim to use as a
handle for site-specific labeling through amide bond formation.10
It was demonstrated that such modification had little effect on
the stability of the resulting PNAÁDNA hybrids and that a range
of labels can be successfully incorporated at any desired posi-
tions.11 Peptoid-like side chains have also been attached to the
nitrogen atom of the aminoethyl group in aegPNA without reduc-
ing the hybridization affinity.12 All of the above-mentioned works
focus on labeling of the original aegPNA system with the amino-
ethylglycine backbone. Recently, we have introduced a conforma-
tionally constrained analogue of PNA consisting of proline/2-
aminocyclopentane carboxylic acid
a/b-dipeptide subunit called
acpcPNA.13–15 In addition to the ability to bind to its complemen-
tary DNA target with higher affinity and sequence specificity than
DNA and aegPNA,16 this acpcPNA exhibited a number of unusual
properties not observed in aegPNA including the high preference
for binding to DNA over RNA and the absence of self-pairing be-
tween two complementary strands.14,15 Although terminal labeling
of acpcPNA had been routinely performed,16,17 there is no general
⇑
Corresponding author. Tel.: +66 055 963 462; fax: +66 055 963 401.
E-mail address: chaturongs@nu.ac.th (C. Suparpprom).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.079

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N. Reenabthue et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6465–6469
it is stable under non-aqueous basic conditions required for Fmoc
O
O
O
deprotection, yet can be readily removed by treatment with an
aqueous base where the Rink amide linker is completely stable.
Syntheses of both enantiomers of the known Fmoc/Boc-protected
trans-APC spacer were carried out following a protocol described
by Gellman et al. (Scheme 3).19 Diastereoselective reduction of
the chiral enamines derived from ethyl 3-oxopyrrolidine-4-carbox-
B
B
*
*
N
N
HN
HN
O
*
*
n
n
N
H
ylic acid (5)20 and (S)-(À)-
a-methylbenzylamine or (R)-(+)-a-
methylbenzylamine as chiral auxiliaries gave the (3R,4S) and
acpcPNA
apcPNA
(3S,4R) amines 6a and 6b, respectively.
Scheme 1. Structure of acpcPNA and apcPNA.
These intermediates were purified from the undesired diaste-
reomers by recrystallization of the HCl salts (>98% of the desired
diastereomer according to 1H NMR and HPLC analysis). Cleavage
of the chiral auxiliary, followed by protecting groups exchange
and activation by pentafluorophenyl trifluoroacetate (PfpOTfa)
provided the trans-(3R,4S)- and (3S,4R)-Pfp-activated spacers 3a
and 3b. On the other hand, when the Boc group was removed by
treatment with trifluoroacetic acid (TFA) prior to the activation
with an excess of PfpOTfa, the Pfp-activated N1-Tfa-protected
(3R,4S)-APC spacer 4a was obtained.
The acpcPNAs which were fully or partially modified with either
enantiomer of the APC spacer were synthesized according to our
previously published protocol.14 The APC spacers 3a and 3b cou-
pled at least as efficiently as the standard ACPC spacer on the solid
support (>98% coupling yield for each step). The Boc group was re-
moved during the cleavage from the resin with TFA. If the acpc/
apcPNA was to be further functionalized on the solid support, the
Tfa-protected APC spacer 4a was employed instead of 3a. After
end-capping by acetylation or benzoylation, the nucleobase side
chain protecting groups was removed by treatment with 1:1 aque-
ous ammonia/dioxane at 60 °C overnight. This condition also
simultaneously removed the N-Tfa protecting group on the Tfa-
protected APC unit. A label such as pyrene-1-carboxylic acid or
4-(pyrene-1-yl)butyric acid was then coupled onto the solid-sup-
ported acpc/apcPNA via HATU activation. After cleavage from the
solid support by treatment with TFA, the acpc/apcPNA oligomers
were purified by RP-HPLC and characterized by MALDI-TOF mass
spectrometry (Table 1).
solution for internal labeling available. We envisioned that the
ability to site-specifically attach a label within the acpcPNA back-
bone would expand its utility further.
In this Letter, we report a new modification of our acpcPNA
backbone by partial or total replacement of the 2-aminocyclopen-
tanecarboxylic acid (ACPC) spacer with an isosteric 3-aminopyrrol-
idine-4-carboxylic acid (APC) spacer (Scheme 1).18 We also
evaluate the DNA binding properties of these novel apcPNA as well
as mixed acpc/apcPNA chimeric oligomers, and compared to the
corresponding unmodified acpcPNA. In addition, we have devel-
oped an orthogonally protected APC monomer and demonstrated
its suitability as a handle for incorporation of fluorescence labels
at the internal positions of acpcPNA via amide bond formation.
The Fmoc-protected, pentafluorophenyl (Pfp)-activated pyrro-
lidinyl PNA monomers 1a–1d and the ACPC spacer (2) were syn-
thesized following the literature procedure.14 Since the trans-
(3R,4S) APC should be isostructural with trans-(1S,2S)-ACPC spacer
in acpcPNA, we decided to focus on this particular stereoisomer,
but the trans-(3S,4R) stereoisomer of APC was also included for
comparison purpose.
These monomers were required in Fmoc-protected, Pfp-acti-
vated forms (Scheme 2) in order to be compatible with our stan-
dard protocol for the solid phase peptide synthesis (SPPS) of
acpcPNA.14 In addition, the pyrrolidine nitrogen atom of the APC
requires a semi-permanent protection that is compatible to the
Fmoc-SPPS. The Boc group was chosen (monomers 3a, 3b) for ini-
tial evaluation of the DNA-binding behaviour of the modified
acpcPNA. However, since the Boc group cannot be selectively re-
moved without concomitant cleavage of the acpcPNA from the so-
lid support equipped with Rink amide linker generally used in
Fmoc-SPPS, an alternative protecting group is required to enable
post-synthetic modifications of the acpcPNA on the solid support.
The trifluoroacetyl (Tfa) group was chosen for this purpose because
The stability of the hybrids formed between the synthesized
acpc/apcPNA with their complementary DNA was determined by
thermal denaturation experiments. The Tm data are summarized
in Table 1 (melting curves shown in Fig. S9). To evaluate the com-
patibility of the APC to the ACPC backbone, the Tm values of the
fully modified homothymine apcPNA nonamers PNA1 (3R,4S) and
PNA2 (3S,4R) with dA9 were compared with the corresponding
unmodified acpcPNA. In accordance with our prediction, the
PNA1 with the (3R,4S)-APC spacer showed a clear melting behav-
iour, albeit with a considerably lower Tm compared to the unmod-
O
O
ified acpcPNA (
D
Tm = À17.0 °C). The PNA2 showed no melting
PfpO
NHFmoc
1a: B = ABz
1b: B = T
B
behaviour down to 20 °C, suggesting that the hybrid must have
been very unstable. Interestingly, the Tm curve of PNA1 was con-
siderably broader than the unmodified acpcPNA, which may sug-
gest the formation of multiple species, which was confirmed in
the first derivative plot (Fig. S9b). The results from UV titration21
(Fig. 1) also confirmed that the PNA1 could form a hybrid with
dA9. They also further revealed that the PNA1 could form both
PNAÁDNA and (PNA)2ÁDNA hybrids as shown by the presence of
two inflection points at ca. 50 and 35 mol % of DNA in the titration
curve. CD spectra of the mixtures of PNA1 and dA9 at 1:1 and 2:1
ratios (Fig. 2) also confirmed the hybrid formation. The negative
band originally at 250 nm in the dA9 was slightly blue-shifted to
247 nm, with an increased intensity upon hybrid formation. In
addition, a new negative band appeared at 267 nm. While other
parts of the spectra remained unchanged, the intensity of this
267 nm band further increased when the ratio of the PNA:DNA
was increased from 1:1 to 2:1 at a fixed concentration of the
PfpO
Fmoc
2
N
1c: B = CBz
1d: B = GIbu
Pyrrolidinyl PNA monomer
(1S,2S)-ACPC spacer
O
O
PfpO
NHFmoc
PfpO
NHFmoc
3a: R = Boc
4a: R = Tfa
3b
N
Boc
N
R
(3R,4S)-APC spacer
(3S,4R)-APC spacer
Scheme 2. Structure of acpcPNA monomers and spacers employed in this study
(Abbreviations: Boc, tert-butoxycarbonyl; ACPC, 2-aminocyclopentanecarboxylic
acid; APC, 3-aminopyrrolidine-4-carboxylic acid; Bz, benzoyl; Fmoc, fluorene-9-
ylmethoxycarbonyl; Ibu, isobutyryl; Pfp, pentafluorophenyl; Tfa, trifluoroacetyl).

N. Reenabthue et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6465–6469
6467
Fmoc
HCl
NH
O
O
NH
O
Ph
i,ii
O
iii,iv,v
78%
OEt
OH
21%
BocN
OEt
BocN
BocN
6a
5
7a
vi,vii
i) (S)-(-)-methylbenzylamine, HOAc, EtOH, rt
ii) a) NaBH3CN, 75 ºC b) 4 N HCl in EtOAc, 0 ºC
iii) LiOH, THF/MeOH/H2O (6:3:1), 0 ºC
iv) H2, Pd/C, EtOH, rt
v) FmocOSu, aq NaHCO3, acetone
vi) TFA, CH2Cl2, rt
vii
60%
60%
Fmoc
Fmoc
NH
O
NH
O
OPfp
OPfp
BocN
TfaN
vii) PfpOTfa, DIEA, CH2Cl2, rt
4a
3a
Scheme 3. Synthesis of the (3R,4S)-APC spacer (steps i–iv are adapted from Ref. 18). The enantiomeric (3S,4R)-APC derivatives 6b, 7b and 3b were prepared in 17%, 66% and
66% yield, respectively starting from 5 and (R)-(+)-methylbenzylamine.
Table 1
Sequence, mass spectrometric and thermal melting data of the acpc/acpPNA
Sequence (N?C)a
m/z (calcd)
m/z (found)
Tm (°C)
DTm (°C)
b
c
Code
PNA1
PNA2
3250.5
3250.5
3250.1
3251.2
55.5d,e
<20d
À17.0
—
Bz-TTTTTTTTT-LysNH2
Bz-TTTTTTTTT-LysNH2
PNA3
PNA4
PNA5
PNA6
PNA7
PNA8
3242.5
3242.5
3621.9
3409.6
3788.9
3893.0
3242.6
3243.7
3622.2
3411.0
3789.4
3893.7
71.1d
28.3d
50.8d
62.2f
44.3f
45.7f
À1.4
À44.2
À1.4
À10.3
À7.9
À6.5
Bz-TTTTTTTTT-LysNH2
Bz-TTTTTTTTT-LysNH2
Bz-GTAGATCACT-LysNH2
Ac-TTTT(Py)TTTTT-LysNH2
Ac-GTAGA(Py)TCACT-LysNH2
Bz-GTAGA(PyBu)TCACT-LysNH2
a
b
c
T = ssACPC-T; T = (3R,4S)-APC-T; T = (3S,4R)-APC-T; (Py) = pyrene-1-carbonyl modification; (PyBu) = 4-(pyrene-1-yl)butyryl modification.
With complementary DNA (dA9 for PNA1–PNA4 and PNA6; dAGTGATCTAC for PNA5, PNA7 and PNA8).
Values refer to the Tm decrease relative to that of unmodified acpcPNA under identical conditions (Tm of hybrids with complementary DNA Ac-TTTTTTTTT-LysNH2:
72.5 °C; Ac-GTAGATCACT-LysNH2: 52.2 °C).
d
Conditions: 10 mM sodium phosphate buffer pH 7.0, 100 mM NaCl, [PNA] = 1.0
Very broad and non-two-state melting curve.
Conditions: 10 mM sodium phosphate buffer pH 7.0, [PNA] = 2.5 lM and [DNA] = 3.0 lM.
l
M and [DNA] = 1.0
lM.
e
f
1.00
0.95
0.90
0.85
0.80
0.75
0.70
15.0
10.0
5.0
0.0
-5.0
-10.0
-15.0
10 20 30 40 50 60 70 80 90
Mol% of DNA
200
220
240
260
280
300
Wavelength (nm)
Figure 1. UV titration plots of PNA1 (s), PNA2 (j), PNA3 (Ç) and their
complementary DNA (dA9). The plots show the ratio of observed A260/calculated
A260 and mole fraction of DNA, and were measured in 10 mM sodium phosphate
Figure 2. CD spectra of (a) dA9 (–j–), PNA1 (–d–), dA9 + 1 equiv PNA1 (–h–), and
dA9 + 2 equiv PNA1 (–s–). The spectra were measured in 10 mM sodium phosphate
buffer, pH 7.0, 100 mM NaCl at 25 °C, [PNA] = 2.5 or 5.0 lM and [DNA] = 2.5 lM.
buffer pH 7.0, 100 mM NaCl at 25 °C, initial PNA concentration = 2 lM.
served in mixtures of PNA2 and dA9 at both 1:1 and 2:1 ratios rel-
ative to the sum of the CD spectrum of each component (Fig. S12).
In addition, the UV titration curve of PNA2 was essentially flat
DNA. This suggests the formation of different hybrids at different
PNA:DNA ratios. No significant change in the CD spectra was ob-

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N. Reenabthue et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6465–6469
without any hypochromicity (Fig. 1), indicating that it cannot form
a stable hybrid at the temperature which the titration was carried
out (25 °C). It can therefore be concluded that only the (3R,4S)-APC,
but not the (3S,4R)-APC, could replace the (1S,2S)-ACPC spacer in
acpcPNA as would be anticipated from their absolute
configurations.
The UV titration curve of the PNA3, singly modified with a
(3R,4S)-APC spacer at the middle of the strand, suggested that it
could form only a duplex with dA9 similar to the unmodified
acpcPNA (Fig. 1). Most importantly, the Tm of the hybrid with com-
plementary DNA of PNA3 was only slightly decreased compared to
tively). The pyrene-1-carboxylic acid-modified homothymine
PNA6 in the single-stranded form exhibited two weak fluorescence
emission bands at 382 and 400 nm. When it was hybridized with
excess of dA9, the fluorescence intensity at 382 nm increased
ꢀ2.6-fold over that of the single-stranded PNA6 (Fig. S13). The
change in the fluorescence could be visualized by naked eyes under
UV irradiation (Fig. S14). Hybridization with a non-complementary
DNA target (dA4TA4) gave barely noticeable change in the fluores-
cence compared to the single stranded PNA6. Consistently, the
fluorescence increase was also observed in the mixed base se-
quence PNA7, although in this case the difference was consider-
ably smaller (ꢀ1.3-fold enhancement upon hybridization)
(Fig. 3a). Hybridization of PNA7 with a single-mismatched DNA
target yielded no significant fluorescence change, indicating the
high specificity of the system.
A more pronounced fluorescence change was observed in PNA8
with the same sequence as PNA7 but with the pyrenebutyryl in-
stead of the pyrenecarbonyl labels. Extending the pyrene from
the acpc/apcPNA backbone by a three-carbon linker resulted in a
4.8 to 5-fold fluorescence enhancement of the 379 nm emission
upon hybridization with the complementary DNA relative to the
single stranded PNA8 and its hybrid with single mismatched
the corresponding unmodified acpcPNA hybrid (
the other hand, the hybrid of PNA4 carrying a single (3S,4R)-APC
modification was much more destabilized (
D
Tm = À1.4 °C). On
D
Tm = À44.2 °C). These
results confirmed the conclusion from the aforementioned studies
on the fully-modified PNA1 and PNA2 that only the (3R,4S)-APC
could substitute the (1S,2S)-ACPC without much destabilizing ef-
fect. This conclusion is also true for mixed sequence acpc/apcPNA
such as PNA5. In this case the Tm was again only slightly decreased
(D
Tm = À1.4 °C) upon substitution of one ACPC by one (3R,4S)-APC
unit. The specificity of DNA recognition by PNA1 was demon-
strated by the absence of a sigmoidal melting curve when the
DNA was changed from dA9 (complementary) to dA4TA4 (single
mismatched). Likewise, the thermal stability of the hybrids formed
between the singly APC-modified PNA3 or PNA5 and their single-
mismatched DNA targets was also markedly decreased (À28.0 °C
and À21.3 °C, respectively) compared to the fully complementary
hybrids (Fig. S10). These results indicated that the high specificity
of the DNA recognition is still retained in the apcPNA and acpc/
apcPNA.
250
a
200
150
100
50
One distinctive feature of the homothymine PNA1, fully-modi-
fied with (3R,4S)-APC spacer, compared to the unmodified acpcPNA
with the same sequence is that it forms a triplex with DNA in addi-
tion to the duplex. This could be explained by the favourable elec-
trostatic attraction between the positively-charged backbone of
the fully-modified PNA and the negatively-charged phosphate
backbone of the DNA. The sharpening of the Tm curve at pH 8
(Fig. S11) supports this hypothesis because at higher pH, the pyr-
rolidine nitrogen (pKa ꢀ8.75)22 would become less protonated,
hence the electrostatic attraction should decrease. The behaviour
of the APC spacer at high pH would therefore be more closely
resemble that of the non-ionizable ACPC spacer. Since the singly-
modified homothymine PNA3 formed only duplex with comple-
mentary DNA, the triplex formation should not be a problem be-
cause for labeling purpose the PNA would need to be modified
with only one or up to a few APC residues. Furthermore, upon
modification by the amide bond formation, the APC residue should
no longer be protonated at neutral pH.
In order to investigate the viability of using (3R,4S)-APC as a
handle for internal modification of acpcPNA with a fluorophore,
three pyrene-labeled acpc/apcPNA (PNA6, PNA7 and PNA8) were
synthesized as models. Pyrene-1-carboxylic acid and 4-(pyrene-
1-yl)-butyric acid were chosen as the labels with the expectation
that they should exhibit different fluorescence in the single
stranded and duplex states. In the single stranded form, the pyrene
should strongly interact with the nucleobases of the PNA resulting
in fluorescence quenching.23 Upon hybridization with the comple-
mentary DNA strand by Watson–Crick base pairing, the pyrene
should interact differently with the nucleobases, leading to a fluo-
rescence change.10,24,25 A similar concept has previously been
demonstrated in DNA system whereby the pyrene was attached
to the nucleobase.26,27 According to Tm analyses, labeling the
acpc/apcPNA with pyrene cause further destabilization of the
0
350
400
450
500
550
Wavelength (nm)
250
b
200
150
100
50
0
350
400
450
500
550
Wavelength (nm)
Figure 3. Fluorescence spectra of pyrene-labeled acpc/apcPNA (a) PNA7 (–h–),
PNA7 + complementary DNA (–s–), PNA7 + single mismatch DNA (––) and (b)
PNA8 (–h–), PNA8 + complementary DNA (–s–), PNA8 + single mismatch DNA (–
–); complementary DNA = (dAGTGATCTAC) and single mismatched DNA = (dAGT-
GACCTAC). The spectra were measured in 10 mM sodium phosphate buffer, pH 7.0
PNAÁDNA duplexes, presumably due to steric effects (
DTm com-
pared to the corresponding unmodified acpcPNAÁDNA hybrids are
À10.3, À7.9 °C and À6.5 °C for PNA6, PNA7 and PNA8, respec-
at 25 °C, [PNA] = 2.5 lM and [DNA] = 3.0 lM, excitation wavelength = 345 nm.

N. Reenabthue et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6465–6469
6469
DNA (Fig. 3b). The better discrimination should be attributed to the
Supplementary data
more effective quenching of the pyrenebutyryl label in the single
stranded PNA8 than the pyrenecarbonyl label in PNA7 as shown
by the smaller fluorescence of PNA8 compared to PNA7. After
hybridization with the complementary DNA, both pyrene-labeled
PNAs yielded similar fluorescence signals. Quenching of pyrene
by nucleobases is known to occur via electron transfer from G, to
T or to C.28,29 The more effective quenching of the pyrenebutyryl
label than the pyrenecarbonyl label can be attributed to at least
two factors. First, the pyrenebutyryl group has a flexible linker,
allowing more effective contact with neighboring nucleobases in
the single stranded PNA. Second, the pyrenebutyryl label has an al-
kyl group, while the pyrenecarbonyl label has a carbonyl group, di-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.079.
References and notes
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The generation of a positive (turn-on) fluorescence signal in this
particular system is an advantage over many other related pyrene-
labeled DNA probes whereby the fluorescence is often quenched
upon hybridization with DNA as a result of intercalation of the pyr-
ene chromophore between the base stack in the DNA duplex.30
Only when the pyrene was linked to the nucleobase via relatively
rigid linkers that a fluorescence enhancement was observed since
no intercalation was possible without disrupting the Waston-Crick
base pairing.27,31,32 The fluorescence enhancement observed, to-
gether with the absence of duplex stabilization of the pyrene-la-
beled acpc/apcPNA relative to the unlabeled acpc/apcPNA,
suggested the absence of intercalation of the pyrene labels be-
tween the base stack in acpc/apcPNAÁDNA hybrids.33 In effect,
the pyrenecarbonyl- or pyrenebutyryl-labeled acpc/apcPNAs be-
have as quencher-free PNA beacons.34 These singly-labeled probes
are more easily synthesized than the corresponding dual-labeled
PNA probes and should be less likely to have problems of aqueous
solubility.5,35 Although the fluorescence enhancement of these pro-
totypic systems is relatively modest because of the high back-
ground fluorescence of the pyrene chromophores in the single
stranded PNA, the excellent specificity warrants potential use of
the system. As the extent of the quenching of pyrene by nucleo-
bases is likely to be sequence-dependent,23,24 work is now in pro-
gress to optimize the positions and structures of the label that will
allow maximum fluorescence change to improve the efficiency of
the DNA sequence determination.
Acknowledgments
32. Hwang, G. T.; Seo, Y. J.; Kim, S. J.; Kim, B. H. Tetrahedron Lett. 2004, 45, 3543.
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111, 6966.
34. For a review on quencher-free DNA beacons see: Venkatesan, N.; Seo, Y. J.; Kim,
B. H. Chem. Soc. Rev. 2008, 37, 648.
We acknowledge the Thailand Research Fund (TRF) and Com-
mission on Higher Education (MRG5180025 to C.S. and
RTA5280002 to T.V.) and the Center of Excellence for Innovation
in Chemistry (PERCH-CIC), Commission on Higher Education, Min-
istry of Education (to N.R.) for financial support.
35. Seitz, O. Angew. Chem., Int. Ed. 2000, 39, 3249.