Synthesis and fluorescence properties of 3,6-diaminocarbazole-modified pyrrolidinyl peptide nucleic acid

Article abstract of DOI:10.1039/c6ra15861g

This work aims to explore a new clickable carbazole-based fluorescent label, its incorporation into pyrrolidinyl peptide nucleic acid (acpcPNA), and the interactions between the labeled PNA probe and DNA by fluorescence spectrophotometry and molecular dynamics simulation. A carbazole derivative, namely 3,6-diaminocarbazole (DAC), was synthesized and incorporated into the internal and terminal positions of azide-modified acpcPNA via a sequential reductive alkylation and click reaction previously developed by our group. The DAC-modified acpcPNA can form a stable hybrid with complementary DNA with somewhat lower stability compared to unmodified acpcPNA. Most importantly, the DAC-modified acpcPNA exhibits a remarkable fluorescence increase in the presence of DNA (up to 35.5 fold with complementary DNA). Non-complementary as well as single mismatched DNA targets gave a smaller fluorescence increase (1.1 to 18.6 fold), and the discrimination could be further improved by increasing the temperature to dissociate the mismatched hybrids. Molecular dynamic simulations revealed that the DAC interacts with adjacent nucleobases in single stranded PNA, resulting in quenching of the fluorescence signal. When the PNA formed a hybrid with DNA, the DAC was pushed away from the duplex, resulting in a fluorescence increase. Thus, the DAC-labeled acpcPNA is a potential candidate as a self-reporting probe for determination of DNA sequences.

Full text of DOI:10.1039/c6ra15861g

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1
Synthesis and Fluorescence Properties of 3,6-Diaminocarbazole-
Modified Pyrrolidinyl Peptide Nucleic Acid
1
2
2
3
Aukkrapon Dangsopon , Nattawee Poomsuk , Khatcharin Siriwong , Tirayut Vilaivan ,
1
Chaturong Suparpprom *
1
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science,
Naresuan University, Muang, Phitsanulok 65000, Thailand
Materials Chemistry Research Center, Department of Chemistry and Center of Excellence for Innovation in
Chemistry, Faculty of Science, Khon Kaen University,
2
Khon Kaen 40002, Thailand
3
Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University,
Phayathai Road, Patumwan, Bangkok 10330, Thailand
*
E-mail: chaturongs@nu.ac.th
Abstract
This work aims to explore a new clickable carbazoleꢀbased fluorescent label, its incorporation
into pyrrolidinyl peptide nucleic acid (acpcPNA), and the interactions between the labeled PNA
probe and DNA by fluorescence spectrophotometry and molecular dynamic simulation. A
carbazole derivative, namely 3,6ꢀdiaminocarbazole (DAC), was synthesized and incorporated
into the internal and terminal positions of azideꢀmodified acpcPNA via a sequential reductive
alkylation and click reaction previously developed by our group. The DACꢀmodified acpcPNA
can form a stable hybrid with complementary DNA with somewhat lower stability compared to
unmodified acpcPNA. Most importantly, the DACꢀmodified acpcPNA exhibits a remarkable
fluorescence increase in the presence of DNA (up to 35.5 fold with complementary DNA). Nonꢀ
complementary as well as single mismatched DNA targets gave smaller fluorescence increase
(1.1 to 18.6 fold), and the discrimination could be further improved by increasing the
temperature to dissociate the mismatched hybrids. Molecular dynamic simulations revealed that
the DAC interacts with adjacent nucleobases in single stranded PNA, resulting in quenching of
the fluorescence signal. When the PNA formed a hybrid with DNA, the DAC was pushed away
from the duplex, resulting in a fluorescence increase. Thus, the DACꢀlabeled acpcPNA is a
potential candidate as a selfꢀreporting probe for determination of DNA sequences.

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2
Introduction
Selfꢀreporting fluorescence oligonucleotides have been extensively used as a probe for
determination of DNA sequences. Several approaches have been explored in order to introduce a
mechanism for fluorescence change upon hybridization of the probe with the correct DNA target.
1
Classically, two labels that can interact by static quenching, Fluorescence Resonance Energy
2
,3,4
5
Transfer (FRET)
or excimer/exciplex formation are used in combination. More recently,
there are attempts to develop singly labeled selfꢀreporting probes by attachment of an
6
intercalative or environment sensitive dye to the oligonucleotide. However, the performance of
these singlyꢀlabeled probes is generally variable, depending on the sequence and therefore still
leaves rooms for improvements.
Carbazole is an electron rich aromatic molecule that showed many interesting properties
such as high thermal stability, excellent photophysical and electroconductive properties and the
ease of synthesis and chemical modification at 3ꢀ, 6ꢀ and 9ꢀ positions. Carbazole derivatives,
both in monomeric and oligomeric forms, have found widespread applications as
7
,8,9,10
11ꢀ12
photoconductivity polymers,
polymeric light emitting devices,
high temperature
1
3
14,15,16
thermostable polymers,
electrochromic materials,
dyeꢀsensitized solar cell and
1
7,18,19
dendrimeric electro phosphorescent devices.
In addition, carbazole derivatives have been
2
0
used as fluorophores in sensor applications, including DNA detection. Carbazole derivatives
with cationic groups at 3,6ꢀpositions have been reported to bind in the DNA groove of ATꢀrich
2
1,22,23,24
sequences.
However, the use of carbazole derivatives in combination with an
oligonucleotide or analogues for sequenceꢀspecific detection of DNA sequences is unknown.
Peptide nucleic acid or PNA is a DNA mimic consisting of nucleobases attached onto a
2
5,26
peptideꢀlike backbone.
PNA can hybridize with DNA according to WatsonꢀCrick base
pairing rules with high affinity and sequence specificity. The absence of phosphate group in the
PNA backbone results in decreasing of the electrostatic repulsion with DNA, and therefore PNA
can form a more stable duplex with DNA than DNA itself. In addition, the binding of PNA and
DNA is highly sequence specific and is not sensitive to ionic strength. These properties, together
with the excellent chemical and biological stabilities, make PNA a promising tool for several
applications in medical and biotechnology fields. Recently, we reported a series of new PNA
system with restricted conformation flexibility as a result of incorporation of cyclic structures
2
7,28,29,30
into the PNA backbone.
Pyrrolidinyl PNA probes labeled with environment sensitive

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3
3
1,32
32,33
34
dyes such as pyrene,
Nile red,
or other DNA binding dyes such as thiazole orange have
been successfully applied as selfꢀreporting fluorescence probes for DNA sequence detection.
From the remarkable photophysical properties and DNA binding ability mentioned
above, carbazole has a potential to be developed into a probe for DNA detection by fluorescence
spectroscopy. In the present work, an electron rich carbazole derivative, namely 3,6ꢀ
diaminocarbazole (DAC), was incorporated at an internal position of acpcPNA in order to
develop a new selfꢀreporting fluorescence hybridization probe. Here we report the synthesis,
thermal stabilities and fluorescence properties of DACꢀmodified acpcPNA probes, as well as
their ability to discriminate between complementary and singleꢀmismatched DNA targets.
Moreover, molecular dynamics simulation was performed to explain the behavior of DACꢀ
labeled acpcPNA probes.
Results and Discussion
Synthesis of diaminocarbazole (DAC)-labeled PNA
A novel clickable 3,6ꢀdiaminocarbazole for labeling of the acpcPNA was first prepared.
9
N ꢀPropargylated, Bocꢀprotectedꢀ3,6ꢀdiaminocarbazole (5) was prepared in 4 steps with 37%
overall yield starting from carbazole (Scheme 1). Controlled nitration of the carbazole (1) with
fuming nitric acid in 1,2ꢀdichloroethane at 45 °C afforded the dinitration product 2 in good yield.
Subsequent reduction of the dinitroꢀcompound 2 with tin(II) chloride in a mixture of acetic acid
and concentrated hydrochloric acid provided the desired diaminocarbazole 3 in excellent yield
after simple precipitation. The amino groups in 3 were protected to avoid undesired side
reactions at this position during subsequent steps. Boc group was chosen as it could be easily
introduced and subsequently cleaved under acidic conditions required for the cleavage of the
modified PNA from the solid support. The Bocꢀprotected DAC (4) was next alkylated with
propargyl bromide in the presence of K CO /KI to get the desired clickable diaminocarbazole
2
3
derivative (5).
AcpcPNA sequences were synthesized manually by Fmoc solid phase peptide synthesis
2
7,35
as described previously.
In order to provide a handle for attachment of the DAC via the
3
4
previously reported reductive alkylationꢀClick chemistry strategy, one of the 2ꢀamino
cyclopentanecarboxylic acid (ACPC) spacer in the middle of the acpcPNA strand was replaced
3
5
with 3ꢀaminopyrrolidineꢀ4ꢀcarboxylic acid (APC) for internal labeling. For terminal labeling,

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4
no modification with APC was required, but the last ACPC residue was omitted from the PNA
sequence. After endꢀcapping by acetylation (this step was skipped for the terminal modification)
and removal of all protecting groups from the nucleobase and the APC spacer, the azide group
was introduced by reductive alkylation with 4ꢀazidobutanal. Finally, the clickable DAC label (5)
was attached to the azideꢀmodified acpcPNA on the solid support by Cuꢀcatalyzed click
chemistry. Four DACꢀmodified acpcPNA sequences were designed to investigate the DNA
binding and fluorescent properties under different sequence context. These include an internallyꢀ
DAC
labeled homothymine (T9 ), two internallyꢀlabeled mix sequences with different flanking
DAC
DAC
bases (A/T and C/G pairs) (M10AT
and M10GC ) and a terminallyꢀlabeled mix sequence
(DAC-M10). All modified PNAs were purified by reverse phase HPLC (to >90% purity). The
identities of the obtained PNA sequences were confirmed by MALDIꢀTOF mass spectrometry,
which showed the mass signals that are in a good agreement with the expected values. The
sequences and characterization data of all PNA involved in this studies are shown in Table 1.
Table 1. Sequence and characterization data of DACꢀlabeled acpcPNA.
tR
min)
Yield
(%)
PNA
Sequence (N → C)
a
b
m/z (calcd) m/z (found)
(
DAC
(DAC)
T9
AcꢀTTTT
TTTTTꢀLysNH
2
32.0
29.5
27.7
29.0
5.8
5.2
4.4
5.4
3513.6
3892.8
3893.8
3738.8
3512.4
3894.1
3893.2
3738.3
DAC
(DAC)
M10AT
M10GC
AcꢀGTAGA
AcꢀGTAGC
TCACTꢀLys NH
2
DAC
(DAC)
GCACTꢀLys NH
2
DAC-M10 DACꢀGTAGATCACTꢀLys NH
2
a
HPLC condition: C18 column 4.6 x 50 mm, particle size 3 µm, gradient 0.1% TFA in H O:MeOH 90:10 for 5 min
2
then linear gradient to 10:90 over 30 min, flow rate 0.5 mL/min.
b
isolated yield after HPLC

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5
NO2
O N
NH2
2
H N
2
i
ii
N
76%
N
H
91%
N
H
H
(
1)
(2)
(3)
NHBoc
NHBoc
iv
55%
BocHN
iii
BocHN
9
9%
N
N
H
(4)
(5)
o
i) 90% HNO , 1,2-dichloroethane, 45 C, 4h
3
ii) SnCl /HOAc/HCl, reflux
2
iii) Boc O, Et N, MeOH, rt
2
3
iv) Propargyl bromide, K CO , KI, DMF, rt
2
3
Scheme 1. Synthesis of clickable 3,6ꢀdiaminocarbazole label (5).
Thermal stability and fluorescent properties of DAC-labeled acpcPNA
To investigate the binding properties with DNA, the stabilities of complementary and
mismatched DACꢀmodified PNA⋅DNA hybrids were determined by thermal denaturation
experiments. Thermal stability (T ) and fluorescent properties of DACꢀlabeled acpcPNA are
m
DAC
summarized in Table 2. The complementary DNA hybrid of T9
sequence showed a T of
m
65.2 °C, which was considerably lower than the corresponding DNA hybrid of unmodified T9
DAC
acpcPNA (>80°C). The T of the DNA hybrid of the M10AT
sequence of 53.4 °C was also
m
reduced compared to the unmodified PNA (57.0 °C). This suggests that the internal modification
with the DAC dye destabilizes the acpcPNA⋅DNA duplex, probably due to steric effect. This
behavior was also observed in our previous studies with the same apc/acpcPNA system with
3
4,35
DAC
different labels.
The mixꢀbase sequences PNA M10GC
and DAC-M10 showed Tm
values of 59.0 and 58.2°C, respectively. The T value of the terminally DACꢀmodified DAC-
m
M10 duplex was in fact larger than that of the unmodified duplex (ꢀT = +1.2 °C). This can be
m
explained by the additional stabilization from the end stacking of the DAC at the terminal base
3
1
pair of the PNA·DNA duplex as observed in the case of pyrene label. All single mismatched
PNA·DNA hybrids exhibited considerably lower T than complementary hybrids (ꢀT = –27.0
m
m
DAC
DAC
DAC
to –41.6 °C for T9
and –18.4 to –28.9 °C for M10GC / M10AT
and less than –38.2°C

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6
for DAC-M10). The ꢀT values are comparable to those of unmodified PNA, which indicated
m
that the base pairing specificity of the DACꢀlabeled acpcPNA remained high.
Next, fluorescence experiments were performed with all PNA sequences and their DNA
hybrids. The responsiveness of the PNA probe was reported in terms of fluorescence intensity
ratio (F/F ; where F = fluorescence intensity of PNA⋅DNA hybrid, F = fluorescence intensity of
0
0
single stranded PNA; λ 315 nm; λem 450 nm). While free 3,6ꢀdiaminocarbazole showed a high
ex
Φ of 0.489, all internallyꢀlabeled single stranded PNA probes exhibited very low Φ values of
F
F
0.01 or less, regardless of the identity of the neighboring nucleobase (Table 2, entries 1, 6, 11).
This suggests that the DAC label is quenched upon incorporation into the PNA, probably by
3
1
DAC
DAC
interactions with the nucleobases similar to pyrene. The sequences M10AT
and T9
DAC
showed lower quantum yields than M10GC , which means that A/T are better quenchers than
C/G bases for DAC label. Nevertheless, judging from the uniformly low quantum yields of all
PNA probes, the difference in quenching efficiency by different nucleobases is much less than
3
1
pyrene, which emphasizes the important feature of the DAC label.
A remarkable increase in fluorescence signal was observed upon hybridization with
complementary DNA, as shown by the fluorescence intensity ratio (F/F ) between 10.2–35.5
0
(Table 2, entries 2, 7, 12). The quantum yield of the DACꢀlabeled PNA·DNA duplexes was also
consistently increased by >10ꢀfold to around 0.08–0.24, which is still considerably lower than
the free DAC (3). This indicates that the interaction between the DAC and the neighboring
nucleobases is reduced in PNA·DNA duplexes due to the formation of base pairs. This behavior
3
1
is similar to pyrene label and a similar mechanism of fluorescence increase is proposed. It
should be noted that free DAC also interact with single stranded DNA, resulting in fluorescence
quenching, which is consistent with the proposed mechanism (See supporting information
DAC
Figure S22). CD spectra of M10GC
and its complementary DNA hybrid (see supporting
information Figure S23) also clearly confirms the formation of PNA·DNA hybrids as shown by
the change in CD signals at 210 and 260 nm. No CD signal was observed in the UV absorption
region of DAC, suggesting that the DAC label was not wellꢀoriented and thus did not
appreciably interact with the PNA·DNA duplex.
Single mismatched PNA·DNA hybrids generally showed smaller fluorescence increase,
DAC
but some variations in the sequence context were observed (Figure 1). The M10AT
showed
DAC
DAC
a better mismatch discrimination than the T9
and M10GC
probes. Possible explanation is

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7
that certain mismatched PNA·DNA hybrids (Table 2, entries 3, 5, 10, 13–15) are still
sufficiently stable at 20 ºC – the temperature at which the fluorescent experiments were carried
out. Completely unrelated DNA target provided a signal that was indistinguishable from the
single stranded probes in all cases. For terminal labeled PNA probe DAC-M10, no fluorescence
change could be observed for both complementary and mismatched DNA targets (Figure 1b).
Since the single stranded PNA probe exhibited low fluorescence similar to internally labeled
PNA probes, it can be concluded that the quenching by neighboring nucleobases still operates.
Tm measurement confirms the strong binding between the DAC-M10 probe and DNA in a
sequence specific fashion (58 ºC for complementary duplexes and <20 ºC for mismatched
duplexes). The absence of fluorescence increase upon duplex formation was therefore explained
by the interaction between the DAC and the terminal GC base pair of the PNA·DNA duplex
3
1
similar to the pyrene label.
These results clearly demonstrate that the fluorescence of internally DACꢀlabeled
acpcPNA is low in single stranded PNA probes due to quenching by neighboring nucleobases,
especially T and A bases. This interaction diminished – resulting in a large fluorescence increase
–
when the PNA·DNA duplexes are formed, provided that the DAC label does not locate at the
end of the duplex which allows interactions with the terminal base pairs.
a
Table 2. Thermal stability (T ) and fluorescence properties of DACꢀlabeled acpcPNA.
m
Φ (ds)/
Φ (ss)
F
c
F
Entry
PNA
DNA (3’→5’)
Tm (°C)
F/F0
ΦF
b
Notes
ss
1
2
3
4
5
6
7
8
9
none
ꢀ
ꢀ
0.009
0.242
0.139
0.041
0.160
0.007
0.083
0.012
0.014
0.034
ꢀ
AAAAAAAAA
AAAACAAAA
AAAAGAAAA
AAAATAAAA
none
65.2
33.7
23.6
38.2
ꢀ
35.5
18.6
6.0
15.7
ꢀ
26.7
15.3
4.5
17.6
ꢀ
comp
smC
smG
smT
ss
DAC
T9
AGTGATCTAC
AGTGCTCTAC
AGTGGTCTAC
AGTGTTCTAC
53.4
29.0
24.5
35.0
13.2
2.7
1.7
6.5
11.9
1.7
2.0
4.8
comp
smC
smG
smT
DAC
M10AT
1
0

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1
1
1
1
1
1
2
3
4
5
none
ꢀ
ꢀ
0.011
0.117
0.099
0.085
0.104
ꢀ
ss
AGTGCGCTAC
AGTGATCTAC
AGTGGTCTAC
AGTGTTCTAC
59.0
30.8
33.4
39.2
10.2
4.9
7.0
9.1
11.1
9.4
8.0
9.9
comp
smA
smG
smT
DAC
M10GC
a
All measurements were performed in 10 mM sodium phosphate buffer (pH 7.0), [PNA] = 1.0 µM, [DNA] = 1.2
b
µM, λ 315 nm, λ 450 nm. Quantum yields were measured in phosphate buffer using quinine sulfate (0.540) as a
ex
em
c
reference. ss = single stranded PNA, comp = complementary, sm = single mismatched.
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
Complementary
Mismatch T
a)
Mismatch C
Mismatch G
Non-complementary
ss PNA
3
50
400
450
500
550
600
650
Wavelength (nm)
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
b)
Complementary
Mismatch C
Mismatch G
Mismatch T
Non-complementary
ssPNA
3
50
400
450
500
550
600
650
Wavelength (nm)

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Figure 1. Fluorescence spectra of DACꢀlabeled acpcPNA with complementary, single mismatch,
DAC
nonꢀcomplementary and single strand form of (a) M10AT
(b) DAC-M10. Conditions: 10
mM sodium phosphate buffer, pH 7.0 at 25 °C, [PNA] = 1.0 ꢁM and [DNA] = 1.2 ꢁM,
excitation wavelength = 315 nm. Complementary DNA = AGTGATCTAC, Mismatch C DNA =
AGTGCTCTAC, Mismatch G DNA = AGTGGTCTAC and Mismatch T DNA =
AGTGTTCTAC.
Improving single mismatch discrimination of the DAC-labeled acpcPNA probes
Although the T measurement indicated the high specificity as shown by a large decrease
m
in the T values between complementary and single mismatch PNA·DNA duplexes (ꢀT
m
m
ranging from –18.4 to –31.5 °C), but the fluorescence spectra of certain mismatch duplexes still
showed relatively high fluorescence signal. Based on a hypothesis that the high fluorescence was
due to the high stability of some mismatched duplexes at room temperature, the temperature was
increased to improve the single mismatch discrimination by fluorescence spectrophotometry.
Single mismatch hybrids should dissociate more easily than complementary hybrids, and
therefore the temperature increase should be able to enhance the mismatch discrimination ability
of DACꢀlabeled acpcPNA. The experiment was carried out only with the complementary hybrids
and mismatched hybrids that does not show satisfactory discrimination in Table 2. The
fluorescence spectra were measured at different temperature from 20 °C to 90 °C (see supporting
information). Upon heating, the duplex dissociated with concomitant decrease in the
fluorescence intensity. The plots between the fluorescence signal at 450 nm and temperature
appeared in the form of reverse Sꢀcurves (Figure 2a).
Since the maximal difference will be obtained at the temperature whereby the
mismatched duplexes were dissociated as much as possible and the complementary duplexes are
still stable, it is important to vary the temperature to find the optimal value case by case. The
DAC
DAC
specific temperature that provided maximum differentiation in the case of T9 , M10AT
DAC
DAC
and M10GC
was 55, 45 and 50°C, respectively. The mismatched hybrids of T9
and
DAC
DAC
M10GC
were more stable than those of M10AT
and therefore required a higher
temperature to complete the separation. At the aforementioned optimal temperature, the
fluorescence ratio between complementary and single mismatch hybrids improved from 1.0–2.5
folds to 6.0–21.1 folds (Figures 2b, 2c and Table 3). The results confirm that the specificity of

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DACꢀlabeled acpcPNA in distinguishing between complementary and singleꢀmismatched DNA
by DACꢀlabeled PNA probes can be improved by simply increasing the temperature without
requiring more complex operations such as enzymatic digestion.
Next, to support the proposed mechanism of fluorescence change of the DACꢀlabeled
PNA probe, molecular dynamics simulation and energy calculation were performed on both
single stranded and duplex PNA probe.
Table 3. Comparison of F/F of DACꢀlabeled acpcPNA at room temperature and high
0
a
temperature.
F/F0
25°C)
Fcomp/Fmm
(25°C)
F/F0
(high temp)
Fcomp/Fmm
(high temp)
Entry
PNA
DNA (3’ → 5’)
b
b
(
1
2
3
4
5
6
7
8
9
AAAAAAAAA
AAAACAAAA
AAAAGAAAA
AAAATAAAA
TTTTTTTTT
35.4
18.6
6.0
ꢀ
35.7
2.5
ꢀ
ꢀ
1.9
5.9
2.3
32.4
ꢀ
14.2
DAC
T9
ꢀ
15.7
1.1
1.7
ꢀ
21.1
ꢀ
AGTGATCTAC
AGTGCTCTAC
AGTGGTCTAC
AGTGTTCTAC
GGGGGGGGGG
AGTGCGCTAC
AGTGATCTAC
AGTGGTCTAC
AGTGTTCTAC
GGGGGGGGGG
13.2
2.6
12.9
1.3
ꢀ
ꢀ
5.2
8.2
2.3
10.9
ꢀ
10.0
DAC
M10AT
1.6
ꢀ
5.7
1.2
ꢀ
10.6
1
1
1
1
1
1
0
1
2
3
4
5
1.2
ꢀ
10.1
5.3
10.2
ꢀ
ꢀ
ꢀ
1.9
1.4
1.1
11.1
DAC
M10GC
7.1
1.0
1.7
ꢀ
10.3
6.0
ꢀ
9.0
0.9
a
All measurements were performed in 10 mM sodium phosphate buffer (pH 7.0), [PNA] = 1.0 µM, [DNA] = 1.2
b
DAC
DAC
DAC
µM, λ 315 nm, λ 450 nm. high temperature: T9
= 55 °C; M10AT
= 45 °C; M10GC
= 50 °C
ex
em

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1
00
Complementary
Mismatch T
Mismatch G
ssPNA
90
80
70
60
50
40
30
20
10
0
2
0
30
40
50
60
70
80
90
o
Temperature ( C)
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
b) RT
Complementary
Mismatch T
Mismatch G
ssPNA
3
50
400
450
500
550
600
650
Wavelength (nm)

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8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
o
c) 50 C
Complementary
Mismatch T
Mismatch G
ssPNA
3
50
400
450
500
Wavelength (nm)
550
600
650
DAC
DAC
Figure 2. a) Fluorescence melting curves of M10GC
at RT c) Fluorescence spectra of M10GC
b) Fluorescence spectra of M10GC
DAC
at 50 °C. All experiments was performed in10 mM
phosphateꢀbuffered pH 7.0, [PNA] = 1.0 µM, [DNA] = 1.2 µM, λ 315 nm, PMT voltage =
ex
medium
Molecular modeling
MD structures: For both simulated systems (ssPNA and PNA·DNA duplex), the
structure of DAC started by pointing to bulk solvent was rolled and close to PNA strand after 0.5
ns and then fluctuated in such position along 10 ns of simulation time, as displayed in Figure 3.
The root mean squared deviations (RMSDs) of the MD structures with respect to their starting
structures were 4.42±0.67 Å and 4.02±0.40 Å for ssPNA and PNA·DNA systems, respectively,
averaged over the last 2,000 structures, see Figure S25 for the RMSD profiles. Clearly, single
stranded PNA provides larger fluctuation because of a less restriction of its structure. The
average structure revealed that the DAC is closer to the nucleobases in the single stranded PNA
than that in the PNA·DNA duplex (Figure 4), which should lead to a larger interaction between
DAC and PNA for the single stranded PNA system.

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Figure 3. MD snapshots of a single stranded PNA and the corresponding PNA·DNA duplex.
Figure 4. Average MD structures of single stranded PNA and PNA·DNA duplex.

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14
Energy Calculation: In order to investigate the DACꢀPNA interactions between DAC
and nucleobases in single stranded PNA as well as PNA·DNA duplexes in more details, we have
calculated the binding energy between the DAC and three closest PNA nucleobases, whereas
other fragments were omitted. The calculations were performed using Gaussian 09 program with
the density functional theory B3LYP/6ꢀ31G* level based on the average MD structures for
ssPNA and PNA·DNA systems. As shown in Table 4, the binding energy of single stranded
PNA system is significantly higher than that of PNA·DNA duplex. This confirms that there are
stronger interactions between DAC and nucleobases in single stranded PNA than in PNA·DNA
duplex, and further supports the mechanism of fluorescence change proposed above.
a
Table 4. The binding energy (ꢀEbinding) between DAC and three closest PNA nucleobases.
System
ꢀEbinding (kcal/mol)
–11.40
ssPNA
PNA·DNA
–3.86
a
The calculation is expressed as ꢀEbinding = EDACꢀPNA – EDAC – EPNA, where EDACꢀPNA
,
EDAC and EPNA are the total energy of DAC and three PNA nucleotides, DAC moiety
and three PNA nucleotides, respectively.
Experimental section
General procedures
Carbazole (reagent grade for synthesis) was purchased from Merck. All reagent grade
chemicals and solvents were purchased from standard suppliers and were used without further
1
13
purification. H and C NMR spectra were recorded on Bruker Avance 400 NMR spectrometer
1
13
operating at 400 MHz for H and 100 MHz for C. RPꢀHPLC experiments were performed on a
Waters Delta 600 HPLC system. Oligonucleotides were obtained from Pacific Science
(Thailand) or BioDesign (Thailand).
9
N -Propargyl-3,6-bis(tert-butoxycarbonylamino)carbazole (5)
A reaction mixture containing 3,6ꢀbis(tertꢀbutoxycarbonylamino)carbazole (4) (0.80 g,
2.00 mmol) (see supporting information), propargyl bromide (0.28 mL, 3.00 mmol), KI (0.33 g,

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2
.00 mmol) and K CO (2.77 g, 20.0 mmol) in dry DMF (5 mL) was stirred under N
2
3
2
atmosphere at room temperature overnight. The reaction mixture was diluted with water and
extracted with CH Cl . The combined organic extracts were washed with water and brine, dried
2
2
over anhydrous Na SO and evaporated to dryness. The crude product was purified by column
2
4
1
chromatography on silica gel to obtain the pure compound as a yellow solid. H NMR (400
MHz, DMSOꢀd ): δ 1.49 (s, 18H), 3.20 (t, J = 2.2 Hz, 1H), 5.18 (s, 2H), 7.41 (d, J = 8.7 Hz, 2H),
6
1
3
7
.49 (d, J = 8.7 Hz, 2H), 8.21 (s, 2H), 9.30 (s, 2H) C NMR (100 MHz, DMSOꢀd ): δ 153.2,
6
136.2, 131.9, 122.3, 118.2, 109.5, 79.2, 78.7, 74.3, 31.9, 28.2, 27.9; IR: 3397, 3254, 2974, 2112
ꢀ1
(
C≡C), 1720, 1702 (C=O), 1524, 1490, 1048, 1023, 864, 792, 578 cm ; HRMS (ESI+): m/z
+
+
+
calcd for C H N O Na : 458.2056 [M+Na] found: 458.2092, m/z calcd for C H N O K :
2
5
29
3
4
25 29
3
4
+
[
M+K] 474.1795 found: 474.1826.
Synthesis, purification and characterization of DAC-modified acpcPNA
The apcꢀmodified acpcPNA was synthesized via solid phase peptide synthesis using the
Bz
Bz
four Fmocꢀprotected pyrrolidine monomers (FmocꢀA ꢀOPfp, FmocꢀTꢀOPfp, FmocꢀC ꢀOPfp,
Ibu
and FmocꢀG ꢀOPfp) and spacers [Fmocꢀ(1S,2S)ꢀACPCꢀOPfp, Fmocꢀ(3R,4S)ꢀAPC(Tfa)ꢀOPfp]
2
7ꢀ28,35
at 1.5 µmol scale on Tentagel SꢀRAM resin according to the previously published protocol.
Lysine was included at the Cꢀterminus in the form of FmocꢀLys(Boc)ꢀOPfp to improve the water
solubility of the PNA. After completion of the PNA synthesis, the Nꢀterminal Fmoc group was
removed and the free amino group was capped by acetylation. The apcꢀmodified acpcPNA was
split to 0.5 µmol portions for further labeling experiments. The nucleobase protecting groups
(Bz, Ibu) and APC spacer protection (Tfa) were removed by heating with 1:1 aqueous
ammonia/dioxane at 60 °C overnight. The apcꢀmodified acpcPNA (0.5 µmol) was alkylated at
the pyrrolidine ring nitrogen of the APC spacer with 4ꢀazidobutanal (15 µmol, 30 equiv) in the
presence of sodium cyanoborohydride (30 µmol, 60 equiv) and acetic acid (30 µmol, 60 equiv)
3
4
in methanol (100 µL) at room temperature overnight. The azideꢀmodified acpcPNA was further
9
reacted with N ꢀpropargylꢀ3,6ꢀbis(Bocꢀamino)carbazole (5) (7.5 µmol, 15 equiv) while still
attached on the solid support in the presence of tris[(1ꢀbenzylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4yl)methyl]amine
(TBTA, 30 µmol, 60 equiv), tetrakis(acetonitrile) copper(I) hexafluorophosphate (15 µmol, 30
t
equiv) and (+)ꢀsodiumꢀLꢀascorbate (60 µmol, 120 equiv) in 3:1 (v/v) DMSO: BuOH at room
3
4
temperature overnight. After the reaction was completed as monitored by MALDIꢀTOF

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16
analyses (12 h), the diaminocarbazoleꢀmodified acpcPNA was cleaved from the resin with 50%
trifluoroacetic acid (TFA) in dichloromethane and 10% triisopropylsilane (TIS) (500 µL×30
min×3). The combined cleavage solution was dried under a stream of nitrogen gas and the crude
PNA was precipitated by addition of diethyl ether. The crude PNA was purified by reverseꢀphase
HPLC with using mobile phase A:B (A: 0.1% TFA in H O; B: 0.1% TFA in MeOH) 9:1 for 5
2
min followed by a linear gradient to A:B 1:9 over 55 min. The purified PNA was characterized
by MALDIꢀTOF mass spectrometry (Microflex, Bruker Daltonics) by using αꢀcyanoꢀ4ꢀhydroxyꢀ
cinnamic acid (CCA) as a matrix.
O
B
O
B
B
B
O
B
B
O
N3
O
O
O
H
Lys
Lys
N
N
N
N
N
N
HN
HN
HN
HN
HN
HN
O
NaBH3CN, AcOH
MeOH, rt, 12 h
O
O
n
O
O
n
O
N
H
N
Reductive Alkylation
n
n
B = nucleobase
N3
BocHN
NHBoc
Cu(I), TBTA,
sodium ascorbate
N
"Click Chemistry"
B
O
B
O
B
B
O
B
B
5
0% TFA/CH Cl2
O
2
O
O
10% Pr SiH
3
H NLys
2
N
Lys
N
N
N
N
HN
HN
HN
N
HN
HN
HN
O
O
O
n
O
O
n
O
N
N
n
n
NH2
NHBoc
N
N
N
N
N
N
N
N
NH2
NHBoc
Scheme 2. Synthesis of DACꢀmodified acpcPNA
Spectroscopic experiments
All samples were prepared at PNA concentration = 1.0 µM and DNA concentration = 1.2
µM in 10 mM phosphate buffer pH 7.0. UVꢀvisible spectra and thermal stabilities (T ) were
m
measured on a CARY 100 Bio UVꢀVis spectrophotometer at 260 nm from 20–90 °C at a rate of
1
°C/min. The melting temperatures were determined from first derivative plots. Fluorescence

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17
spectra were measured on Cary Eclipes Fluorescence Spectrophotometer at excitation
wavelength of 315 nm. Both excitation and emission slits were set to 5 nm and PMT voltage =
medium.
Fluorescence quantum yield (Φ ) measurement
F
The fluorescence quantum yield (Φ ) of 3,6ꢀdiaminocarbazole (3), single strandedꢀPNA
F
DAC
DAC
DAC
and PNA⋅DNA duplexes [T9 , M10AT ) and M10GC ] were calculated using quinine
3
6
sulfate (Φ = 0.540) as a reference. Absorption and fluorescence spectra were measured on
F
CARY 100 Bio UVꢀVis spectrophotometer (Varian) and CARY Eclipse Fluorescence
spectrophotometer (Varian/Agilent Technologies), respectively. Seven concentrations (1.0, 2.0,
2.5, 5.0, 7.5, 10.0, 12.0 µM) of quinine sulfate samples in phosphate buffer were prepared from a
stock solution (1.0 mM quinine sulfate in 0.1 M H SO ). The absorbance at 315 nm was less than
2
4
0.1 for all concentrations. The magnitudes of integrated fluorescence intensity were plotted
against the absorbance of the solution absorbance. The quantum yield of sample was then
3
1
calculated according to equation (1).
2
2
Φ (sample) = Φ (standard)[m(sample) / m(standard)] [η(sample) / η(standard) ]…...(1)
F
F
where m is the slope from the plot of integrated fluorescence intensity vs. absorbance and η is
the refractive index of the solvent.
Molecular dynamics (MD) simulation: MD simulations were performed on single stranded
acpcPNA with a sequence of GTAGATCACT whereby the DAC was linked to the apc spacer at
the underlined position, and the corresponding acpcPNA·DNA duplex. Because the threeꢀ
dimensional structures of ssPNA and PNA·DNA duplex were not available, the starting structure
of PNA·DNA duplex was built up based on the corresponding sequence of DNA·DNA duplex
(dGTAGATCACT⋅dAGTGATCTAC) which was generated using the NAB module of the
3
7
AMBER 12 package. The first strand of the duplex was replaced with PNA strand in an
3
8
antiparallel direction yielding the PNA⋅DNA duplex. The structure of diaminocarbazole (DAC)
linked to backbone of PNA was first optimized with B3LYP/6ꢀ31G* calculation using
3
9
Gaussian 09 before adding to the PNA strand. The initial structure of single stranded acpcPNA
was obtained in a similar manner. The force field parameters of the PNA and DAC were
3
8
generated using the standard procedure. To setup the simulations, the starting structure of each

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18
4
0
system was immersed in a rectangular water box with TIP3P water molecules extended by 10
+
Å in each direction from the solute and then neutralized by adding Na counterions (for
PNA⋅DNA system). The MD simulations were carried out using AMBER 12 suite of program
with parm10 force field complemented with the prepared parameters of PNA and DAC, SHAKE
4
1
algorithm employed for all hydrogen atoms, a 9 Å cutoff applied to the nonꢀbonding Lennardꢀ
4
2
Jones interactions, the particle mesh Ewald method used to account for longꢀrange interactions,
time step of 1 fs and NPT ensemble (P = 1 atm and T = 300 K). The simulation protocol consists
3
8
of heating, equilibration and production steps, and was described in details elsewhere. The MD
trajectories were generated for 10 ns and the coordinates were stored every 1 ps, totally 10,000
structures.
Conclusion
In conclusion, we successfully synthesized novel internally and terminally 3,6ꢀdiaminoꢀ
carbazoleꢀlabeled acpcPNA and evaluated their binding and fluorescence properties. Thermal
denaturation experiments revealed the high stability (T 53.4–65.2 °C) and high specificity
m
(
ꢀT > 20 °C) of PNA⋅DNA hybrid similar to unmodified acpcPNA. Single stranded DACꢀ
m
labeled acpcPNA showed low fluorescence signals due to quenching of the DAC label with
neighboring nucleobases. After hybridization with DNA, the internally DACꢀlabeled acpcPNA
showed moderate to large fluorescence increase (10.1–35.4 folds) compared to the single
stranded PNA. The specificity was further improved to allow discrimination between
complementary and single mismatched DNA targets by increasing the temperature. Terminally
DACꢀlabeled acpcPNA showed no significant fluorescence change after hybridization with
DNA. A model was proposed to explain the mechanism of fluorescence change, which was
further supported by MD simulations and energy calculation. These results demonstrate the
potential of DAC as a fluorescence label to be used in combination with oligonucleotide probes
for detection of specific DNA sequences.
Acknowledgement
We acknowledge Naresuan University (NU grant No.P2558C371 to CS) and The
Thailand Research Fund (DPG5780002, to TV and KS) for financial supports and National eꢀ
Science Infrastructure Consortium (URL: http://www.eꢀscience.in.th) for computing resources.

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Partial technical supports by Ms. Boonsong Ditmangklo (Chulalongkorn University) are also
gratefully acknowledged.
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Diaminocarbazole-labeled acpcPNA as a self-reporting probe
for determination of DNA sequences