Clickable styryl dyes for fluorescence labeling of pyrrolidinyl PNA probes for the detection of base mutations in DNA

Article abstract of DOI:10.1039/c9ob01492f

Fluorescent hybridization probes are important tools for rapid, specific and sensitive analysis of genetic mutations. In this work, we synthesized novel alkyne-modified styryl dyes for conjugation with pyrrolidinyl peptide nucleic acid (acpcPNA) by click chemistry for the development of hybridization responsive fluorescent PNA probes. The free styryl dyes generally exhibited weak fluorescence in aqueous media, and the fluorescence was significantly enhanced (up to 125-fold) upon binding with DNA duplexes. Selected styryl dyes that showed good responses with DNA were conjugated with PNA via sequential reductive alkylation-click chemistry. Although these probes showed little fluorescence change when hybridized to complementary DNA, significant fluorescence enhancements were observed in the presence of structural defects including mismatched, abasic and base-inserted DNA targets. The largest increase in fluorescence quantum yield (up to 14.5-fold) was achieved with DNA carrying base insertion. Although a number of probes were designed to give fluorescence response to complementary DNA targets, probes that are responsive to mutations such as single nucleotide polymorphism (SNP), base insertion/deletion and abasic site are less common. Therefore, styryl-dye-labeled acpcPNA is a unique probe that is responsive to structural defects in the duplexes that may be further applied for diagnostic purposes.

Full text of DOI:10.1039/c9ob01492f

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Clickable Styryl Dyes for Fluorescence Labeling of Pyrrolidinyl PNA ProbesDfOoIr: 10th.10e39/C9OB01492F
Detection of Base Mutations in DNA
Boonsong Ditmangklo,a Jaru Taechalertpaisarn,a,b Khatcharin Siriwong,c Tirayut Vilaivana,*
aOrganic Synthesis Research Unit, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand
bNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science
and Technology Development Agency, Pathumthani 12120, Thailand
cMaterials Chemistry Research Center, Department of Chemistry and Center of Excellence
for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002,
Thailand
*e-mail: vtirayut@chula.ac.th
Electronic Supplementary Information (ESI) Available. See DOI: xxx

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Abstract
Fluorescence hybridization probes are important tools for rapid, specific and sensitive
analysis of genetic mutations. In this work, we synthesized novel alkyne-modified styryl dyes
for conjugation with pyrrolidinyl peptide nucleic acid (acpcPNA) by click chemistry for the
development of hybridization responsive fluorescence PNA probes. The free styryl dyes
generally exhibited weak fluorescence in aqueous media, and the fluorescence was
significantly enhanced (up to 125-fold) upon binding with DNA duplexes. Selected styryl
dyes that showed good responses with DNA were conjugated with the PNA via sequential
reductive alkylation-Click chemistry. While these probes showed little fluorescence change
when hybridized to complementary DNA, significant fluorescence enhancements were
observed in the presence of structural defects including mismatched, abasic and base-inserted
DNA targets. The largest fluorescence increase (up to 14.5-fold) was achieved with DNA
carrying base insertion. While a number of probes were designed to give fluorescence
response to complementary DNA targets, probes that are responsive to mutations such as
single nucleotide polymorphism (SNP), insertion and deletion/or abasic site are less common.
Therefore, the styryl-dye-labeled acpcPNA is a unique probe that is responsive to structural
defects in the duplexes that may be further applied for diagnostic purposes.

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Introduction
Detection of genetic variations or mutations including single nucleotide
polymorphism (SNP), base deletion, abasic site, insertion and tandem repeats has important
applications in many areas.1 Although the continuously developed sequencing-based methods
can provide very detailed information under a reasonable timescale and cost in the past few
years, it still requires a large investment in terms of instrument and skilled operators, and
often gives too much information that requires complicated analyses. On the other hand, the
use of fluorescence hybridization oligonucleotide probes that are capable of detection of
specific nucleic acid targets in homogeneous solution is still of importance due to its
simplicity and efficiency in terms of sensitivity and specificity.2 In addition, it can provide
information about the concentration and location of the nucleic acid target in real-time and
non-invasive fashions that cannot be equaled by other methods. The first generation
hybridization probes consist of an oligonucleotide strand carried two dyes that interact
differently in response to the hybridization-induced conformational change, leading to a
fluorescence change in response to the presence or absence of the target.3 Later, singly-
labeled probes have been developed whereby the dye must be able to change its fluorescence
upon the hybrid formation.4 This can be achieved by employing interaction of the dye with
double-stranded DNA (dsDNA) via intercalation or groove binding, or by sensing of the local
environment change such as polarity. Examples of such environment-sensitive dyes which
have been successfully used to develop hybridization responsive oligonucleotide probes are
pyrene5 and thiazole orange (TO)6,7,8 or related cyanine dyes.9
Styryl dye is a class of fluorescent dye that consists of an electron-deficient N-
alkylated heterocyclic ring and an electron-rich aromatic ring connected to each other via a
double bond to create an extended -conjugated system.10,11 The excellent fluorescence
properties of styryl dyes that are dependent on the environment make them useful as optical
sensitizer,12 as sensors for various ions or small molecule targets such as cyanide,13,14 silver,15
hydrogen peroxide,16,17 and thiols,18 for biomolecular labeling,19,20 and bioimaging
applications.21,22,23 While certain styryl dyes are known to bind to DNA with concomitant
fluorescence enhancement,24,25 their use has been limited to DNA staining and very few of
these dyes have been further developed into fluorescence hybridization probes. In this
context, Wagenknecht group had prepared a clickable quinoline-indole cyanine (CyIQ) dye
and incorporated it into DNA probes, but the singly-labeled probe showed only small
response to DNA targets.26 Therefore, an additional dye such as thiazole red (TR) was

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required and the fluorescence resonance energy transfer (FRET) principle was employed to
create a wavelength-shifting probe that can change its color from yellow (CyIQ) to red (TR)
upon hybridization with complementary DNA due to the change in FRET efficiencies
between the two dyes.27 A similar wavelength-shifting probe has been applied for the
detection of adenosine using labeled adenosine aptamer.28
However, oligonucleotide probes are readily degraded by nucleases, and their target
specificities are not always ideal.29,30 Several synthetic oligonucleotide analogs have been
developed in order to address these problems. Peptide nucleic acid (PNA) is a remarkable
example of oligonucleotide analog in which the deoxyribose-phosphate backbone of DNA is
completely replaced by a peptide-like backbone. PNA not only offers excellent chemical and
biological stabilities, but it also recognizes complementary nucleic acid targets with even
greater affinity and specificity than natural nucleic acids.31,32 PNA is therefore a promising
candidate for the development of fluorescent probes for homogeneous detection of nucleic
acid both in vitro and for real-time detection in living cells.33 Pyrrolidinyl PNA with /-
dipeptide backbone deriving from proline/2-aminocyclopentane carboxylic acid (acpcPNA) is
a new generation of PNA that binds to DNA with higher affinity and specificity than aegPNA
because of the more rigid conformation.34 Several singly-labeled fluorogenic acpcPNA
probes have been developed by using fluorescence nucleobases35,36 or by backbone
modification with pyrene,37 carbazole,38 thiazole orange39 and nile red.40 Only one example
of styryl-dye-labeled acpcPNA probe was reported, and it was used in combination with
another label on the DNA strand for monitoring of strand displacement and invasion into
DNA duplexes.41
The main objective of this study is to investigate the potential of using styryl-dye-
labeled acpcPNA as a new fluorescence probe for DNA sequence analysis. Alkyne-
functionalized styryl dyes were synthesized and conjugated to the backbone of acpcPNA via
the sequential reductive alkylation-Click chemistry previously developed in our group.39
Next, the optical properties of the styryl-dye-labeled acpcPNA probes were investigated in
the presence of various DNA targets. In contrast to our expectation, the styryl-dye-labeled
acpcPNA probes were more responsive to the presence of structural defects, most notably
base insertion, than to perfectly complementary DNA targets. This suggests potential
applications of the developed styryl-dye-labeled acpcPNA probes for the detection of base
insertion in the DNA targets.

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Results and Discussion
Synthesis of Clickable Styryl Dyes
A library of styryl dyes bearing a propargyl group for further conjugation to the azide-
modified acpcPNA backbone via click chemistry39 was first synthesized. Synthesis of the
styryl dyes required two building blocks: an N-alkylated heterocyclic ring system carrying an
acidic methyl group (2-methylbenzothiazolium and 4-methylquinolinium salts), and an
aromatic aldehyde carrying different aromatic ring systems (benzene with various electron-
donating or -withdrawing substituents, thiophene, carbazole, coumarin, and pyrene). In the
first step, 2-methylbenzothiazole and 4-methylquinoline were reacted with propargyl bromide
or methyl iodide (when the propargyl group was to be attached elsewhere in the dye
molecule) to afford the N-methyl/N-propargylbenzothiazolium (a, b, c) and N-propargyl/N-
methylquinolinium (d, e) salts. Next, these salts were condensed with an appropriate aromatic
aldehyde via an aldol-type reaction to afford the alkyne-modified styryl dyes in 15–97%
yield. The reactions were carried out either in acetic anhydride24 or in refluxing ethanol.
-
Counterion exchange to PF6 was performed when the bromide or iodide salts of the products
did not spontaneously precipitate. The primary amino-substituted dye 5a was prepared and
stored in the N-Boc-protected form (5), but the optical properties were measured after
removal of the Boc group by treatment with trifluoroacetic acid (TFA). With the exception of
a known compound 3,19 all other alkyne-modified styryl dyes have not been previously
reported in the literature. The synthetic scheme of these novel clickable benzothiazolium (1–
17) and quinolinium (18–19) styryl dyes is shown in Scheme 1.

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O
NMe
NEt2
S
N
Ar
ArCHO
S
N
Ar =
O
methods A or B
iv
I
I
a (92%)
2, 4, 6
2: A (32%)
4: A (70%)
6: A (69%)
S
N
v
NMe2
NHBoc
TFA
NH2
NPh2
7: B (58%)
ArCHO
3: A (81%)
9: A (67%)
5: B (82%)
5a: (quant.)
Ar
S
i-iii
S
N
MeO
methods A or B
N
NEt2
Ar =
OBz
OMe
NO2
Br
Br
O
S
N
O
b (58%)
O
11: A (84%)
12: A (70%)
3, 5, 7, 9, 11-17
13: A (60%)
1 (15%, 3 steps)
N
S
vi
14: A (38%)
15: B (49%)
16: B (73%)
17: B (97%)
ArCHO
S
Ar
S
Ar =
N
N
OMe
methods A or B
N
PF6
PF6
c
(80%)
NMe2
8, 10
8: A (78%)
10: A (60%)
NEt2
O
ArCHO
ArCHO
methods A or B
vi
iv
N
N
methods A or B
N
I
N
Br
N
Br
IH
e
(91%)
d
(98%)
18
: B (74%)
19
: B (81%)
Scheme 1. Synthesis of alkyne-modified styryl dyes 1–19.
Reagent and conditions: i) 4-hydroxybenzaldehyde, Ac2O, 90 C, 24 h, ii) 10% NaOH 80 C,
5 h, iii) propargyl bromide, K2CO3, DMF, 50 C, 8 h, iv) MeI, MeCN, reflux, 8 h, v)
propargyl bromide, 95 C, 2 days, vi) NH4PF6, MeOH; Method A: Ac2O, 90 C, 8–10 h.
Method B: EtOH, reflux, 8 h.
Optical Properties of Free Styryl Dyes
Dependency of optical properties of structurally-related styryl dyes to both polarity
and viscosity of the medium has been well-recognized.42,43,44,45 The absorption properties of 3
as a representative styryl dye in solvents with different polarities (water, MeOH, MeCN,
DMSO, EtOAc and THF) clearly show its solvatochromic behavior (Figure 1A, Table S1).
The plot of the absorption energy versus the ET(30) solvent parameter shows a good
correlation (Figure S33). The observed negative solvatochromism (blue-shifted absorption
maxima in more polar solvents) indicates better stabilization of the ground state dye relative
to the excited state, which is typical for n* donor-acceptor dyes.46 On the other hand, no
clear correlation between the emission energies and solvent polarity could be deduced. This
may be explained by the presence of multiple twisted excited states that can contribute to the
fluorescence emission.47,48 It should also be noted that the ET30 parameter reflects the

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combination of solvent properties based on a single reference dye which may not necessarily
be the same as the styryl dyes being investigated. Hence a multi-parameters correlation such
as the Kamlet-Taft solvatochromic scale may be necessary for more precise analyses,46 but
this is beyond the scope of the present work.
400
0.10
A
B
0.05
0.00
0.10
0.05
0.00
0.10
0.05
0.00
0.10
0.05
0.00
0.10
0.05
0.00
0.10
0.05
0.00
THF
EtOAc
DMSO
MeCN
MeOH
water
THF
350
300
250
200
150
100
50
EtOAc
DMSO
MeCN
MeOH
water
0
350 400 450 500 550 600 650 700 750
550
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
Figure 1. (A) Absorption and (B) fluorescence spectra of styryl dye 3 in various solvents.
Conditions: [3] = 1 M, ex = 525 nm. The spectra in water was recorded in 10 mM sodium
phosphate buffer pH 7.0.
The fluorescence quantum yield of 3 as the free dye was generally small (F = 0.001–
0.02) which could be ascribed to the rapid internal rotation leading to the enhanced
radiationless decay of the excited states. This was supported by the strong fluorescence
increase of the dye in high viscosity media such as glycerol (Figure S34). Significant
fluorescence increase together with red-shifting of the absorption maxima was also observed
in non-viscous low-polarity solvents such as ethyl acetate and THF (Figure 1B, Table S1).
The fluorescence increase in these cases may also involve a local change in micropolarity that
can affect the rate of non-radiative decay of the excited states of the dye molecules.49 While
quenching due to self-aggregation of the dyes can be proposed as an alternative fluorescence
change mechanism,26,50 the absorption maxima and molar absorptivity of 3 in aqueous buffer
solution were essentially constant over the concentration range of 0.1 to 5 µM (Figure S35),
indicating no aggregation of the dye at the experimental concentrations. Such sensitivity of
absorption and fluorescence properties of styryl dyes to the environment suggest their

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potential utility as a label for the development of hybridization responsive fluorescence
probe.
The fluorescence quantum yields and fluorescence enhancements of all clickable
styryl dyes in the presence of a random sequence 30 base pair DNA duplex (dsDNA) were
determined in aqueous phosphate buffer. The ratio between the dye and DNA was first
optimized by titration of 3 into a fixed concentration of a 30bp dsDNA (1 M) (Figure S36).
The fluorescence was rapidly increased until the concentration of 3 reached 2 M (equivalent
to 15 bp/dye molecule) which indicated saturation of all available binding sites. Therefore, all
subsequent experiments were performed at a concentration dye = 2 M and dsDNA = 1 M.
Based on the non-linear fitting of the titration curve of 3 with dsDNA, a binding constant of
6×104 M-1 was estimated, which was in good agreement with the values of related dyes
reported in the literature.51,52
Table 1. Optical properties of alkyne-modified styryl dyes: absorption maxima max(abs),
extinction coefficients (), emission maxima max(em), fluorescence quantum yield () and
fluorescence enhancement ratio (F/F0)a
Brightnessd
c


max (nm)
F
F(DNA)
 F(dye)
n.d.
b
Dye
F/F0
(104 M-1·cm-1)
abs
297
399
533
486
477
519
516
578
387e
413
439
373e
551
479
281e
292
468
541
537
em
475
498
605
590
579
584
671
628
447
514
529
450
639
632
552
376
595
702
668
dye
n.d.
n.d.
+DNA
n.d.
n.d.
0.032
0.142
0.036
0.134
0.002
0.083
n.d.
0.003
n.d.
n.d.
0.162
0.011
n.d.
dye
n.d.
n.d.
81
+DNA
n.d.
1
2
3
4
5a
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1.3
3.5
6.2
4.1
3.5
6.9
3.1
2.3
2.7e
3.0
0.8
2.2e
7.6
2.8
1.2e
2.8
1.7
4.4
3.3
1.0
0.5
n.d.
n.d.
25.6
23.4
14.3
41.1
17.9
20.8
1.2
0.001
0.009
0.003
0.003
<0.001
0.005
n.d.
1.9×103
32.0
15.8
12.0
44.7
>20
16.6
n.d.
1.0
n.d.
n.d.
3.8
1.1
3.6×102 4.6×103
1.1×102 1.2×103
2.1×102 9.2×103
~3
60
1.0×102 1.8×103
n.d.
81
n.d.
77
1.0
0.8
1.3
3.4
1.0
0.8
1.1
4.3
0.003
n.d.
n.d.
0.043
0.010
n.d.
n.d.
n.d.
n.d.
n.d.
3.3×103 1.2×104
2.8×102 2.9×102
n.d.
n.d.
n.d.
26
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>24
n.d.
n.d.
n.d.
n.d.
0.024
0.102
37.8
124.8
<0.001
0.001
1.0×103
2.8×103
40
102
aconditions; [Dye] = 2 M, [DNA] = 1 M; [DNA(in bp)]:[Dye] = 15:1; all measurements were performed in 10
mM sodium phosphate buffer pH 7.0 unless otherwise stated.
dsDNA =
5-CGCGGCGTACAGTGATCTACCATGCCCTGG-3 +
3-GCGCCGCATGTCACTAGATGGTACGGGACC-5
bfluorescence intensity with dsDNA (F) and without dsDNA (F0)
csee ESI for details of the fluorescence quantum yield (ΦF) calculation
dbrightness = ×F
ein MeOH
n.d. = not determined due to the very weak fluorescence intensity or small fluorescence response with DNA

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160
140
120
100
80
160
Dye 3
3 + dsDNA
Dye 5a
5a + dsDNA
A
B
140
120
100
80
60
40
20
0
60
40
20
0
550
600
650
700
750
800
500
550
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
1000
800
600
400
200
0
1000
C
D
Dye 6
6 + dsDNA
Dye 19
19 + dsDNA
800
600
400
200
0
550
600
650
700
750
800
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
Figure 2. Fluorescence spectra of styryl dyes in the absence and presence of DNA: (A) 3 (B)
5a (C) 6 and (D) 19; Conditions [dsDNA] = 1 M, [Dye] = 2 M in 10 mM sodium
phosphate buffer pH 7.0, ex = 525 (3,6), 487 (5a), ex 568 (19) nm
Next, the optical properties including absorption maxima [max(abs)], molar extinction
coefficients (), fluorescence emission [max(em)] and fluorescence quantum yield (F) of all
alkyne-modified styryl dyes in aqueous phosphate buffer were investigated in the absence
and presence of dsDNA (Table 1). While most of the styryl dyes are weakly emissive in the
free state as shown by the very low fluorescence quantum yields, the fluorescence emission
intensities of the dyes 38 and 1819 were significantly increased (14 to 125-fold) in the
presence of dsDNA (Table 1, Figure 2). In contrast, no or very small fluorescence
enhancement was observed for the dyes 12 and 917 (Table 1). The increase in
fluorescence emissions/quantum yields upon binding of the styryl dyes to dsDNA can be

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explained by both the restriction of internal bond rotation and the change in local
micropolarity around the dye molecule as a result of intercalative or groove binding. To
further confirm that the fluorescence increase of styryl dye is selective to DNA, the dye 3 was
tested with two other anionic polymers including heparin and poly(styrenesulfonate) at a
similar dye/negative charge ratio. No fluorescence change was observed with heparin, and a
smaller response was observed in the case of poly(styrenesulfonate) (Figure S37).
Considering the presence of both negative charge and hydrophobic/aromatic moiety in
poly(styrenesulfonate), such non-specific interaction is not entirely unexpected and has been
observed in other DNA-binding dyes.53 Nevertheless, the results clearly indicate that the dye
responds to DNA in a fairly selective fashion.
Conjugation of Styryl Dyes to acpcPNA Probes by Click Chemistry
O
O
O
O
Base
N
Base
N
H
H
O
O
N
N
Ar
S
N
m
m
O
O
Base
Base
N
N
H
H
O
O
N
N
N
N
O
O
Base
N
Base
N
Cu(I)/TBTA
"Click"
H
H
N3
N
N
O
O
N
N
N
n
n
O
O
Base
Base
N
N
N
H
H
S
N
N
Ac
Ac
Ar
Scheme 2. Conjugation of styryl dyes to the backbone of acpcPNA by click chemistry
Since styryl dyes bind to any DNA sequences without specificity, combination with a
probe such as PNA was necessary to target specific DNA sequences. The dyes that showed a
good response with dsDNA including 38 and 1819 were therefore selected for PNA
labeling to create a hybridization responsive probe, with the assumption that the dyes that
show good responses to the DNA duplex would bind to PNA·DNA duplexes in the same
way. The selected dyes were post-synthetically conjugated onto the backbone of azide-
modified acpcPNA by click chemistry in the presence of tetrakis(acetonitrile) copper(I)
hexafluorophosphate/tris((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amine (TBTA) (Scheme
2).39 The progress of the click reaction was conveniently monitored by MALDI-TOF MS

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(Figure S38). Two model acpcPNA sequences (T9 and M10) bearing various styryl dye
labels were synthesized and characterized by MALDI-TOF MS (Table 2). It should be noted
that acidolytic cleavage of the PNA labeled with the dye 5 from the solid support resulted in
the simultaneous deprotection of the Boc group to give the dye 5a carrying a free NH2 group.
The yields of isolated products (ranging from 4 to 19% at 0.5 mole scale) were somewhat
lower than the typical range (10–30%) for the synthesis of unlabeled acpcPNA. This could be
attributed to the inefficiency of the click reaction because some styryl dyes were partially
decomposed under the reaction conditions, as well as to some loss during reverse-phase
HPLC purification at such small scale syntheses.
Table 2. Yield of isolated products and mass (MALDI-TOF) data of styryl-dye-labeled
acpcPNAs obtained via sequential reductive alkylation-click reaction
PNA
T9-3
tR (min)a Yieldb
m/z (calcd)
3598.0
3569.9
3656.1
3650.1
3592.0
3650.0
3977.4
3977.4
3949.3
4035.5
3971.4
4029.4
m/z (found)
3599.8
3570.9
3656.5
3648.3
3594.6
3651.6
3978.4
3980.7
3952.9
4034.7
3971.4
4029.1
Sequence (NC)
Ac-TTTT(3)TTTTT-LysNH2
Ac-TTTT(5a)TTTTT-LysNH2
Ac-TTTT(6)TTTTT-LysNH2
Ac-TTTT(8)TTTTT-LysNH2
Ac-TTTT(18)TTTTT-LysNH2
Ac-TTTT(19)TTTTT-LysNH2
Ac-GTAGA(3)TCACT-LysNH2
Ac-GTAGA(4)TCACT-LysNH2
Ac-GTAGA(5a)TCACT-LysNH2
Ac-GTAGA(6)TCACT-LysNH2
Ac-GTAGA(18)TCACT-LysNH2
Ac-GTAGA(19)TCACT-LysNH2
33.3
32.3
34.4
32.4
33.7
34.1
31.2
30.4
31.3
31.0
33.9
33.4
19
14
11
10
15
11
9
T9-5a
T9-6
T9-8
T9-18
T9-19
M10-3
M10-4
M10-5a
M10-6
M10-18
M10-19
12
12
16
4
14
aHPLC conditions: C18 column, 4.6150 mm, 3 , gradient 0.1% TFA in H2O:MeOH 90:10 for 5 min then
linear gradient to 10:90 over 30 min, flow rate 0.5 mL/min, 260 nm.
bpurified by HPLC (>90% purity), spectrophotometrically determined based on the scale of the synthesis
Optical Properties and DNA Binding Studies of Styryl-dyes-labeled acpcPNA Probes
All synthesized PNA probes and their respective hybrids with complementary, single
mismatched, base-inserted and abasic DNA targets were evaluated in terms of thermal
stabilities and optical properties. The thermal stability data showed that the styryl-dye-labeled
acpcPNA retained the ability to strongly hybridize with complementary DNA (Table 3). The
specificity of the recognition of DNA target by the PNA probe was confirmed by the large
decrease in thermal stability in the presence of non-complementary DNA targets carrying
single base mismatch (–23 to –30 °C for T9 and –17 to –25 °C for M10), base insertion (–13

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to –26 °C for T9 and –6 to –19 °C for M10) and abasic site (–18 to –25 °C for T9 and –16 to
–26 °C for M10). Interestingly, DNA targets with base insertion appeared to give a
consistently smaller Tm decrease when compared to other types of mismatches. The effects
can be most clearly observed in the probes M10-5a, M10-18 and M10-19 (Table 3).
Table 3. Thermal stability and fluorescence data of styryl-dye-labeled PNA probes with
various DNA targetsa,b
Tm (Tm) (ºC)
Single Base-
mentary mismatched inserted
F(ds)/F(ss)c
Single Base-
mentary mismatched inserted
c
ex
em
F
(ss)
PNA
Comple-
Comple-
(nm) (nm)
Abasic
Abasic
T9-3
T9-5a
T9-6
525
487
525
565
560
568
525
510
487
525
560
568
595
571
586
622
682
668
602
588
577
592
692
663
0.018
0.027
0.040
0.001
0.006
0.023
0.019
0.065
0.026
0.049
0.007
0.025
73
73
73
68
73
71
52
57
59
59
59
60
45 (–28)
46 (–27)
43 (–30)
45 (–23)
45 (–28)
41 (–30)
35 (–17)
41 (–17)
38 (–21)
35 (–24)
34 (–25)
35 (–25)
50 (–23) 49 (–24)
53 (–20) 50 (–23)
56 (–17) 48 (–25)
55 (–13) 50 (–18)
57 (–16) 48 (–25)
45 (–26) 50 (–21)
33 (–19) 36 (–16)
41 (–17) 38 (–19)
0.7
0.9
0.8
0.7
1.4
0.7
3.2
1.5
1.8
1.5
1.6
1.6
2.1
6.2
1.1
1.4
2.9
1.0
4.1
2.4
4.2
2.9
2.3
2.9
5.6
8.7
1.4
1.2
4.8
1.1
11.9
3.1
14.5
4.5
3.6
2.0
6.8
8.6
1.1
1.1
3.8
0.8
3.6
3.3
3.6
3.3
2.2
2.4
T9-8
T9-18
T9-19
M10-3
M10-4
M10-5a
M10-6
M10-18
M10-19
54 (–6)
40 (–19)
42 (–17) 40 (–19)
45 (–14) 33 (–26)
49 (–11) 37 (–23)
aDNA sequences for T9 series: complementary = dAAAAAAAAA, mismatched = dAAAATAAAA, base-
inserted = dAAAACAAAAA, abasic = dAAAAXAAAA; for M10 series: complementary = dAGTGATCTAC,
mismatched = dAGTGCTCTAC, base-inserted = dAGTGACTCTAC, abasic = dAGTGXTCTAC
bConditions: phosphate buffer pH 7.0, [PNA] = 1.0 µM, [DNA] = 1.2 µM
cSee ESI for details of the fluorescence quantum yield (ΦF) calculation.
The single-stranded PNA probes were generally more fluorescence by an order of
magnitude when compared to the free dyes (Table 3). For example, the fluorescence
quantum yields (F) of the dye 3 in the free form and when incorporated into the PNA probe
T9-3 were 0.001 and 0.018, respectively. This can be explained by the interaction between
the dye and the PNA molecule, which resulted in a change of local microenvironment and
restriction of the internal rotation of the dye. This proposal was supported by the significant
red-shifting of the absorption maxima of the dyes in the single-stranded PNA probes relative
to the free dyes under the same condition (Tables 1 and S2). Such dye-PNA interactions had
been previously observed54 and were indeed the basis of the working principle of many
singly-labeled PNA beacons.33
The fluorescence quantum yields of the single-stranded PNA probes changed only
little in the presence of complementary DNA target for all dyes tested (3, 4, 5, 6, 8, 18 and
19) regardless of the PNA sequence (0.7–1.4 folds for T9 and 1.5–3.2 folds for M10). This is
in sharp contrast to our original expectation and could reflect the different interactions
between the dye and PNA·DNA vs DNA·DNA duplexes (vide infra). On the other hand,
significant increases in the fluorescence were unexpectedly observed in certain PNA probes

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including T9-3, T9-5a, M10-3 and M10-5a in the presence of DNA targets carrying
mismatch, base insertion and abasic site (Table 3). For example, the probe T9-3 gave 2.1, 5.6
and 6.8-fold increase in quantum yields for the mismatched, base-inserted and abasic DNA
targets, respectively, while the quantum yield was decreased by 0.7 fold with the
complementary DNA. For both PNA probe sequences, the dyes 3 and 5a which bear
structural resemblance showed the most significant fluorescence change in the presence of
these "defective" DNA targets. In the case of mix-sequence PNA probes M10-3 and M10-5a,
the best responses were observed for the base-inserted DNA, which showed the increase of
fluorescence quantum yields of 11.7–14.5 folds (Figure 3, Table S2). These changes were
much higher than the mismatched and abasic targets, and the fluorescence change can be
visible by naked eyes under UV light (Figure 3C). The structurally related dyes 4 and 6
whereby the position of the alkyne attachment was on the benzene ring rather than the
benzothiazole ring showed strong fluorescence enhancement as a free dye upon binding to
DNA, but not when attached to the PNA probes. This suggests the crucial role of the position
of the dye attachment. Likewise, despite their good responsiveness to dsDNA as the free
dyes, the rather sterically hindered dyes 8, 18 and 19 when incorporated into the PNA probes
showed little fluorescence change towards all types of DNA targets.
Since these styryl dyes exhibit solvatochromic properties, additional insightful
information about the local environment of the dyes in different types of PNA·DNA duplexes
could be obtained from the absorption spectra. Pronounced red-shifting in the absorption
maxima of the PNA probes (13–22 nm for M10-3 and 28–29 nm for M10-5a) was observed
upon binding with DNA targets carrying a single base insertion or abasic site located near the
position of the dye (Figures 3A, B). This indicates that the dye in these defective PNA·DNA
duplexes experienced a less polar environment than the complementary duplex, whereby such
large red-shifting was not observed. Importantly, when the base insertion or the abasic site
was located away from the position of the dye, only small red-shifting of the absorption
maxima (Figure 3A) along with very small fluorescence change were observed (Figure 3A,
Table S2). The experiments with DNA carrying different base insertion 5-
AGTGANTCTAC-3 (N = C, A, T, G) confirmed that the large fluorescence increase and the
red-shifting of the absorption spectra were general for all base-inserted DNA targets (Table
S2). Negligible fluorescence change was observed in the case of non-complementary DNA
target (1.2 fold), thus further confirming the specificity of the fluorescence enhancement.

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300
300
250
200
150
100
50
0.08
0.06
0.04
0.02
0
A
B
0.08
0.06
0.04
0.02
0
free probe
complementary
mismatched
base inserted
abasic
indirect insert
indirect abasic
free probe
complementary
mismatched
base inserted
abasic
250
200
150
450
500
550
600
650
350 400 450 500 550 600
Wavelength (nm)
Wavelength (nm)
100
50
0
0
500
550
600
650
700
750
800
550
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
15
C
M10-5a
mismatched
abasic
complementary
base inserted
10
5
0
complementary mismatched
base inserted
abasic non-complementary
DNA
Figure 3. Fluorescence and absorption spectra (insets) of (A) M10-3 (B) M10-5a.
Conditions: 10 mM sodium phosphate buffer pH 7.0; [PNA] = 1 M, [DNA] = 1.2 M. (C)
Fluorescence enhancement ratio of M10-5a in the presence of various DNA. Conditions: 10
mM sodium phosphate buffer pH 7.0; [PNA] = 0.1 M, [DNA] = 0.12 M. Error bars
represent standard deviations of three experiments. Inset shows photographs under UV light
(315 nm) at [PNA] = 5 µM, [DNA] = 6 µM; See Table 3 for the DNA sequences.
The results presented so far revealed that the styryl-dye-labeled PNA probes gave
distinctive optical responses to the base insertion, and smaller extents for the abasic site and
single base mismatch, but not to complementary DNA targets as most conventional
hybridization probes do. The conformation of different types of PNA·DNA duplexes was
further investigated by circular dichroism (CD) experiments (Figure 4). The CD spectra
revealed that in all cases the hybrid was formed as shown by a marked difference between the

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spectra of the duplex and sums of the spectra of the free probe and DNA target. Interestingly,
the CD spectrum of the duplex with the base-inserted DNA target showed a positive band
between 250 and 300 nm as a distinctive feature that was not observed in other types of
PNA·DNA duplexes suggesting subtle structural difference among these duplexes. No
induced CD signal in the region of dye absorption could be observed in all cases. Strong
induced CD has been observed in certain styryl dyes-DNA complexes and was attributed to
the formation of chiral aggregates,65 but there are also styryl dyes-DNA complexes that show
very small65 or no induced CD at all.64 Although the reason for is unclear at present, the
absence of induced CD signal in this case is not unique and should warrant further studies.
15
10
5
15
10
5
free probe
PNA M10-5a
A
B
complementary
mismatched
base inserted
abasic
DNA (base inserted)
PNA+DNA (expt)
PNA+DNA (sum)
0
0
-5
-5
-10
200
-10
200
250
300
350
400
250
300
350
400
Wavelength (nm)
Wavelength (nm)
Figure 4. CD spectra of (A) M10-5a + base-inserted DNA in comparison with the sum of the
two components (B) M10-5a in the absence and presence of complementary, mismatched,
base-inserted and abasic DNA targets. Conditions: 10 mM sodium phosphate buffer pH 7.0 at
20 C; [PNA] = 2.5 M, [DNA] = 3.0 M; See Table 3 for the DNA sequences.
Applications of Styryl-dyes-labeled acpcPNA Probes for the Detection of Base Insertion
To demonstrate potential applications of the styryl-dye-labeled PNA probe for the
detection of base insertion mutation, the sequence of Liddle's syndrome whereby a single
cytosine base insertion in the -subunit of human epithelial sodium channel gene was chosen
as a test case.55 We investigated the optical properties of acpcPNA probe M12-5a (Ac-
CCCAGT(5a)GTTGGG-LysNH2) in the presence of wild type DNA (5-
CCCAACACTGGG-3, Tm = >80 C) which are fully complementary to the probe, and
Liddle’s syndrome sequence which contained a single G base insertion (5-

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CCCAACGACTGGG-3, Tm = 61.7 C). The fluorescence data showed that the M12-5a
probe gave a larger increase in fluorescence quantum yield by 6-fold (F = 0.157) relative to
the single-stranded probe ( = 0.026) after hybridized with the Liddle’s syndrome DNA
(Figure 5A). Smaller increase of the fluorescent quantum yield was observed with the wild
type DNA (2.4-fold,  = 0.063). Moreover, the absorption spectra showed a more
pronounced red-shifting in the case of Liddle’s syndrome sequence (25 nm, max = 525 nm)
than the wild type DNA (5 nm, max = 505 nm) relative to the free PNA probe (max = 500
nm) (Figure 5B). Due to the small fluorescence signal difference between the base-inserted
and complementary DNA targets, a quantitative measurement of the signal with appropriate
calibration may be necessary for practical applications. Nevertheless, this example clearly
showcases the potential applicability of the styryl-labeled PNA probes in distinguishing
between base insertion mutation and wild type DNA targets.
0.06
0.05
0.04
0.03
0.02
0.01
0
120
100
80
60
40
20
0
A
B
free probe
wild type
Liddle's syndrome
free probe
wild type
Liddle's syndrome
Liddle’s
syndrome
Wild type
Probe
-0.01
350
400
450
500
550
600
650
500
550
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
Figure 5. (A) Fluorescence spectra and (B) UV-vis spectra of PNA probe M12-5a in the
presence of wild type and Liddle’s syndrome DNA in 10 mM sodium phosphate buffer pH
7.0; [PNA] = 1.0 M, [DNA] = 1.2 M, ex = 487 nm. Inset shows photographs under UV
light (315 nm) at [PNA] = 10 µM, [DNA] = 12 µM

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Structural Insights in the Interaction of Styryl Dyes with PNA·DNA and DNA·DNA
Duplexes
To gain further understanding in the interaction of styryl dyes and PNA·DNA vs
DNA·DNA duplexes, fluorescence experiments between free styryl dyes (3 and 18) and
various unlabeled acpcPNA and DNA hybrids were investigated. The experiments showed
that the fluorescence of both dyes was almost unchanged in the presence of complementary
PNA·DNA hybrid (Figure S39). This behavior is in contrast with DNA·DNA duplexes
whereby significant fluorescence increases were observed for both dyes (Table 1), indicating
the difference in binding of styryl dyes to DNA·DNA and PNA·DNA duplexes. This is
consistent with the fact that very little fluorescence change was observed with styryl dye-
labeled acpcPNA probes, thus making these probes less useful for the detection of
complementary DNA target as originally intended. On the other hand, significant
fluorescence increases of both dyes were observed with the hybrids of PNA and mismatched,
base-inserted and abasic DNA (Figure S39), indicating that the dye can only interact with the
PNA·DNA duplexes when there is a defect in the structure that may facilitate the interaction
between the dye and the duplex. It is known that typical intercalators and groove binders
interact less strongly with aegPNA·DNA than DNA·DNA duplexes.56 A cyanine dye
DiSC2(5) can aggregate within the minor groove aegPNA·DNA duplex templates,57 but the
same aggregation was not observed with aegPNA variants containing a substituent that can
block the minor groove, and therefore prevent the dye binding.58
To explain the unexpectedly small response of the styryl-dye-labeled acpcPNA probe
towards its complementary DNA target, the interactions between DNA·DNA and
acpcPNA·DNA duplexes with the styryl dye 3 were explored by molecular modeling. The
minor groove of DNA·DNA duplex (PDB ID = 4c64)59 was wider (~6 Å) and much deeper
(~5 Å) than the corresponding acpcPNA·DNA duplex60 (width ~2–3 Å and very shallow)
(Figure S40A). The dimension of the groove suggests that the styryl dyes should fit nicely
into the minor groove of DNA·DNA duplexes. A docking simulation with Autodock Vina61,62
revealed that both 3 and 18 fit well into the minor groove of DNA·DNA duplex, whereas the
backbone of acpcPNA blocked styryl dyes from binding in the minor groove of
acpcPNA·DNA duplex (Figure S40B). Collectively, these in silico studies were in agreement
with experiments in which fluorescence intensities of styryl dyes significantly enhanced in
the presence of complementary DNA·DNA but not PNA·DNA duplexes.

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A
B
Figure 6. Averaged MD structures of M10-5a binding to (A) base-inserted and (B)
complementary DNA. See Table 3 for the DNA sequences.
While previous experimental studies with structurally-related styryl dyes indicated
that they predominantly interact with dsDNA by groove binding,63,64 the intercalative binding
mode has also been observed.52,65 Molecular dynamics (MD) simulations were performed to
gain further understanding of the interactions between the dyes and the PNA·DNA duplexes.
The MD structure averaged along 10000 structures of the last 10 ns MD trajectory for each
system illustrated in Figure 6 revealed that, for the M10-5aDNA duplex carrying a base
insertion, the styryl dye was located in the PNADNA duplex and partially stacked with the
adjacent base pairs, while the inserted cytosine base pointed outside the duplex. It can thus be
concluded that the presence of a defect in the structure creates a binding site that allows the
dye to fit in, resulting in the observed fluorescence enhancement. Similar effects had been
previously observed in other acpcPNA probes, although with a decrease in fluorescence35,37

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or a much smaller degree of fluorescence increase.40 In contrast, only part DoOfI: 10th.10e39d/Cy9eOB01492F
molecule was placed in the minor groove of the complementary M10-5aDNA duplex. This
leads to a larger fluctuation of the dye molecule in the complementary compared to the base-
inserted duplexes as confirmed by the root-mean-square deviation (RMSD) of atomic
positions. The RMSDs of dye structures in the MD trajectory with respect to its averaged
structure, which refers to a change of the simulated dye structures along a simulation time,
were 0.5±0.2 and 0.8±0.3 Å for the base-inserted and complementary duplexes, respectively.
Such a large degree of flexibility is in good agreement with the observed small response of
the probe to complementary DNA.
Conclusion
In this work, we successfully synthesized novel alkyne-modified styryl dyes and
conjugated them onto the conformationally rigid pyrrolidinyl PNA by click chemistry. The
alkyne-modified styryl dye exhibited low fluorescence in the free state but yielded large
fluorescence enhancement (up to 125-fold) in the presence of DNA. The styryl-dye-labeled
acpcPNA probes retained the ability to bind to their complementary DNA target, but the
fluorescence change was small. On the other hand, the probes yielded strongly enhanced
fluorescence with mutated DNA carrying base mismatch, abasic site and, most notably, base
insertion. Based on spectroscopic studies and modeling, we propose that the defect in the
PNA·DNA duplex structure provides a binding site that can accommodate the styryl dye
located nearby, thus resulting in the observed fluorescence enhancement. The unique
fluorescence response of the styryl-dye-labeled acpcPNA probes can be considered as a
valuable addition to the only few reported examples of fluorescence oligonucleotide probes
that are responsive to base insertion66 or abasic sites67,68,69 in the DNA targets. As a result, the
present styryl-dye-labeled acpcPNA probes are potentially useful for the detection of specific
types of mutated DNA targets such as base insertion mutations that generally do not respond
to conventional probes.

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Experimental
Synthesis of Clickable Styryl Dyes
All styryl dyes were synthesized by condensation of 2-methylbenzothiazolium or 4-
methylquinolinium salts and an appropriate aldehyde in acetic anhydride at 90 C for 8–10 h
(Method A for 1–4, 6, 8–14) or in refluxing ethanol for 8 h (Method B for 5, 7, 15–19). They
are generally isolated with the same counterion as the benzothiazolium or 4-
methylquinolinium salt used (iodide for N-methyl and bromide for N-propargyl salts). In
-
some cases when the product was not precipitate, the counterion was exchanged to PF6 by
treatment of the bromide or iodide salts with ammonium hexafluorophosphate in methanol.
(E)-2-(2-(4-(2-Propynyloxy)phenyl)ethenyl)benzo[d]thiazole (1)
1
Yield 15% (3 steps, from 2-methylbenzothiazole); H NMR (400 MHz, CDCl3): δ 7.90 (d, J
= 8.0 Hz, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 16.4 Hz, 1H)
7.38 (t, J = 7.7 Hz, 1H), 7.21 (d, J = 16.2 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 4.65 (s, 2H), 2.47
(s, 1H); 13C NMR (100 MHz, CDCl3): δ 167.3, 158.6, 153.8, 137.2, 134.2, 129.0, 128.9,
126.3, 125.2, 122.8, 121.4, 120.4, 115.4, 78.2, 75.9, 55.9; HRMS (MALDI-TOF): m/z calcd
for C18H14NOS+: 292.0796 [M+H]+ found: 292.0796.
(E)-3-Methyl-2-(2-(4-(2-propynyloxy)phenyl)ethenyl)benzo[d]thiazolium iodide (2)
Yield 32%; 1H NMR (400 MHz, DMSO-d6): δ 8.39 (d, J = 8.1 Hz, 1H), 8.24 (d, J = 9.1 Hz,
1H), 8.20 (d, J = 16.2 Hz, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 15.8 Hz, 1H), 7.84 (t, J
= 7.8 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 7.8 Hz, 2H), 4.93 (s, 2H), 4.32 (s, 3H),
13
3.65 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 172.0, 160.7, 148.4, 142.0, 131.9, 129.3,
128.3, 127.6, 127.4, 124.2, 116.7, 115.6, 111.7, 78.8, 78.6, 56.0, 55.9, 36.3, 18.5; HRMS
(MALDI-TOF): m/z calcd for C19H16NOS+: 306.0947 [M]+ found: 306.0952.
(E)-2-(2-(4-(Dimethylamino)phenyl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium bromide (3)
Yield 81%; 1H NMR (400 MHz, DMSO-d6): δ 8.32 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 15.2 Hz,
1H), 8.13 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 8.8 Hz, 2H), 7.82 (t, J = 7.8 Hz, 1H), 7.74–7.67
(m, 2H), 6.89 (d, J = 8.8 Hz, 2H), 5.78 (s, 2H), 3.72 (s, 1H), 3.15 (s, 6H); 13C NMR (100
MHz, DMSO-d6): δ 189.8, 171.4, 153.9, 151.6, 140.4, 133.4, 129.0, 127.5, 126.6, 124.1,
121.5, 115.6, 112.1, 111.0, 105.2, 78.1, 76.0, 37.6; HRMS (MALDI-TOF): m/z calcd for
C20H19N2S+: 319.1263 [M]+ found: 319.1265.

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(E)-3-Methyl-2-(2-(4-(N-methyl-N-2-propynyl)phenyl)ethenyl)benzo[d]thiazolium iodide (4)
Yield 70%; 1H NMR (400 MHz, DMSO- d6): δ 8.34 (d, J = 7.9 Hz, 1H), 8.14 (d, J = 8.8 Hz,
1H), 8.10 (d, J = 15.8 Hz, 1H), 7.95 (d, J = 8.8 Hz, 2H), 7.81 (t, J = 7.7 Hz, 1H), 7.75–7.67
13
(m, 2H), 6.96 (d, J = 8.9 Hz, 2H), 4.34 (s, 2H), 4.27 (s, 3H), 3.24 (s, 1H), 3.09 (s, 3H); C
NMR (100 MHz, DMSO-d6): δ 171.6, 152.2, 149.8, 141.9, 132.4, 128.9, 127.6, 127.0, 123.8,
122.7, 116.1, 113.0, 107.7, 79.4, 74.8, 41.0, 38.0, 35.8; HRMS (MALDI-TOF): m/z calcd for
C20H19N2S+: 319.1263 [M]+ found: 319.1270.
(E)-2-(2-(4-(N-tert-Butoxycarbonylamino)phenyl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium
bromide (5)
Yield 82%; 1H NMR (400 MHz, DMSO- d6): δ 9.93 (s, 1H), 8.46 (d, J = 8.1 Hz, 1H), 8.31–
8.23 (m, 2H), 8.04–7.99 (m, 3H), 7.91 (t, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.67 (d, J
13
= 8.5 Hz, 2H), 5.93 (s, 2H), 3.79 (s, 1H), 1.51 (s, 9H); C NMR (100 MHz, DMSO-d6): δ
172.6, 152.4, 150.3, 144.3, 140.5, 131.5, 129.6, 128.4, 127.7, 124.5, 118.0, 116.4, 110.6,
80.0, 78.7, 75.7, 38.4, 28.0; HRMS (MALDI-TOF): m/z calcd for C23H23N2O2S+: 391.1480
[M]+ found: 391.1471.
(E)-2-(2-(4-(Diethylamino)-2-(2-propynyloxy)phenyl)ethenyl)-3-methylbenzo[d]thiazolium
iodide (6)
Yield 69%; 1H NMR (400 MHz, DMSO-d6): δ 8.24 (d, J = 7.4 Hz, 1H), 8.06 (d, J = 15.2 Hz,
1H), 8.05 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 9.2 Hz, 1H), 7.75 (t, J = 7.9 Hz, 1H), 7.64 (t, J =
7.7 Hz, 1H), 7.54 (d, J = 15.2 Hz, 1H), 6.54 (dd, J = 9.1, 2.0 Hz, 1H), 6.34 (d, J = 2.0 Hz,
1H), 5.08 (s, 2H), 4.15 (s, 3H), 3.75 (s, 1H), 3.53 (q, J = 7.0 Hz, 4H), 1.19 (t, J = 7.0 Hz, 6H);
13C NMR (100 MHz, DMSO-d6): δ 171.1, 159.7, 153.4, 143.9, 142.0, 133.0, 128.7, 127.0,
126.3, 123.6, 115.6, 111.1, 106.5, 105.0, 95.0, 79.0, 78.7, 56.3, 44.5, 35.2, 12.6; HRMS
(MALDI-TOF): m/z calcd for C23H25N2OS+: 377.1682 [M]+ found: 377.1684.
(E)-2-(2-(4-(N,N-Diphenylamino)phenyl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium bromide
(7)
1
Yield 58%; H NMR (400 MHz, DMSO-d6): δ 8.41 (d, J = 8.0 Hz, 1H), 8.26–8.20 (m, 2H),
7.94 (d, J = 8.9 Hz, 2H), 7.92–7.85 (m, 2H), 7.78 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.8 Hz, 4H),
7.27 (t, J = 7.5 Hz, 2H), 7.23 (d, J = 7.5 Hz, 4H), 6.93 (d, J = 8.8 Hz, 2H), 5.86 (s, 2H), 3.76
13
(s, 1H); C NMR (100 MHz, DMSO-d6): δ 172.2, 151.8, 150.3, 145.3, 140.5, 132.2, 130.0,

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129.4, 128.2, 127.4, 126.3, 126.0, 125.6, 124.4, 118.8, 116.2, 109.1, 78.5, 75.7, 38.1; HRMS
(MALDI-TOF): m/z calcd for C30H23N2S+: 443.1576 [M]+ found: 443.1579.
(E)-3-(2-Propynyl)-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-
yl)ethenyl)benzo[d]thiazolium hexafluorophosphate (8)
Yield 78%; 1H NMR (400 MHz, DMSO-d6): δ 8.23 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.4 Hz,
1H), 7.94 (d, J = 14.7 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.52 (s, 1H),
7.49 (d, J = 14.7 Hz, 1H), 5.67 (s, 2H), 3.70 (s, 1H), 3.41 (m, 4H), 2.74 (m, 4H), 1.91 (m,
4H); 13C NMR (100 MHz, DMSO-d6): δ 169.9, 151.4, 148.6, 140.4, 131.0, 128.8, 127.0,
126.1, 123.8, 121.5, 120.8, 115.0, 102.8, 77.8, 76.2, 49.8, 37.0, 26.8, 20.4; HRMS (MALDI-
TOF): m/z calcd for C24H23N2S+: 371.1576 [M]+ found: 371.1579.
(E)-2-(2-(4-(Benzoyloxy)phenyl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium bromide (9)
Yield 67%; 1H NMR (400 MHz, DMSO-d6): δ 8.51 (d, J = 8.0 Hz, 1H), 8.40 (d, J = 15.7 Hz,
1H), 8.35 (d, J = 8.5 Hz, 1H), 8.23–8.17 (m, 5H), 7.96 (t, J = 7.8 Hz, 1H), 7.86 (t, J = 7.8 Hz,
1H), 7.80 (t, J = 7.5 Hz, 1H), 7.66 (t, J = 7.7 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 5.98 (s, 2H),
13
3.82 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 172.7, 164.2, 153.8, 149.0, 140.6, 134.3,
131.8, 131.4, 129.9, 129.8, 129.0, 128.8, 128.6, 128.1, 124.7, 123.0, 116.8, 113.5, 78.9, 75.6,
38.7; HRMS (MALDI-TOF): m/z calcd for C25H18NO2S+: 396.1058 [M]+ found: 396.1054.
(E)-2-(2-(4-Methoxy)phenyl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium
hexafluorophosphate (10)
1
Yield 60%; H NMR (400 MHz, DMSO-d6): δ 8.44 (d, J = 8.1 Hz, 1H), 8.34–8.26 (m, 2H),
8.08 (d, J = 8.6 Hz, 2H), 7.99 (d, J = 15.6 Hz, 1H), 7.91 (t, J = 7.6 Hz, 1H), 7.81 (t, J = 7.7
13
Hz, 1H), 7.17 (d, J = 8.6 Hz, 2H), 5.91 (s, 2H), 3.90 (s, 3H), 3.78 (s, 1H); C NMR (100
MHz, DMSO-d6): δ 172.8, 163.4, 150.4, 140.5, 132.5, 129.6, 128.4, 127.6, 126.8, 124.5,
116.4, 115.0, 110.4, 78.6, 75.7, 55.8, 38.4; HRMS (MALDI-TOF): m/z calcd for
C19H16NOS+: 306.0947 [M]+ found: 306.0962.
(E)-2-(2-(2,4-Dimethoxyphenyl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium bromide (11)
1
Yield 84%; H NMR (400 MHz, DMSO-d6): δ 8.40 (d, J = 8.1 Hz, 1H), 8.32–8.25 (m, 2H),
8.12 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 15.6 Hz, 1H), 7.89 (t, J = 7.8 Hz, 1H), 7.78 (t, J = 7.7
Hz, 1H), 6.79 (d, J = 8.8 Hz, 1H), 6.76 (s, 1H), 5.86 (s, 2H), 4.01 (s, 3H), 3.93 (s, 3H), 3.79

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13
(s, 1H); C NMR (100 MHz, DMSO-d6): δ 173.0, 165.7, 161.4, 144.8, 140.6, 1D3O2I.:410,.1103299/C.59O,B01492F
128.2, 127.3, 124.4, 116.3, 115.5, 109.9, 107.8, 98.4, 78.6, 75.6, 56.4, 56.0, 38.2; HRMS
(MALDI-TOF): m/z calcd for C20H18NO2S+: 336.1053 [M]+ found: 336.1058.
(E)-2-(2-(4-Nitrophenyl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium bromide (12)
1
Yield 70%; H NMR (400 MHz, DMSO-d6): δ 8.56 (d, J = 8.1 Hz, 1H), 8.48–8.38 (m, 5H),
8.35 (d, J = 8.7 Hz, 2H), 7.99 (t, J = 7.8 Hz, 1H), 7.89 (t, J = 7.8 Hz, 1H), 6.04 (s, 2H), 3.84
13
(s, 1H); C NMR (100 MHz, DMSO-d6): δ 172.1, 148.9, 146.6, 140.6, 139.8, 130.7, 130.6,
130.1, 129.1, 128.6, 124.9, 124.2, 117.2, 117.0, 79.1, 75.5; HRMS (MALDI-TOF): m/z calcd
for C18H13N2O2S+: 321.0692 [M]+ found: 321.0685.
(E)-2-(2-(7-(Diethylamino)-2-oxo-2H-chromen-3-yl)ethenyl)-3-(2-
propynyl)benzo[d]thiazolium bromide (13)
Yield 60%; 1H NMR (400 MHz, DMSO-d6): δ 8.65 (s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.29 (d,
J = 8.5 Hz, 1H), 8.12 (m, 2H), 7.88 (t, J = 7.8 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.61 (d, J =
9.1 Hz, 1H), 6.90 (dd, J = 9.1, 1.8 Hz, 1H), 6.70 (s, 1H), 5.75 (s, 2H), 3.81 (s, 1H), 3.56 (q, J
13
= 7.0 Hz, 4H), 1.18 (t, J = 7.0 Hz, 6H); C NMR (100 MHz, DMSO-d6): δ 172.3, 159.4,
157.4, 153.6, 148.8, 146.0, 140.6, 132.1, 129.5, 128.2, 127.4, 124.5, 116.1, 111.9, 111.1,
110.3, 109.1, 96.6, 78.7, 75.3, 44.7, 38.7, 12.4; HRMS (MALDI-TOF): m/z calcd for
C25H23N2O2S+: 415.1475 [M]+ found: 415.1468.
(E)-3-(2-Propynyl)-2-(2-(1-pyrenyl)ethenyl)benzo[d]thiazolium bromide (14)
Yield 38%; 1H NMR (400 MHz, DMSO-d6): δ 9.31 (d, J = 15.1 Hz, 1H), 9.00 (d, J = 8.3 Hz,
1H), 8.91 (d, J = 9.2 Hz, 1H), 8.55–8.31 (m, 9H), 8.20 (t, J = 7.8 Hz, 1H), 7.97 (t, J = 7.7 Hz,
13
1H), 7.88 (t, J = 7.4 Hz, 1H), 6.08 (s, 2H), 3.83 (s, 1H); C NMR (100 MHz, DMSO-d6): δ
172.2, 145.9, 140.6, 134.1, 130.8, 130.6, 130.2, 130.1, 129.8, 129.7, 128.7, 128.2, 127.4,
127.2, 127.2, 127.0, 126.9, 125.8, 125.5, 124.6, 124.0, 123.5, 122.7, 116.7, 114.6, 78.9, 75.8,
38.7; HRMS (MALDI-TOF): m/z calcd for C28H18NS+: 400.1154 [M]+ found: 400.1149.
(E)-2-(2-(1-Naphthyl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium bromide (15)
Yield 49%; 1H NMR (400 MHz, DMSO-d6): δ 9.06 (d, J = 15.5 Hz, 1H), 8.58–8.53 (m, 2H),
8.48 (d, J = 7.4 Hz, 1H), 8.39 (d, J = 8.5 Hz, 1H), 8.30 (d, J = 15.5 Hz, 1H), 8.25 (d, J = 8.1
Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.98 (t, J = 7.8 Hz, 1H), 7.88 (t, J = 7.8 Hz, 1H), 7.80–7.75

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(m, 2H), 7.69 (t, J = 7.4 Hz, 1H), 6.04 (s, 2H), 3.84 (s, 1H); 13C NMR (100 MHz,DDOIM: 10S.1O03-9d/C69)O:B01492F
δ 172.4, 146.1, 140.6, 133.4, 133.2, 131.0, 130.6, 129.9, 129.0, 128.8, 128.3, 127.8, 127.6,
126.8, 125.8, 124.7, 123.5, 116.8, 115.4, 78.9, 75.6, 38.8; HRMS (MALDI-TOF): m/z calcd
for C22H16NS+: 326.0998 [M]+ found: 326.1001.
(E)-2-(2-(5-Phenylthiophen-2-yl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium bromide (16)
Yield 73%; 1H NMR (400 MHz, DMSO-d6): δ 8.58 (d, J = 15.3 Hz, 1H), 8.47 (d, J = 8.1 Hz,
1H), 8.29 (d, J = 8.3 Hz, 1H), 8.04 (d, J = 3.5 Hz, 1H), 7.92 (t, J = 7.9 Hz, 1H), 7.83 (m, 5H),
7.54 (t, J = 7.3 Hz, 2H), 7.50–7.45 (t, J = 7.3 Hz, 1H), 5.92 (s, 2H), 3.82 (s, 1H);
13C NMR (100 MHz, DMSO-d6): δ 171.8, 151.9, 142.4, 140.5, 138.4, 137.8, 132.5, 129.6,
129.6, 129.5, 128.5, 127.7, 126.2, 126.0, 124.6, 116.4, 110.8, 78.8, 75.7, 38.4; HRMS
+
(MALDI-TOF): m/z calcd for C22H16NS2 : 358.0719 [M]+ found: 358.0717.
(E)-2-(2-(9-Methyl-9H-carbazol-3-yl)ethenyl)-3-(2-propynyl)benzo[d]thiazolium bromide
(17)
1
Yield 97%; H NMR (400 MHz, DMSO-d6): δ 8.91 (s, 1H), 8.49 (d, J = 15.5 Hz, 1H), 8.41
(d, J = 8.8 Hz, 1H), 8.29–8.23 (m, 3H), 8.10 (d, J = 15.5 Hz, 1H), 7.87 (t, J = 8.3 Hz, 1H),
7.81 (d, J = 8.5 Hz, 1H), 7.76 (t, J = 7.9 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.56 (t, J = 7.7 Hz,
1H), 7.34 (t, J = 7.7 Hz, 1H), 5.91 (s, 2H), 3.96 (s, 3H), 3.77 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ 172.5, 152.4, 143.5, 141.4, 140.5, 129.4, 128.2, 128.2, 127.4, 126.9, 125.1,
124.4, 124.2, 122.8, 122.0, 120.4, 116.2, 110.3, 110.2, 109.3, 78.6, 75.8, 38.3, 29.4; HRMS
(MALDI-TOF): m/z calcd for C25H19N2S+: 379.1263 [M]+ found: 379.1258.
(E)-4-(2-(4-(Dimethylamino)phenyl)ethenyl)-1-(2-propynyl)quinolinium bromide (18)
Yield 74%; 1H NMR (400 MHz, DMSO-d6): δ 9.19 (d, J = 6.8 Hz, 1H), 9.07 (d, J = 8.4 Hz,
1H), 8.41–8.38 (m, 2H), 8.29–8.22 (m, 2H), 8.03 (d, J = 15.6 Hz, 1H), 7.95 (t, J = 7.6 Hz,
1H), 7.90 (d, J = 8.9 Hz, 2H), 6.84 (d, J = 8.9 Hz, 2H), 5.82 (s, 2H), 3.89 (s, 1H), 3.09 (s,
6H); 13C NMR (100 MHz, DMSO-d6): δ 154.4, 152.6, 146.1, 145.5, 137.5, 134.9, 131.8,
128.6, 126.7, 126.0, 123.0, 118.8, 114.0, 113.0, 111.9, 80.1, 76.3, 45.6, 39.7; HRMS
+
(MALDI-TOF): m/z calcd for C22H21N2 : 313.1699 [M]+ found: 313.1696.
(E)-4-(2-(4-(Diethylamino)-2-(propynyloxy)phenyl)ethenyl)-1-methylquinolinium bromide
(19)

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Yield 81%; 1H NMR (400 MHz, DMSO-d6): δ 9.00 (d, J = 6.8 Hz, 1H), 8.85 (d,DJOI=: 108.1.0039H/C9zO,B01492F
1H), 8.32 (d, J = 8.4 Hz, 1H), 8.24–8.15 (m, 3H), 8.06 (d, J = 15.6 Hz, 1H), 7.97 (t, J = 8.1
Hz, 1H), 7.87 (d, J = 9.0 Hz, 1H), 6.50 (dd, J = 9.0, 2.1 Hz, 1H), 6.38 (d, J = 2.1 Hz, 1H),
5.06 (s, 2H), 4.42 (s, 3H), 3.78 (s, 1H), 3.50 (q, J = 7.0 Hz, 4H), 1.19 (t, J = 7.0 Hz, 6H);
13C NMR (100 MHz, DMSO-d6): δ 159.0, 153.3, 151.6, 146.5, 139.5, 138.8, 134.4, 132.4,
128.4, 125.8, 125.6, 119.0, 113.2, 112.8, 112.2, 105.6, 95.4, 79.2, 78.8, 56.0, 44.2, 43.8, 12.6;
HRMS (MALDI-TOF): m/z calcd for C25H27N2O+: 371.2118 [M]+ found: 371.2117.
Synthesis of Styryl-Dye-Labeled acpcPNA Probes
The (3R,4S)-3-aminopyrrolidine-4-carboxylic acid (APC)-modified acpcPNAs were
synthesized manually on Tentagel S-RAM resin from the respective Fmoc-protected
monomers (ABz, CBz, T, G) and spacer (APCTfa, ACPC) at 1.5 mol scales as described
earlier.39 After completion of the synthesis, the N-terminal Fmoc group was removed and
capped by acetylation. The resin was split into 0.5 µmol portions and the side-chain
protecting groups of nucleobase A and C (Bz) and APC spacer (Tfa) were removed by
treatment with 1:1 ammonia-dioxane at 65 ºC overnight. The deprotected APC-modified
acpcPNA was reacted with the 4-azidobutanal (30 equiv.) in the presence of NaBH3CN (60
equiv.) and HOAc (60 equiv.) in MeOH (100 L) at room temperature overnight.39 The
azide-modified acpcPNA on the solid support was split into 0.5 mol portions for labeling
experiments. This azide-modified acpcPNA was reacted with alkynyl-modified styryl dye (15
equiv.) in the presence of tetrakis(acetonitrile) copper(I) hexafluorophosphate (30 equiv.) in
DMSO/tBuOH (3:1) at room temperature overnight. After the reaction was completed as
monitored by MALDI-TOF mass spectrometry, the PNA was cleaved from the solid support
by treatment with trifluoroacetic acid (TFA) (500 L, 30 min, 3 times). The cleavage solution
was combined and dried under a stream of nitrogen and the crude styryl-dye-labeled
acpcPNA was precipitated by the addition of diethyl ether and purified by reverse-phase
HPLC. The HPLC experiments were performed on Water Delta 600TM system equipped with
a gradient pump by using 0.1% TFA in H2O:acetonitrile (9:1 to 1:9) as a mobile phase. The
purified styryl-dye-labeled acpcPNA were characterized by MALDI-TOF mass spectrometry
on a Microflex MALDI-TOF mass spectrometer (Bruker Daltonics) using -cyano-4-
hydroxycinnamic acid (CCA) as a matrix.
MD Simulation

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To investigate a positioning of styryl dye in the styryl-dye-labeled acpcPNA·DNA
duplex, molecular dynamics (MD) simulations of M10-5a binding to the base-inserted and
complementary DNA targets were carried out in an aqueous solution with the OL15 force
field.70 The starting structures of the simulated systems, atomic point charges as well as
missing force field parameters of styryl dye and acpcPNA units, and the setup of the
simulations were constructed in a similar manner as previous work.38,60,71 MD simulations
were performed using Sander module in the AMBER 2017 suite program.72 The production
runs were carried out for 20 ns and the coordinates were stored every 1 ps, totally 20000
simulated structures.
Acknowledgements
The authors acknowledge financial supports from the Thailand Research Fund (DPG5780002
and RGU6180001 to T.V.), Chulalongkorn University (The 90th Anniversary of
Chulalongkorn University Scholarship, to B.D.), the Science Achievement Scholarship of
Thailand (to B.D.) and National Science and Technology Development Agency (NSTDA)
(Research Chair Grant #P1850116). The National e-Science Infrastructure Consortium (URL:
http://www.e-science.in.th) is also acknowledged for providing computing resources.

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