Synthesis and Fluorescence Properties of 1,1-Dimethyl-1,4-Dihydrodibenzo[b,h][1,6]naphthyridinium Iodides: Turn-on Type Detection of DNA

Article abstract of DOI:10.1002/ejoc.201701277

1,4-Dihydrodibenzo[b,h][1,6]naphthyridines were synthesized by the reaction of 2-acetylaminobenzaldehyde with acetophenones in the presence of NaOH. N-Methylation of the dihydrodibenzonaphthyridines with MeI gave 1,1-dimethylammonium iodides. These salts

Full text of DOI:10.1002/ejoc.201701277

A Journal of
Accepted Article
Title: Synthesis and Fluorescence Property of 1,1-Dimethyl-1,4-
Dihydrodibenzo[b,h][1,6]naphthyridinium Iodides: Turn-on Type
Detection of DNA
Authors: Kentaro Okuma, Akinori Oba, Risa Kuramoto, Hidefumi
Iwashita, Noriyoshi Nagahora, Kosei Shioji, Ryoma Noguchi,
and Masatora Fukuda
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201701277
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10.1002/ejoc.201701277
European Journal of Organic Chemistry
COMMUNICATION
Synthesis and Fluorescence Property of 1,1-Dimethyl-1,4-
Dihydrodibenzo[b,h][1,6]naphthyridinium Iodides: Turn-on Type Detection
of DNA
Kentaro Okuma,* Akinori Oba, Risa Kuramoto, Hidefumi Iwashita, Noriyoshi Nagahora, Kosei Shioji,
Ryoma Noguchi, and Masatora Fukuda
Thus, we focused on compounds that produce strong emission
when intercalated into DNA (turn on type). In the course of
synthesizing compound 1, we found a new type of fluorescent
reagent, the 1,4-dihydrodibenzonaphthyridine derivatives. The
Abstract:
1,4-Dihydrodibenzo[b,h][1,6]naphthyridines
were
synthesized by the reaction of 2-acetylaminobenzaldehyde with
acetophenones in the presence of NaOH. The N-methylation of the
dihydrodibenzonaphthyridines with MeI gave 1,1-dimethylammonium
iodides. These salts showed UV/vis absorbance maxima at 235 and
418 nm and very weak fluorescence at 530 nm. The fluorescence
intensities were enhanced 3–10 times when these salts were treated
with double-stranded DNA via intercalation, which enabled the
detection of DNA/RNA in PAGE gels.
methylation
of
these
compounds
produced
1,1-
dimethyldibenzonaphthyridinium salts 2. We report herein a
series of highly DNA staining quinoline-based fluorophores,
aromatic substituted dihydrodibenzonaphthyridines, for high-
contrast and high-brightness imaging in DEAE gels.
Firstly,
we
attempted
to
synthesize
Treatment
1,4-
2-
dihydrodibenzonaphthyridines.
of
acetylaminobenzaldehyde 3a with acetophenone 4a in the
presence of NaOH (1 eq.) in refluxing ethanol for 2 h followed by
the addition of NaOH (10 eq.) for an additional 4 h resulted in the
formation of 1,4-dihydrodibenzonaphthyridine in 61% yield. 1,4-
Dibenzodihydro[1,6]naphthyridine 5a was easily oxidized in
solution to afford dibenzonaphthyridine, the isolation of which
could be accomplished in 10 minutes. The other reactions were
carried out in a similar manner (Table 1).
It is necessary to acquire an insight into the selective staining of
DNA to achieve a full understanding of biological functions and
processes.1
For example, ethidium bromide, a quinoline
derivative, is widely used for the detection of DNA because of its
ability to intercalate into the DNA double helix.2 Ethidium bromide
inserts itself into the spaces between the base pairs of the double
helix (turn on). Generally, the binding of an analyte to a probe
causes fluorescence enhancement (turn on) or fluorescence
quenching (turn off).3 Other quinolines, 1,8-naphthyridines, and
1,6-naphthyridines are also important compounds because of
their interesting fluorescence properties and biological activities.4
A cascade reaction in which a reactive intermediate from one step
directly undergoes further transformations is very important in
organic synthesis, because such sequential processes not only
rapidly build up complex molecules but also efficiently enhance
chemo- and regioselectivity of the overall transformation.5 We
Table 1. Synthesis of dihydrodibenzonaphthyridines 5
R
H
O
1) NaOH (1 eq), 2h
2) NaOH (10 eq), 4h
O
2
+
N
H3C
EtOH
reflux
NHAc
N
H
R
have
reported
the
synthesis
of
6-methyl-1,6-
4a, R= H
3
dibenzonaphthyridinium triflates 1 via a cascade approach.6
These compounds produce fluorescence at 420 nm when
irradiated at 360 nm, which have the ability to intercalate into DNA.
However, these compounds have some disadvantages compared
with ethidium bromide. Compound 1 produces an emission at 440
nm in solution, but when it intercalates into DNA, its emission
intensity decreases (turn off), rendering DNA detection difficult
4b, R= Me
5a, R= H
4c, R= OMe
4d, R= Cl
4e, R= Br
5b, R= Me
5c, R= OMe
5d, R= Cl
5e, R= Br
Yield/% a
Entry
4
4a
4a
4b
4c
4d
4e
5
5a
5a
5b
5c
5d
5e
1
2
3
4
5
6
61
63
65
71
70
65
(Figure 1).
R
Me
Et
H2N
N
N
N
a) Reactions were carried out by using 3 (3.0 mmol), 4 (1.5 mmol),
5.0 M NaOH (0.3 mL, 1.5 mmol), and 20 M NaOH (0.75 mL, 15 mmol).
N
N
Me Me
The structure of 5a was confirmed by spectroscopic analysis (1H
NMR, 13C NMR, MS) and elemental analysis. The 1H NMR
spectrum of 5a shows signals for methylene (4.1 ppm) and
aromatic groups (6.8–9.4 ppm). The reaction is speculated to
I
Br
Ethidium Bromide
OTf
1
2
Figure 1. DNA intercalation reagents.
proceed
as
follows:
The
aldol
reaction
of
[*]
K. Okuma, A. Oba, R. Kuramoto, H. Iwashita, N. Nagahora, K.
Shioji, R. Noguchi, and M. Fukuda
Department of Chemistry
Fukuoka University
Jonan-ku, Fukuoka 814-0180
acetylaminobenzaldehyde
3
with acetophenone
4
gave
unsaturated ketone a, which was hydrolyzed and condensed to
give corresponding imine b. The intramolecular aza-Morita-
Baylis-Hillman reaction and the subsequent intramolecular
condensation gave 1,2-dihydronaphthyridine c. Proton transfer of
this compound finally afforded 1,4-dihydronaphthyridine
(Scheme 1).
E-mail: kokuma@fukuoka-u.ac.jp
http://www.sc.fukuoka-u.ac.jp
5
Supporting information for this article is given via a link at the end
of the document.
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10.1002/ejoc.201701277
European Journal of Organic Chemistry
COMMUNICATION
R
R
R
Figure 2 shows the excitation and emission spectra of 6a and 2a
in CH2Cl2 solution. N-methyldihydronaphthyridine 6a was found
to have an excitation wavelength at 420 nm, whereas upon
excitation at its absorption maxima, 1,1-dimethylnaphthyridinium
iodide 2a exhibited relatively weak emission and low fluorescence
quantum yield in CH2Cl2, which could be attributed to the very
rapid nonradioactive decay caused by the strong and dynamic
adhesive interactions with EtOH and water molecules (Figure 2).
O
O
H
H
O
H
NaOH
Aldol
HO
+
H
H
NHAc
Hydrolysis
NH2
O
NHAc
a
H3C
4
3
H
NHAc
O
O
R
R
O
HO
aza-Morita
-Baylis-Hillmann
NHAc
H
N
N
b
NHAc
H
N
N
R
R
N
N
Me
Me
I
Me
O
R
6a
2a
HO
N
N
NH
N
N
H
N
H
0,2
0,1
0
6a (abs.)
2a (abs.)
6a (F.I.)
2a (F.I.)
200
150
100
50
c
5
Scheme 1. Reaction Mechanism.
Dihydronaphthyridine 5a was treated with an excess amount of
methyl iodide in refluxing acetone for 15 h followed by the addition
of aq. NaOH to afford 1-methyldihydrodibenzonaphthyridine (6a)
in 68% yield. Further methylation by adding excess methyl iodide
in refluxing acetone afforded dimethylammonium iodide 2a in
57% yield. The other methylation reactions were carried out in a
similar manner (Table 2).
0
300
400
500
wavelength (nm)
600
700
R
R
R
Figure 2. UV and fluorescence spectra of 6a and 2a in CH2Cl2.
Other ammonium iodides 2b–2e showed a similar tendency as well
1) MeI
2) aq. NaOH
(Table 3, Figure S-1).
MeI
N
N
R
N
acetone
reflux
acetone
reflux
N
N
N
H
Me Me
Me
I
5
6
2
N
Table 2. Synthesis of N-methyl-1,4-
dihydro[b,h][1,6]dibenzonaphthyridines 6 and N,N-dimethyl-1,4-
dihydro[b,h][1,6]dibenzonaphthyridium iodides 2
N
Me
Me
I
λ
=
2a-2e
Entry
R
6
Yield/%
2
Yield/%
Table 3. UV/vis and fluorescence spectral data for 2a–2e
1
2
3
4
5
H
6a
6b
68
2a
2b
2c
2d
2e
57
2
R
Stokes




shift (cm-1)
Me
62
69
2a
2b
2c
2d
2e
H
Me
OMe 422
Cl
Br
418
415
11800 581 6710
11500 572 6610
12300 577 6370
11200 587 6550
12000 577 6710
0.32
0.36
0.28
0.32
0.31
OMe 6c
69
42
Cl
Br
6d
6e
49
50
54
52
424
416
1
The structures of 6a and 2a were fully characterized by H NMR
and 13C NMR measurements and elemental analysis. The 1H
NMR spectrum of 6a shows signals for N-methyl (3.3 ppm) and
aromatic protons (6.7-8.7 ppm), and that of 2a shows N-methyl
(4.0 and 4.1 ppm) and aromatic signals (6.9–8.3 ppm).
Compound 2a-2e are reddish orange stable crystals and can be
stored on the shelf for more than 2 months. As can be seen in
Table 2, yields of 2a-2e were in the range of 42-69%, which
indicated that no substituent effects of R were observed.
We then examined the intercalation properties of 6a and 2a with
the expectation that these compounds would behave as an
ethidium bormide-like DNA staining reagent. As shown in Figure
3, a titration experiment revealed a decrease in the fluorescence
intensity of 6a with the addition of plasmid DNA, whereas another
titration experiment showed
a
gradual increase in the
fluorescence intensity of 2a as plasmid DNA concentration was
increased (Figure 4, also see Figure S-2). These results can be
explained by considering the ability of the nitrogen-containing
Because
the
product,
1,4-dihydro-1-
methyldibenzonaphthyridine 6a, produced strong flurorescence,
we further investigated the photophysical properties of 6a and 2a.
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10.1002/ejoc.201701277
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COMMUNICATION
410 nm. b) Excitation of 2a with black light in the absence (left) or
presence (right) of plasmid DNA.
planar ring to coordinate to DNA, because the structure of the ring
system is very similar to that of ethidium bromide. Interestingly,
such a positive effect of the intercalation of 2a on the fluorescence
is similar to that observed in ethidium bromide, whose
fluorescence is strongly enhanced by the intercalation into DNA.
The results indicate the successful development of a new turn-on-
type DNA staining reagent. The mechanism for fluorescent signal
induction is possibly due to the suppression of free rotation of the
aromatic fragment and the vibrational motions of molecule when
it is bound inside the pocket of the DNA substrate.
The similarity of the fluorescence properties of 2a and
ethidium bromide was visually confirmed when DNA fragments
electrophoresed on acrylamide gels were stained with those two
reagents. When 2a-stained gel was observed under UV
irradiation, DNA fragments were visible as bright bands against
the dark background of the gel (Figure 5). As typical DNA
intercalators, such as ethidium bromide and acridinium
derivatives, have planar aromatic rings, the interaction mode of
2a is predicted to be an intercalation. The fluorescence of 2a was
enhanced with increasing DNA concentration, as was observed
1000
in several intercalators.
Therefore, one of the possible
6a
mechanisms for this fluorescence enhancement is the energy
transfer between the compound and nucleic bases and/or the
immersion of ethidium bromide in a hydrophobic region.7
900
800
6a + DNA
700
600
500
400
300
200
100
0
420
470
520
570
wavelength (nm)
Figure 3. Titration experiment of 6a. Plasmid DNA (1.5 g/L) in Tris
buffer (0.01 mM, pH 7.6) was added to a solution of 6a (1 L) in Tris
buffer (0.01 mM, pH 7.6) containing 0.1% DMSO.
Figure 5. DNA staining of 2a.100 bp DNA ladder fragments
electrophoresed on 8% acrylamide gels were stained with ethidium
bromide (left) and 2a (right). Fluorescence was observed by
excitation with 254 nm UV light.
25
0 nM
1 nM
2 nM
3 nM
4 nM
5 nM
6 nM
7 nM
To confirm that the fluorescence was a result of intercalation, we
conducted a competition assay using ethidium bromide-stained
DNA.8 As shown in Figure 6, the fluorescence peak of ethidium
bromide was gradually decreased by adding compound 2a in
buffer solution.
20
15
10
5
8 nM
9 nM
10 nM
12 nM
14 nM
16 nM
18 nM
20 nM
22 nM
24 nM
11 nM
13 nM
15 nM
17 nM
19 nM
21 nM
23 nM
25 nM
7
0 nM
90 nM
47 nM
129 nM
198 nM
257 nM
308 nM
353 nM
392 nM
426 nM
457 nM
485 nM
165 nM
229 nM
284 nM
331 nM
373 nM
409 nM
442 nM
471 nM
498 nM
6
5
4
3
2
1
0
0
450
500
550
600
650
wavelength (nm)
N
N
430
480
530
wavelength (nm)
580
630
Me
Me
I
2a
Figure 6. Fluorescence changes for the titration of 2a with plasmid
DNA-ethidium bromide system ([2a] = 0–498 nM). Arrows show the
intensity change upon increasing 2a concentration.
Figure 4. a) Titration experiment of 2a with DNA. Plasmid DNA (1.5
g/L) in Tris buffer (0.01 mM, pH 7.6) was added to a solution of 2a
(1 L) in Tris buffer (0.01 mM, pH 7.6) containing 0.1% DMSO. ex
=
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10.1002/ejoc.201701277
European Journal of Organic Chemistry
COMMUNICATION
We
naphthyridines
acetylaminobenzaldehyde
basic conditions. 1,1-Dimethyl-1,6-dibenzonaphthyridinium
iodides were easily synthesized by treatment of with
methyl iodide in refluxing acetone, and showed significant
fluorescence when treated with double-stranded DNA.
have
synthesized
in one pot by reacting 2-
with acetophenone under
1,4-dihydrodibenzo[b,h][1,6]-
5
3
4
2
5
A
new turn-on type fluorescence reagents was developed.
Further studies of the novel features of these reagents are in
progress.
Keywords: Dibenzonaphthyridine • 2-acetylaminobenzaldehyde
• acetophenone • DNA • intercalation
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Author(s), Corresponding Author(s)*
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Title
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R
R
K. Okuma,* A. Oba, R. Kuramoto, H.
Iwashita, N. Nagahora, K. Shioji, R.
Noguchi, M. Fukuda
R
DNA
H
Intercalation
One-pot
O
MeI
NaOH
N
N
2
+
NHAc
N
N
Page No. – Page No.
H3C
H
O
Me Me
I
The reaction of 2-acetylaminobenzaldehyde with acetophenones under basic
conditions gave dihydronaphthyridines in good yields. The reaction of the
dihydronaphthyridines with methyl iodide gave dimethylammonium iodides.
Treatment of ammonium iodides with plasmid DNA produced bright fluorescence
at 420 nm.
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