Synthesis, crystal structures, and spectral properties of double N-alkylated dimethine cyanine dyes and their interactions with biomolecules and living cells

Article abstract of DOI:10.1039/c4ra10741a

Six new double N-alkylated dimethine cyanine dyes were synthesized and the crystal structures of two of the dyes were analyzed by X-ray diffraction. The investigation of the spectral properties of the dyes in different solvents indicated that the absorption maxima of the dyes decreased with the increase of the basicity of the heterocyclic nucleus, and the increase of the solvent dielectric constants of the protonic solvents and non-protonic solvents. The six dyes all emitted fluorescence, and had a larger Stokes shift in water. The interaction between the dye molecules and the six biological molecules showed that one dye exhibited a larger enhancement of fluorescent quantum yield in the presence of DNA. Investigation of the cytotoxicity and cell staining of the selected dye showed that the dye had virtually no toxicity at the application dose and duration used and that it could stain cytoplasm, suggesting its potential application as a fluorescent reagent with which to observe and analyze the characteristics of living cells.

Full text of DOI:10.1039/c4ra10741a

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PAPER
Synthesis, crystal structures, and spectral
properties of double N-alkylated dimethine cyanine
dyes and their interactions with biomolecules and
living cells†
Cite this: RSC Adv., 2015, 5, 4681
a
a
a
b
a
*
Hongliang Jia, Ying Lv, Shu Wang, Dan Sun and Lanying Wang
Six new double N-alkylated dimethine cyanine dyes were synthesized and the crystal structures of two of
the dyes were analyzed by X-ray diffraction. The investigation of the spectral properties of the dyes in
different solvents indicated that the absorption maxima of the dyes decreased with the increase of the
basicity of the heterocyclic nucleus, and the increase of the solvent dielectric constants of the protonic
solvents and non-protonic solvents. The six dyes all emitted fluorescence, and had a larger Stokes shift
in water. The interaction between the dye molecules and the six biological molecules showed that one
dye exhibited a larger enhancement of fluorescent quantum yield in the presence of DNA. Investigation
of the cytotoxicity and cell staining of the selected dye showed that the dye had virtually no toxicity at
the application dose and duration used and that it could stain cytoplasm, suggesting its potential
application as a fluorescent reagent with which to observe and analyze the characteristics of living cells.
Received 19th September 2014
Accepted 27th November 2014
DOI: 10.1039/c4ra10741a
www.rsc.org/advances
cumbersome, and the yields are usually low.21 Compared with
monomethine cyanine dyes, dimethine cyanine dyes, which are
1. Introduction
prepared from a heterocyclic quaternary salt having an active
methyl group via a condensation reaction with an aromatic
heterocyclic aldehyde, are easily synthesized and puried with
high yields and are produced by an environmentally friendly
pathway. More importantly, the dimethine cyanine dyes have
similar properties to monomethine cyanine dyes, such as good
spectral properties and affinity for biological molecules.11,22,23
Therefore, research on the synthesis and application of dime-
thine cyanine dyes is of great interest and importance. Wang
and Chang24 reported on the combinatorial synthesis of benzi-
midazolium dyes, studied their interaction with nucleotides,
and discovered the rst turn-on uorescent guanosine
triphosphate sensor. In addition, the research team synthesized
a type of dimethine cyanine dye,25 which had low cell cytotox-
icity, low phototoxicity, high photostability, and found that it
was suitable for detecting an in vitro RNA response and for live
cell nuclear imaging. Abd El-Aal et al.26 synthesized b-
substituted dimethine cyanine dyes, and discussed the rela-
tionship between the properties and structures of the dyes.
Deligeorgiev et al.27 described three new general procedures for
the synthesis of dimethine cyanine dyes using uncatalysed
Knoevenagel condensation, which had several virtues, such as
high yields, highly pure nal dyes, short reaction time and an
environmentally friendly method, and investigated the spectral
properties of the novel dyes. In previous work,23,28 we synthe-
sized a series of dimethine cyanine dyes and investigated, both
experimentally and theoretically, the absorption and
Cyanine dyes, a class of synthetic functional dyes with two
heteroaromatic rings joined by a methine chain, are widely used
in materials,1–4 biomedicine5–8 and other elds9 because of their
high extinction coefficients, tunable absorption/emission
spectra, and a moderate-to-high uorescence quantum yield.
Monomethine cyanine dyes are important in the family of
cyanine dyes, and have also been used in the biomedical eld
because of their high affinity towards certain biological mole-
cules,10 such as in nucleic acid detection,11–13 as a protein-based
uoromodule,14 for DNA sequencing15–17 and cell imaging18,19
and so on. Monomethine cyanine dyes are usually prepared by
the condensation reaction of a heterocyclic quaternary salt
bearing an active methyl group, with another quaternary salt of
2- or 4-alkylthio heterocyclic compounds,20 and the reaction is
accompanied by the formation of a thiol molecule. This will
pollute the environment and does not comply with the concept
of green chemistry. In addition, the processes of separation and
purication of the products are relatively complex and
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of
Education, College of Chemistry and Materials Science, Northwest University, Xi'an
710069, P. R. China. E-mail: wanglany@nwu.edu.cn; Fax: +86 29 81535026; Tel:
+86 29 81535026
bInstitute of Photonics & Photon-Technology, Northwest University, Xi'an 710069, P. R.
China
† Electronic supplementary information (ESI) available. CCDC 851574 and
822789. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c4ra10741a
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uorescence properties of these dyes. They were also studied as
uorescent dyes for DNA or bovine serum albumin (BSA)
detection, and it was found that uorescent intensity was
increased when the dyes were bound to DNA. Recently, the
synthesis of a series of dimethine cyanine dyes was reported
and their properties as uorescent probes for living cell imaging
and ow cytometry were investigated. The results showed that
some dyes could penetrate an intact cell membrane and stain
the cell nucleus, and they could also be applied in ow cytom-
etry.29 However, from the research mentioned previously it was
found that the reported dimethine cyanine dyes, with hetero-
cyclic nitrogen links with hydrogen, are susceptible to the
external environment30 and have a small Stokes shi.
In this paper, the synthesis of a series of new double N-
alkylated dimethine cyanine dyes including N-methylation and
N-benzylation (Scheme 1), which would have a large Stokes shi
and resistance to environmental interference as well as good
stability, was described. Their structures were identied using
ultraviolet-visible (UV-vis) spectroscopy, proton nuclear
magnetic resonance (1H-NMR) spectroscopy, infrared spec-
troscopy (IR) and high-resolution mass spectroscopy (HRMS)
and the structures of dyes C2 and C6 were characterized and
analyzed by X-ray diffraction (XRD). The absorption and uo-
rescent spectra of these dyes in different solvents and their
interaction with six biological molecules was investigated, as
well as their cytotoxicity and cell staining of the selected dye C3
for their prospective uses as uorescent reagents in the
biomedical eld.
2. Experimental
2.1 General
Commercially available reagents were used without additional
purication. All solvents were of analytical grade. Melting
points were measured using a XT-4 micromelting apparatus and
were used uncorrected. IR spectra (cmꢀ1) were recorded on a
Bruker Equinox 55 spectrometer. The absorption spectra were
recorded on a Purkinje General TU-1900 UV-vis spectrometer.
1H-NMR spectra were recorded at 400 MHz on a Varian Inova
400 spectrometer and chemical shis were reported relative to
the internal standard, tetramethylsilane. HRMS was recorded
on a Bruker micrOTOF-Q II electrospray ionisation – quadru-
pole time-of-ight (ESI-QTOF) liquid chromatography-mass
spectroscopy (LC-MS) spectrometer. High performance liquid
chromatography (HPLC) was performed using a Shimadzu LC-
20AD. Single crystal XRD data were collected using a Bruker
SMART APEX II charge coupled device (CCD) detector for X-ray
crystallography. Fluorescence imaging was carried out on a
Nikon A1 laser scanning confocal microscope. The water
soluble tetrazolium (WST) assay was conducted using a BioTek
ELx 800 microplate reader. Cell lines were cultured in a Thermo
Scientic Forma 3111 water-jacketed carbon dioxide incubator.
2.2 Preparation of the intermediates A1–A6 and B1
2.2.1 Preparation of the intermediates A1–A6. 2,3-Dimethyl
benzothiazolium iodide salt (A1), 1,2,3-trimethyl benzimidazo-
lium iodide salt (A2), 7-chloro-1,2-dimethyl quinolinium iodide
Scheme 1 Synthesis of dyes C1–C6.
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salt (A4), and 1,2-dimethyl quinolinium iodide salt (A5) were MHz, DMSO-d6) d (ppm): 4.15 (s, 6H, NCH3, N+CH3), 5.57 (s,
synthesized according to methods in the literature.29 1,4- 2H, NCH2Ph), 7.18 (d, 1H, J ¼ 16.0 Hz, CH]CH), 7.30–7.37 (m,
Dimethyl quinolinium iodide salt (A3),31 and 1,2,3,3- 7H, ArH), 7.64–7.68 (m, 3H, ArH), 8.04 (d, 1H, J ¼ 16.0 Hz,
tetramethyl-4,5-benzindolium iodide salt (A6)32 were also CH]CH), 8.00–8.06 (m, 2H, ArH), 8.21–8.23 (m, 1H, ArH),
prepared according to methods in the literature.
8.33 (s, 1H, pyrrole-H). IR (KBr) n: 3016 (m, n]C–H), 2931 (m,
2.2.2 Preparation of the intermediate B1. N-Benzyl-3-indole nC–H), 1598, 1502, 1457 (s, nC]C, nC]N), 1349, 1186 (m, dC–H
)
formaldehyde (B1) was prepared according to a modied 964, 738 (m, d]C–H) cmꢀ1. HRMS (TOF-MS ESI) calculated for
procedure from the literature.33 Indole-3-carboxaldehyde (0.14
C
26H24N3+: 378.1965; found: 378.1941.
g, 1.0 mmol) was dissolved in 10.0 mL of N,N-dimethylforma-
1-Methyl-4-[1-benzyl-3-indole-(E)-ethenyl]quinolinium
iodide
mide (DMF) at room temperature, and 40% of sodium hydride salt (C3). UV-vis (MeOH) lmax: 503.0 nm. 1H-NMR (400 MHz,
(0.06 g, 1.0 mmol) was added with stirring at 0–5 ꢁC for 0.5–1 h, DMSO-d6) d (ppm): 4.46 (s, 3H, N+CH3), 5.59 (s, 2H, NCH2Ph),
then the benzyl chloride (0.25 g, 2.0 mmol) was added. The 7.31–7.37 (m, 7H, ArH), 7.65–7.67 (m, 1H, ArH), 8.01 (d, 1H,
resulting mixture was stirred at room temperature for 24 h, then J ¼ 8.0 Hz, ArH), 8.04 (d, 1H, J ¼ 16.0 Hz, CH]CH), 8.21–8.24
ltered and the DMF was evaporated off. The product was (m, 1H, ArH), 8.28–8.30 (m, 1H, ArH), 8.35 (d, 1H, J ¼ 8.0 Hz,
recrystallized from ethanol (EtOH) to give B1 (0.21 g, 87.5%) as ArH), 8.46 (d, 1H, J ¼ 8.0 Hz, ArH), 8.51 (s, 1H, pyrrole-H), 8.55
ꢁ
faint yellow crystals, melting point (mp) 106–107 C (literature (d, 1H, J ¼ 16.0 Hz, CH]CH), 8.96 (d, 1H, J ¼ 8.0 Hz, ArH), 9.15
mp 107 ꢁC).
(d, 1H, J ¼ 8.0 Hz, ArH). IR (KBr) n: 3058 (m, n]C–H), 2921 (w,
nC–H), 1556, 1515, 1482 (m, nC]C, nC]N), 1394, 1295, 1157 (m,
dC–H), 964, 738 (m, d]C–H) cmꢀ1. HRMS (TOF-MS ESI) calculated
2.3 Synthesis of double N-alkylated dimethine cyanine dyes
(C1–C6)
+
for C27H23N2 : 375.1856; found: 375.1840.
7-Chloro-1-methyl-2-[1-benzyl-3-indole-(E)-ethenyl]quinolinium
iodide salt (C4). UV-vis (MeOH) lmax: 491.0 nm. 1H-NMR
(400 MHz, DMSO-d6) d (ppm): 4.44 (s, 3H, N+CH3), 5.61 (s, 2H,
NCH2Ph), 7.33–7.40 (m, 7H, ArH), 7.56 (d, 1H, J ¼ 16.0 Hz,
CH]CH), 7.68–7.69 (m, 1H, ArH), 7.90 (d, 1H, J ¼ 8.0 Hz, ArH),
8.26 (d, 2H, J ¼ 8.0 Hz, ArH), 8.60–8.64 (m, 3H, ArH, pyrrole-H),
8.66 (d, 1H, J ¼ 16.0 Hz, CH]CH), 8.81 (d, 1H, J ¼ 8.0 Hz, ArH).
IR (KBr) n: 3002 (w, n]C–H), 2921 (w, nC–H), 1581, 1506, 1457 (s,
nC]C, nC]N), 1398, 1346, 1299, 1164 (m, dC–H), 956, 844, 740 (m,
d]C–H) cmꢀ1. HRMS (TOF-MS ESI) calculated for C27H22N2Cl+:
409.1466; found: 409.1448.
2.3.1 Preparation of dyes C1–C6. Dimethine cyanine dyes
(C1–C6) were prepared as shown in Scheme 1. Equimolar
amounts of heterocycle intermediates A1–A6 (1.0 mmol) and B1
(1.0 mmol) were dissolved in anhydrous EtOH. A few drops of
piperidine were added and the reaction mixture was stirred
under reux for 3–6 h. The progress of the reaction was moni-
tored using UV-vis spectroscopy. Aer cooling, the resulting
precipitate was ltered off and recrystallized from methanol
(MeOH) (C2) or EtOH (C1, C3–C6). The reaction details and
yields of dyes C1–C6 are listed in Table 1.
2.3.2 Structural conrmation
1-Methyl-2-[1-benzyl-3-indole-(E)-ethenyl]quinolinium
iodide
salt (C5). UV-vis (MeOH) lmax: 479.0 nm. 1H-NMR (400 MHz,
DMSO-d6) d (ppm): 4.49 (s, 3H, N+CH3), 5.61 (s, 2H, NCH2Ph),
7.33–7.38 (m, 7H, ArH), 7.59 (d, 1H, J ¼ 16.0 Hz, CH]CH),
7.67–7.69 (m, 1H, ArH), 7.84–7.88 (m, 1H, ArH), 8.08–8.12 (m,
1H, ArH), 8.25–8.27 (m, 2H, ArH), 8.46 (d, 1H, J ¼ 8.0 Hz, ArH),
8.56 (s, 1H, pyrrole-H), 8.62–8.66 (m, 2H, ArH, CH]CH), 8.84
(d, 1H, J ¼ 8.0 Hz, ArH). IR (KBr) n: 3064, 3019 (m, n]C–H), 2925
(m, nC–H), 1585, 1511, 1455 (s, nC]C, nC]N), 1347, 1299, 1157,
1106 (m, dC–H), 958, 838, 750 (m, d]C–H) cmꢀ1. HRMS (TOF-MS
ESI) calculated for C27H23N2+: 375.1856; found: 375.1846.
1,3,3-Trimethyl-2-[1-benzyl-3-indole-(E)-ethenyl]-4,5-benzindolium
iodide salt (C6). UV-vis (MeOH) lmax: 489.0 nm. 1H-NMR (400
MHz, DMSO-d6) d (ppm): 2.05 (s, 6H, C(CH3)2), 4.16 (s, 3H,
1-Methyl-2-[1-benzyl-3-indole-(E)-ethenyl]benzothiazolium
iodide salt (C1). UV-vis (MeOH) lmax: 471.0 nm. 1H-NMR
(400 MHz, deuterated dimethyl sulfoxide (DMSO-d6)) d
(ppm): 4.28 (s, 3H, N+CH3), 5.60 (s, 2H, NCH2Ph), 7.33–7.38
(m, 7H, ArH), 7.52 (d, 1H, J ¼ 15.6 Hz, CH]CH), 7.68–7.72
(m, 2H, ArH), 7.79–7.82 (m, 1H, ArH), 8.14 (d, 1H, J ¼ 8.0
Hz, ArH), 8.29–8.34 (m, 2H, ArH), 8.42 (d, 1H, J ¼ 15.6 Hz,
CH]CH), 8.58 (s, 1H, pyrrole-H). IR (KBr) n: 3045, 3009 (m,
n]C–H), 2927 (m, nC–H), 1536, 1448 (s, nC]C, nC]N), 1399,
1366, 1343, 1220, 1107 (m, dC–H), 944, 837, 747, 719 (m,
d]C–H
25H21N2S+: 381.1420; found: 381.1397.
1,3-Dimethyl-2-[1-benzyl-3-indole-(E)-ethenyl]benzimidazolium
)
cmꢀ1
.
HRMS (TOF-MS ESI) calculated for
C
1
iodide salt (C2). UV-vis (MeOH) lmax: 376.0 nm. H-NMR (400
Table 1 The molecular formulas, reaction times, melting points, yields and appearance of C1–C6
Dye
Formula
Reaction time (h)
mp (ꢁC)
Yield (%)
Appearance
C1
C2
C3
C4
C5
C6
C25H21N2SI
4.0
6.0
3.5
4.0
3.5
5.0
261–262
>300
245–246
236–237
269–270
207–208
59
77
80
50
84
83
Orange solid
Yellow acicular crystals
Red solid
Dark red power
Red solid
Red acicular crystals
C
26H24N3I
C27H23N2I
27H22N2ICl
C27H23N2I
32H29N2I
C
C
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N+CH3), 5.66 (s, 2H, NCH2Ph), 7.26 (d, 1H, J ¼ 16.0 Hz, CH]CH), cell and the slit width of excitation and emission spectra were
7.35–7.44 (m, 7H, ArH), 7.65 (d, 1H, J ¼ 8.0 Hz, ArH), 7.77 (d, 2H, J 1 cm ꢂ 1 cm ꢂ 4 cm and 5.0 nm, respectively. The absorption
¼ 8.0 Hz, ArH), 8.05 (d, 1H, J ¼ 8.8 Hz, ArH), 8.18 (d, 1H, J ¼ 8.0 and uorescence spectral data are listed in Table 6.
Hz, ArH), 8.24 (d, 1H, J ¼ 8.8 Hz, ArH), 8.33 (d, 1H, J ¼ 8.0 Hz,
2.5.2 Measurements of the spectral properties of dyes in
ArH), 8.40 (d, 1H, J ¼ 8.0 Hz, ArH), 8.76 (d, 1H, J ¼ 16.0 Hz, the presence of biomolecules. The dye stock solutions with a
CH]CH), 8.84 (s, 1H, pyrrole-H). IR (KBr) n: 3091, 3033 (m, n]C–H), concentration of 5 ꢂ 10ꢀ3 mol Lꢀ1 were prepared by dissolving
2995 (m, nC–H), 1563, 1498, 1457 (m, nC]C, nC]N), 1394, 1347, the dyes in DMSO and were then further diluted with TE buffer
1249, 1205, 1160, 1118 (m, dC–H), 939, 792, 744 (m, d]C–H) cmꢀ1
.
consisting of 10 mmol Lꢀ1 Tris–HCl, 1 mmol Lꢀ1 EDTA at pH
+
HRMS (TOF-MS ESI) calculated for C32H29N2 : 441.2325; found: 7.5. The biomolecule stock solutions (200 mg mLꢀ1) were
441.2307.
prepared by dissolving biomolecules (DNA, BSA, muramidase,
amylase, bovine hemoglobin or chymotrypsin) in TE buffer.
Working solutions of complexed biomolecule–dye were prepared
by mixing dye stock solutions and biomolecule stock solutions,
and further diluting them with TE buffer. The working concen-
trations of the dye solutions were 1 ꢂ 10ꢀ5 mol Lꢀ1 and 5 ꢂ 10ꢀ5
mol Lꢀ1 for dyes C1 and C2, and 1 ꢂ 10ꢀ5 mol Lꢀ1 and 3 ꢂ 10ꢀ5
mol Lꢀ1 for dyes C3, C4 and C5, and 1 ꢂ 10ꢀ5 mol Lꢀ1 and
5 ꢂ 10ꢀ6 mol Lꢀ1 for dye C6. The biomolecule concentrations in
working solutions were 0, 4 and 10 mg mLꢀ1. All working solu-
tions were prepared immediately before use. The measurement
method was the same as that described previously. The results
are shown in Table 7.
2.5.3 Preparation of dye–DNA solutions. The dye stock
solution (1 ꢂ 10ꢀ4 mol Lꢀ1) was prepared by dissolving dye C1
in DMSO and diluting it further with TE buffer. The stock
solution of DNA with a concentration of 200 mg mLꢀ1 was
prepared by dissolving DNA in TE buffer. Working solutions of
complexed DNA–dye were prepared by mixing an aliquot of the
dye stock solution with an aliquot of the DNA stock solutions,
and diluting it further with TE buffer to obtain the required
concentrations (DNA: 0, 2, 4, 6, 8, 10 and 12 mg mLꢀ1, dye C1:
1 ꢂ 10ꢀ5 mol Lꢀ1).
2.4 Crystal data
Crystals suitable for X-ray analysis were obtained by the slow
evaporation of a solution of the dyes in MeOH (C2) or EtOH
(C6). All the measurements of the crystals were carried out on
a Bruker SMART APEX II (CCD) detector for crystallography,
equipped with graphite monochromated Mo Ka radiation
(l ¼ 0.71073 nm), by using the u scan technique at room
temperature. The structures were solved by direct methods
using SHELXS-97 soware,34 and rened using the full-matrix
least-squares method on F2 with anisotropic thermal
parameters for all non-hydrogen atoms using SHELXL-97.35
Hydrogen atoms were generated geometrically. Fig. 1 and 2
show the molecular structures with numbering systems of
dyes C2 and C6, respectively. The crystal data, details con-
cerning data collection and structure renement for dyes C2
and C6 are summarized in Table 2. The bond lengths, bond
angles and torsion angles for dyes C2 and C6 are listed in
Tables 3 and 4, respectively. The geometry of D–H/A
hydrogen bonds for dyes C2 are listed in Table 5. Parameters
in crystallographic information framework (CIF) format are
available as ESI.†
2.6 Determination of cytotoxicity and cell staining
2.5 Measurements of the spectral properties of dyes
2.6.1 Cytotoxicity of dye C3. The WST assay was performed
to evaluate the toxicity of dye C3 to human glioma (A172) cells.
The cells were seeded into 96-well plates at a cell density of 5 ꢂ
103 cells per well in 180 mL of culture medium and maintained
for 24 h. Then a solution of dye C3 with different concentrations
2.5.1 Measurements of the spectral properties of dyes in
different solvents. The dye stock solutions (1 ꢂ 10ꢀ2 mol Lꢀ1 in
dimethyl sulfoxide (DMSO)) were diluted with different solvents
and resulted in working solutions of dyes (1 ꢂ 10ꢀ5 mol Lꢀ1).
The absorption and uorescence emission spectra of these
solutions were scanned at room temperature. The size of quartz
Fig. 1 The molecular structure diagram of dye C2.
4684 | RSC Adv., 2015, 5, 4681–4692
Fig. 2 The molecular structure diagram of dye C6.
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Table 2 Crystal data and structure refinement for dyes C2 and C6
Dyes
C2
C6
Empirical formula
Formula weight
Temperature (K)
Wavelength (A)
Crystal system
Space group
C
26H24IN3
C33H31IN2O
598.50
296(2)
0.71073
Monoclinic
P2(1)/n
505.38
298(2)
0.71073
Monoclinic
Pc
ꢀ
Unit cell dimensions
ꢀ
a (A)
9.676(2)
5.8938(13)
19.693(4)
12.3860(14)
10.3493(13)
22.395(3)
ꢀ
b (A)
ꢀ
c (A)
a (deg)
b (deg)
g (deg)
90
94.577(3)
90
1119.5(4)
90
100.695(2)
90
2820.9(6)
ꢀ3
Volume (A )
Z
2
4
Calculated density (mg mꢀ3
)
1.499
1.448
508
1.409
1.163
1216
Absorption coefficient (mmꢀ1
F(000)
)
Crystal size (mm3)
0.35 ꢂ 0.27 ꢂ 0.17
0.32 ꢂ 0.26 ꢂ 0.13
1.85–25.10
ꢀ14/14, ꢀ12/8, ꢀ26/24
q range for data collection
Limiting indices h, k, l
Renement method
Reections collected/unique/Rint
Completeness to q ¼25.10ꢁ
Data/restraints/parameters
Goodness-of-t on F2
2.07–25.10
ꢀ11/11, ꢀ3/7, ꢀ22/23
Full-matrix least-squares on F2
5256/3649/0.0298
99.8%
3649/2/273
1.120
13 504/4921/0.0343
97.9%
4921/6/328
1.043
Final R indices [I > 2s(I)]
R1
wR2
0.0417
0.1194
0.0521
0.1346
R indices (all data)
R1
0.0455
0.0785
wR2
0.1336
0.666 and ꢀ0.842
0.1477
1.066 and ꢀ0.827
ꢀꢀ3
Largest difference peak/hole (e A
)
was added to 96-well plates and incubated for another 12 h. 3-indole formaldehyde and quaternary ammonium salts having
Following this, the medium was removed and washed three an active methyl group, with high yields of 59–84%, in short
times with phosphate buffered saline (PBS). Finally, 100 mL of reaction times, and using an environmentally friendly method.
fresh culture medium and 10 mL of WST-1 reagent were added to The products were easily puried by recrystallization from EtOH
each well. Aer incubation for 1 h, cell viability was measured or MeOH. In the synthetic process, it was found that the
using the WST assay according to the manufacturer's suggested sequence of the reaction activity of various heterocyclic
procedure. All the experiments were conducted in triplicate. quaternary salts was benzimidazole quaternary salt < benzin-
Data are expressed as mean ꢃ standard deviation.
dole quaternary salt < benzothiazole quaternary salt < quinoline
2.6.2 Fluorescence imaging. A172 cells were seeded into 24- quaternary salt. This was because the reactivity of hydrogen in
well plates at a density of 1 ꢂ 104 cells per well and incubated for the active methyl groups differed from each other. The higher
12 h, and then dye C3 was added. Aer further incubation for 10 the reactivity of hydrogen, the easier it was to form a carbanion
min, the culture medium was washed three times with PBS. The from the active methyl under basic conditions, and the easier
uorescence images were acquired using confocal laser scanning the nucleophilic addition–elimination reaction of the quater-
microscopy with an argon laser at an excitation wavelength of 488 nary salts and N-benzyl-3-indole formaldehyde was. However,
nm and an emission wavelength of 570–620 nm.
the reactivity of hydrogen in an active methyl group was affected
by the positive charge density of 2- or 4-carbon in a heterocyclic
quaternary salts: the larger the positive charge density of the 2-
or 4-carbon, the higher the reactivity of hydrogen. The electron-
withdrawing ability of the group linked with a 2- or 4-carbon was
–N(CH3)– < –C(CH3)2– < –S– < –CH]CH–; therefore the positive
charge density of the 2- or 4-carbon was benzimidazole
3. Results and discussion
3.1 Synthesis
Six double N-alkylated dimethine cyanine dyes were synthesized
via the nucleophilic addition–elimination reaction of N-benzyl-
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ꢁ
ꢀ
Table 3 Selected bond lengths (A), bond angles and torsion angles ( ) Table 5 The geometry of D–H/A hydrogen bond of dye C2
for dye C2
d(D–H)
(A)
d(H/A)
(A)
d(D/A)
(A)
:(DHA)
(ꢁ)
ꢀ
ꢀ
ꢀ
ꢀ
Bond lengths (A)
D–H/A
N(1)–C(7)
N(1)–C(1)
N(1)–C(25)
N(2)–C(7)
N(2)–C(6)
N(2)–C(26)
N(3)–C(17)
N(3)–C(16)
1.355(9)
1.384(8)
1.460(10) C(8)–C(9)
1.351(9)
1.407(8)
1.462(9)
1.345(8)
1.371(9)
N(3)–C(18)
C(7)–C(8)
1.473(9)
1.455(8)
1.333(9)
1.419(9)
1.400(10)
1.461(9)
1.431(10)
1.522(11)
C(17)–H(17)/I(1)
0.93
2.88
3.798(7)
168
C(9)–C(10)
C(10)–C(17)
C(10)–C(11)
C(11)–C(16)
C(18)–C(19)
was benzimidazole quaternary salt < benzindole quaternary salt
< benzothiazole quaternary salt < quinoline quaternary salt.
3.2 Characterization
Bond angles (ꢁ)
C(7)–N(1)–C(1)
C(7)–N(1)–C(25)
C(1)–N(1)–C(25)
C(7)–N(2)–C(6)
C(7)–N(2)–C(26)
C(6)–N(2)–C(26)
C(17)–N(3)–C(16)
C(17)–N(3)–C(18)
C(16)–N(3)–C(18)
C(6)–C(1)–N(1)
C(1)–C(6)–N(2)
C(5)–C(6)–N(2)
108.7(5)
127.9(6)
123.3(6)
108.9(5)
127.9(5)
123.2(5)
110.1(5)
124.1(6)
125.8(6)
108.1(5)
106.2(5)
131.7(6)
N(2)–C(7)–N(1)
N(2)–C(7)–C(8)
N(1)–C(7)–C(8)
108.1(5)
130.0(6)
121.8(6)
127.9(6)
123.7(6)
127.5(6)
106.1(6)
126.4(7)
105.2(6)
108.4(5)
110.1(6)
114.2(7)
The purity of the prepared dyes C1–C6 was established using
HPLC and HPLC data obtained at particular wavelengths are
presented in the ESI Fig. S1.† For each compound, the detection
wavelength was set to 254 nm, 370 nm and its lmax. As can be
seen from the ESI,† the purity of the prepared dyes C1–C6 is
mostly above 99%. The structures of the six dyes were conrmed
from UV-vis absorption spectra, IR, 1H-NMR and HRMS. The IR
spectra of the six dyes showed typical aromatic absorption
(n]C–H, 3091–3002 cmꢀ1, nC]C, 1598–1536, d]C–H, 964–719
cmꢀ1) and resonance conjugated unsaturated stretching modes
(nC]N, nC]C, 1515–1448 cmꢀ1). Each dye also had a moderate
absorption peak in the range of 964–939 cmꢀ1, which belonged
to the trans-RCH]CRH chain. It indicated that these dyes had a
C(9)–C(8)–C(7)
C(8)–C(9)–C(10)
C(17)–C(10)–C(9)
C(17)–C(10)–C(11)
C(9)–C(10)–C(11)
C(16)–C(11)–C(10)
N(3)–C(16)–C(11)
N(3)–C(17)–C(10)
N(3)–C(18)–C(19)
Torsion angles (ꢁ)
N(1)–C(1)–C(6)–N(2)
C(6)–N(2)–C(7)–C(8)
0.0(6)
179.5(6)
C(10)–C(11)–C(16)–N(3) 0.0(6)
C(9)–C(10)–C(17)–N(3)
ꢀ178.1(6)
1
trans-spatial conguration.36 As can be seen from the H-NMR
C(7)–C(8)–C(9)–C(10) 178.8(6)
spectra data, the coupling constants (3JHH) of hydrogen of the
CH]CH chain were between 15.6–16.8 Hz, which further
conrmed that these dye molecules had a trans-conguration.
1H-NMR spectra of dyes C1–C6 showed the chemical shi of H
in N+CH3 (5.57–5.66 ppm), NCH2 (4.15–4.49 ppm), CH]CH
(7.16–8.04 ppm) and an aromatic heterocycle (7.30–9.15 ppm).
The resonance absorption corresponding to C(CH3)2 (2.05 ppm)
quaternary salt < benzindole quaternary salt < benzothiazole
quaternary salt < quinoline quaternary salt, and the sequence of
the reaction activity of the various heterocyclic quaternary salts
ꢁ
ꢀ
Table 4 Selected bond lengths (A), bond angles and torsion angles ( ) for dye C6
ꢀ
Bond lengths (A)
N(1)–C(15)
N(1)–C(8)
N(1)–C(7)
N(2)–C(18)
N(2)–C(19)
N(2)–C(32)
C(8)–C(13)
C(13)–C(14)
C(14)–C(15)
1.332(5)
1.398(5)
1.472(5)
1.315(5)
1.411(5)
1.465(5)
1.407(5)
1.456(5)
1.384(6)
C(14)–C(16)
C(16)–C(17)
C(17)–C(18)
C(18)–C(29)
C(19)–C(28)
C(28)–C(29)
C(29)–C(30)
C(29)–C(31)
1.405(5)
1.367(6)
1.408(5)
1.534(5)
1.363(5)
1.513(5)
1.537(6)
1.541(6)
Bond angles (ꢁ)
C(15)–N(1)–C(8)
C(18)–N(2)–C(19)
N(1)–C(8)–C(13)
C(8)–C(13)–C(14)
C(15)–C(14)–C(16)
C(15)–C(14)–C(13)
C(16)–C(14)–C(13)
N(1)–C(15)–C(14)
108.8(3)
111.7(3)
107.7(3)
106.5(3)
122.9(4)
105.3(3)
131.7(4)
111.6(3)
C(17)–C(16)–C(14)
C(16)–C(17)–C(18)
N(2)–C(18)–C(17)
N(2)–C(18)–C(29)
C(17)–C(18)–C(29)
C(28)–C(19)–N(2)
C(19)–C(28)–C(29)
C(28)–C(29)–C(18)
127.3(4)
126.3(4)
122.7(4)
109.3(3)
128.0(4)
109.1(3)
109.2(3)
100.6(3)
Torsion angles (ꢁ)
C(15)–N(1)–C(8)–C(13)
C(16)–C(14)–C(15)–N(1)
C(14)–C(16)–C(17)–C(18)
ꢀ1.0(4)
C(16)–C(17)–C(18)–N(2)
C(18)–N(2)–C(19)–C(28)
ꢀ179.7(4)
ꢀ2.5(4)
176.1(4)
ꢀ179.4(4)
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Table 6 The absorption and fluorescence spectral characteristics of dyes C1–C6 in different solvents
Solvents
H2O
Dyes
lmax (nm)
lex (nm)
lem (nm)
Stokes shi (nm)
3 ꢂ 10ꢀ4 (L molꢀ1 cmꢀ1
)
Fa
C1
C2
C3
C4
C5
C6
C1
C2
C3
C4
C5
C6
C1
C2
C3
C4
C5
C6
C1
C2
C3
C4
C5
C6
C1
C2
C3
C4
C5
C6
455.0
368.0
473.0
468.0
458.0
483.0
471.0
378.0
503.0
492.0
481.0
491.0
471.0
376.0
503.0
491.0
479.0
489.0
475.0
380.0
509.0
494.0
483.0
493.0
496.0
384.0
513.0
515.0
503.0
522.0
455.0
368.0
473.0
468.0
458.0
483.0
471.0
378.0
503.0
492.0
481.0
491.0
471.0
376.0
503.0
491.0
479.0
489.0
475.0
380.0
509.0
494.0
483.0
493.0
496.0
384.0
513.0
515.0
503.0
522.0
525.0
468.2
574.0
561.6
554.2
560.0
534.8
469.8
596.6
570.4
565.0
560.8
536.8
471.2
581.6
564.2
558.4
558.4
534.4
460.6
582.4
563.2
561.4
563.2
555.8
471.4
570.0
577.2
568.0
573.4
70.0
100.2
101.0
93.6
96.2
77.0
63.8
91.8
93.6
78.4
84.0
69.8
65.8
95.2
78.6
73.2
79.4
69.4
59.4
80.6
73.4
69.2
78.4
71.2
59.8
87.4
57.0
62.2
65.0
51.4
1.98
0.89
1.91
2.80
3.52
3.24
3.17
0.87
2.09
2.98
3.21
3.26
3.30
0.97
2.28
3.25
4.13
3.53
3.35
0.98
2.33
3.66
3.31
3.44
3.27
0.78
2.82
3.29
2.16
2.34
0.0676
0.0042
0.0332
0.0157
0.0264
0.0843
0.5705
0.0115
0.4572
0.1206
0.2103
0.2758
0.1643
0.0043
0.1507
0.0501
0.1261
0.0765
0.2309
0.0040
0.3614
0.1496
0.2644
0.1120
0.1672
0.0024
0.2573
0.1367
0.1669
0.0368
DMSO
MeOH
EtOH
CHCl3
a
The uorescence quantum yields of the dyes were determined using the reference standard (rhodamine B FF ¼ 0.56 in EtOH at 25 ꢁC).40
was also found in dye C6. The HRMS data of dyes C1–C6 showed respectively, which showed that the indole ring, vinyl chains and
that the relative molecular mass of the six dyes was consistent benzimidazole ring were basically in the same plane. It could also
with the theoretical calculation results, and the error was less be seen that the two methyl groups and the imidazole ring were in
than 5 ppm.
the same plane, while the benzene ring in benzyl group was dis-
torted from coplanarity with the indole ring. For dye C6, the torsion
angles of C(13)–C(14)–C(16)–C(17), C(15)–C(14)–C(16)–C(17),
C(16)–C(17)–C(18)–N(2) and C(16)–C(17)–C(18)–C(29) were 6.8ꢁ,
ꢀ170.6ꢁ, ꢀ179.7ꢁ and 3.6ꢁ, respectively, which showed that the
indole ring and benzindole ring were slightly reversed, and not
entirely in the same plane.
3.3.2 Crystal packing. The crystal packing of dye C2 along
the a axis and C6 along the b axis are shown in Fig. 3 and 4.
Dyes C2 and C6 crystallized in the monoclinic Pc space groups
with two dye molecules per unit cell and monoclinic P21/n
space groups with four dye molecules and one water molecule
per unit cell, respectively. For dye C2, in the crystal packing
along the a axis, there was one type of hydrogen bond between
the molecules, that is C(17)–H(17)$I(1), formed by C(17)–H(17)
in the indole ring from one molecule and I(1) from another
molecule (Table 5), which made dye molecules form a layered
structure in the bc plane. As can be seen from the packing
diagrams of C2 (Fig. 3), adjacent molecules were stacked
through the p–p interaction force, with face-to-face distances
3.3 Crystal structure
3.3.1 Description of crystal structure. The molecular
structures and atom numbering of dyes C2 and C6 are shown in
Fig. 1 and 2, and their bond lengths, bond angles and torsion
angles are listed in Tables 3 and 4. From the tables, it was found
that the carbon–carbon bond lengths of the molecular frame-
work of dyes C2 and C6 were basically intermediate between
typical C–C single (1.54 A) and C]C double (1.34 A) bonds and
the carbon–nitrogen bond lengths were also basically interme-
diate between typical C–N single (1.47 A) and C]N double
ꢀ
ꢀ
ꢀ
ꢀ
(1.27 A) bonds. It meant that the bond lengths of the carbon–
carbon and carbon–nitrogen on the dye molecular skeleton had a
tendency of averaging, and the p electrons in the dye molecular
framework were delocalized. All bond angles were close to 108ꢁ in
ve-membered rings and close to 120ꢁ in benzene rings,
which illustrated that the heterocycle skeleton of both C2 and C6
had no deformation. For dye C2, the torsion angles of
C(8)–C(9)–C(10)–C(11), C(8)–C(9)–C(10)–C(17), N(2)–C(7)–C(8)–C(9)
and N(1)–C(7)–C(8)–C(9) were ꢀ178.8ꢁ, ꢀ1.0ꢁ, ꢀ0.8ꢁ and 177.0ꢁ,
ꢀ
of 3.492 A and the conjugated molecules were arranged in a
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Table 7 The fluorescence quantum yields of dyes C1–C6 with different biomoleculesa
Dye concentrations Biomolecules
Dyes (mol Lꢀ1 concentrations (mg mLꢀ1
DNA
BSA
Muramidase Amylase Chymotrypsin Bovine
)
)
F/Ffree F/Ffree F/Ffree
F/Ffree
F/Ffree
hemoglobin F/Ffree
C1
C2
C3
C4
C5
C6
1 ꢂ 10ꢀ5
0
4
10
0
4
10
0
4
10
0
4
10
0
4
10
0
4
10
0
4
10
0
4
10
0
4
10
0
4
10
0
4
10
0
4
10
1.00
6.83
15.36
1.00
0.84
1.45
1.00
0.63
0.71
1.00
0.69
0.66
1.00
0.79
3.75
1.00
1.11
0.40
1.00
1.16
5.43
1.00
0.34
0.28
1.00
3.76
8.94
1.00
2.97
3.65
1.00
0.99
1.35
1.00
1.20
1.44
1.00
0.97
0.87
1.00
0.85
0.85
1.00
0.80
0.72
1.00
0.98
0.93
1.00
0.92
0.80
1.00
0.97
0.79
1.00
0.74
0.77
1.00
0.53
0.48
1.00
0.91
1.08
1.00
0.84
0.91
1.00
0.81
0.92
1.00
0.96
0.99
1.00
1.13
0.93
1.00
1.00
0.98
1.00
0.33
0.28
1.00
0.47
0.36
1.00
0.96
0.88
1.00
0.98
0.77
1.00
0.87
0.77
1.00
0.74
0.63
1.00
1.11
0.92
1.00
1.17
1.70
1.00
0.90
0.83
1.00
0.98
0.98
1.00
0.98
0.74
1.00
0.96
0.92
1.00
0.39
0.36
1.00
0.28
0.34
1.00
0.94
0.73
1.00
1.06
1.00
1.00
0.80
0.59
1.00
0.90
0.75
1.00
1.16
1.62
1.00
2.96
1.16
1.00
0.93
1.18
1.00
1.05
1.14
1.00
1.23
0.93
1.00
0.92
0.94
1.00
0.33
0.31
1.00
0.27
0.27
1.00
0.94
0.88
1.00
0.94
0.90
1.00
0.86
0.76
1.00
0.64
0.47
1.00
0.98
0.94
1.00
0.75
0.83
1.00
0.85
0.80
1.00
1.06
0.91
1.00
0.97
0.90
1.00
0.87
0.84
1.00
0.35
0.16
1.00
0.39
0.33
1.00
0.88
0.78
1.00
0.81
0.96
1.00
0.80
0.75
1.00
0.46
0.47
1.00
0.98
1.04
1.00
2.28
0.77
1.00
0.59
0.79
1.00
0.97
0.96
5 ꢂ 10ꢀ5
1 ꢂ 10ꢀ5
5 ꢂ 10ꢀ5
1 ꢂ 10ꢀ5
3 ꢂ 10ꢀ5
1 ꢂ 10ꢀ5
3 ꢂ 10ꢀ5
1 ꢂ 10ꢀ5
3 ꢂ 10ꢀ5
5 ꢂ 10ꢀ6
1 ꢂ 10ꢀ5
a
F: the uorescence quantum yields of dye in the presence of biomolecule; Ffree the uorescence quantum yields of pure dye.
head-to-head fashion along the a axis. The coulombic attrac- coefficient of dyes C1–C6 were located in the range of
tion between Iꢀ in one molecule and N+ in adjacent molecules 368.0–522.0 nm and 0.78 ꢂ 104 to 4.13 ꢂ 104 L molꢀ1 cmꢀ1
,
stabilized the molecular packing, whose distance of Iꢀ and N+ respectively. The sequence of the lmax of six dyes was C6 > C3,
ꢀ
ꢀ
of adjacent dye molecules was 5.133 A and 4.885 A, respec- C4, C5 > C1 > C2 in the same solvent. The reason for this was
tively. In this manner, molecular interactions, including that the lmax was related to the size of the conjugated system of
hydrogen bonds, p–p interaction force and coulombic dyes: the greater the conjugated system, the greater the lmax. In
attraction, made the dye molecules form a steric conguration the donor–p–acceptor (D–p–A) conjugated structure of dyes
of a three-dimensional framework. For dye C6, in the same C1–C6, their electron donor was the same, whereas the electron
layer of the packing crystal structure, the dye molecules were acceptor was different and their size of conjugated fragments
stacked face-to-face along the b axis through coulombic was benzoindoleninium > quinolinium > benzothiazolium >
+
attraction between Iꢀ and N , with the distance of 3.909 A and benzimidazolium. Therefore, the size of the conjugated system
ꢀ
ꢀ
4.940 A, respectively. In summary, the existence of coulombic for dyes C1–C6 was in the order C6 > C3, C4, C5 > C1 > C2,
attraction stabilized the steric conguration.
leading to a ranking for lmax of C6 > C3, C4, C5 > C1 > C2 in the
same solvent. The experimental results also found that the lmax
of dyes C1–C6 was increased with a decrease in the basicity of
the heterocyclic nucleus (the sequence of the basicity of
heterocyclic nucleus was benzindole < quinoline < benzothia-
zole < benzimidazole), which was consistent with results in the
literature.37
3.4 Spectral properties
3.4.1 Spectral properties of dyes C1–C6 in different
solvents. The absorption spectral data of six dyes in different
solvents are listed in Table 6. It was found that the maximum
absorption wavelengths (lmax
) and the molar extinction
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Fig. 5 The lmax of dyes C1–C6 in different solvents.
Fig. 3 Crystal packing of dye C2 along the a axis.
DMSO, and had considerable solubility in H2O, MeOH, EtOH
and CHCl3.
The uorescence spectral characteristics of dyes C1–C6 in
different solvents are shown in Table 6. As indicated in the
table, all six dyes uoresced in H2O, MeOH, EtOH, CHCl3 and
DMSO. The uorescence maximum emission wavelength (lem
)
ranged from 460.6 to 596.6 nm in different solvents, and a
bathochromic shi had occurred at 51.4–101.0 nm (Stokes
shi), when compared with the corresponding absorption
maxima; furthermore, the Stokes shi of the six dyes was largest
in H2O, followed by DMSO, which was benecial for the bio-
logical study. The sequence of the uorescence quantum
yields40 in three protonic solvents was F(EtOH) > F(MeOH) >
F(H2O). This was because the electron cloud in the conjugated
system was dispersed as the solvent polarity increased, which
resulted in a strong intramolecular charge transfer, leading to
uorescence quenching.41
Fig. 4 Crystal packing of dye C6 along the b axis.
3.4.2 The interaction of dyes C1–C6 with biomolecules.
The interaction between the dye molecules and six biological
molecules including DNA, BSA, muramidase, amylase, bovine
hemoglobin and chymotrypsin, was investigated to explore the
possibility of their use as biological uorescent probes. The
detailed spectral properties of dyes C1–C6 with different
biomolecules are shown in Table 7. It was found that the uo-
rescence quantum yields of six dyes had amplitude changes
when six kinds of biological molecules were added, but the
uorescence quantum yield of dye C1 at 1 ꢂ 10ꢀ5 mol Lꢀ1
showed a signicant increase when adding 10 mg mLꢀ1 DNA.
Therefore, the interaction between dye C1 and DNA is now
discussed in detail.
The UV-vis absorption and uorescence spectral data of
dye C1 with DNA and the changes of uorescence intensity
with the concentration of DNA are presented in Table 8 and
Fig. 6, respectively. It can be seen that the molar extinction
coefficient of dye C1 was decreased in the presence of DNA
(Table 8), and it also showed a hypochromic effect, which
indicated that the dye C1 was inserted into the DNA mole-
cule.42 As can be seen from Fig. 6 and Table 8, the lem of the
dye displayed a red-shi in the presence of DNA, and the
Fig. 5 shows the lmax of dyes C1–C6 in different solvents
(their dielectric constant: chloroform (CHCl3) 4.8, EtOH 24.6,
MeOH 32.6, DMSO 48.9, H2O 78.4). It could be seen that the
sequence of the lmax of the same dye in protonic solvent and
non-protonic solvent was lmax(EtOH) > lmax(MeOH) > lmax(H2O)
and lmax(CHCl3) > lmax(DMSO). That is, with the increase of the
dielectric constant of solvents, the lmax of dyes was
hypsochromic-shied. The reason for this was that there was
strong interaction between the solvent molecules with a large
dielectric constant and the dye molecules, making the ground
state energy of the dye molecules lower and the transition
energy of dye molecules increase, consequently resulting in a
blue shi in the lmax of dyes.38
At the same time, from the absorption spectra of dyes C1–C6
in different solvents (see ESI†), it was also found that six dyes
mainly existed in the monomer form (M) in H2O, MeOH, EtOH
and DMSO, and dyes C1 and C4–C6 happened to aggregate in
CHCl3, especially C1 which was mainly in H-aggregated states,
which were attributed to the structure of the dye and the
viscosity of the solvent.39 And from the preparation of the dye
solutions it was found that six dyes had good solubility in
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Table 8 The spectroscopic data of dye C1 in the presence of DNA
Absorption
Fluorescence
DNA concentration
lmax
Hypochromic
effect (%)
Dye
(mg mLꢀ1
)
(nm)
3 ꢂ 10ꢀ4 (L molꢀ1 cmꢀ1
)
lex (nm)
lem (nm)
Stokes shis (nm)
FDNA/Ffree
C1
0
2
4
6
8
445.0
454.0
460.0
466.0
468.0
463.0
466.0
0.0
11.3
33.6
23.9
23.3
29.6
19.8
3.18
2.82
2.11
2.42
2.44
2.24
2.55
455.0
455.0
455.0
455.0
455.0
455.0
455.0
521.6
526.6
525.0
525.8
525.2
525.4
525.4
66.6
71.6
70.0
70.8
70.2
70.4
70.4
1.00
4.00
6.83
9.46
12.77
15.36
15.57
10
12
molecules become weak and the uorescence intensity was
increased.43 Because of the packing of p electrons between
base pairs and dye molecules, and the coupling effect of the
empty p* orbital of dye and the p orbital of base pairs, the
energy level of the p* orbital of the dye molecules was low-
ered, and the transition energy of p* / p was reduced,
leading to the red-shi of the lem of dye.44,45
3.5 Cytotoxicity and cell staining of dye C3
For biomedical applications such as uorescent probes and
cellular imaging, the toxicity of the dye was a major concern. To
assess the cytotoxicity of the selected dye C3, a WST assay was
performed with A172 cell lines. The results (Fig. 7) indicated
that the cell viability of dye C3 was more than 87% at the
application dose and duration, suggesting a very low cytotox-
icity of dye C3.
Fig. 6 Fluorescence spectra of dye C1 (1 ꢂ 10ꢀ5 mol Lꢀ1) with
different concentrations of DNA.
The cell staining of dye C3 was also investigated by uores-
cence imaging. A172 cells were incubated with dye C3 at a
concentration of 20 mmol Lꢀ1 for 10 min and the reaction was
observed using uorescence microscopy. As shown in Fig. 8,
strong uorescence (excitation at 488 nm, emission wavelength
of 570–620 nm) was observed in A172 cells, suggesting that dye
C3 could stain A127 cells, and that the dye was mainly distrib-
uted in the cytoplasm. The results indicated that dye C3 would
be able to act as a potential uorescent reagent to clearly
observe cells, for the determination of the characteristics of
cells and the developmental stages of cells. Further
uorescence intensity and uorescence quantum yield of the
dye were increased linearly with increasing concentration of
DNA. This was because when the aromatic ring plane of the
dye was inserted into the base pairs of DNA, the dye molecules
were protected by the DNA hydrophobic effect, which made
the uorescence quenching of dye caused by the solvent
Fig. 7 The viability of A172 cells incubated with dye C3 at various Fig.
concentrations for 12 h. Error bars are based on triplicate samples.
8 Confocal fluorescence microscopic images of A172 cells
incubated with dye C3 at 37 ꢁC for 10 min. Scale bar: 50 mm.
4690 | RSC Adv., 2015, 5, 4681–4692
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investigation of cytotoxicity and cell staining of the other dyes
synthesized is in progress.
7 S. Dash, M. Panigrahi, S. Baliyarsingh, P. K. Behera, S. Patel
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4. Conclusions
9 J. Kabatc, A. Bajorek and R. Dobosz, J. Mol. Struct., 2011, 985,
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Six new double N-alkylated dimethine cyanine dyes were
synthesized using, as starting materials, N-benzyl-3-indole
formaldehyde and heterocyclic quaternary ammonium salts
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using UV-vis, IR, HRMS, 1H-NMR. The single XRD of C2 and C6
revealed that the two dyes crystallized in the monoclinic Pc with
ˇ
ˇ ´
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I. I. Timcheva and T. G. Deligeorgiev, Bioorg. Med. Chem.,
2009, 17, 4747–4755.
ˆ
11 P. Hanczyc, B. Norden and B. Akerman, J. Phys. Chem. B,
2011, 115, 12192–12201.
ꢀ
ꢀ
ꢀ
ꢀ3
a ¼ 9.676 A, b ¼ 5.8938 A, c ¼ 19.693 A, V ¼ 1119.5 A , Z ¼ 2 and
12 S. Kaloyanova, I. Crnolatac, N. Lesev, I. Piantanida and
T. G. Deligeorgiev, Dyes Pigm., 2012, 92, 1184–1191.
13 T. G. Deligeorgiev, A. Vasilev and K. Drexhage, Dyes Pigm.,
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14 N. I. Shank, H. H. Pham, A. S. Waggoner and B. A. Armitage,
J. Am. Chem. Soc., 2013, 135, 242–251.
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18 C. Holzhauser, R. Liebl, A. Goepferich, H. A. Waqenknecht
and M. Breunig, Chem. Biol., 2013, 8, 890–894.
19 Y. Kam, A. Rubinstein, A. Nissan, D. Halle and E. Yavin, Mol.
Pharmaceutics, 2012, 9, 685–693.
20 T. G. Deligeorgiev, A. Vasilev and K. Drexhage, Dyes Pigm.,
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21 Y. L. Fu, B. R. Zhang, S. Wang, X. X. Gao and L. Y. Wang, Bull.
Korean Chem. Soc., 2013, 2, 489–494.
ꢀ
ꢀ
ꢀ
monoclinic P21/n with a ¼ 12.3860 A, b ¼ 10.3493 A, c ¼ 22.395 A,
ꢀ3
V ¼ 2820.9 A , Z ¼ 4 space groups, respectively, and the crystal
packing was stabilized by hydrogen bonds, p–p stacking inter-
actions and coulombic attractions. The absorption maxima and the
molar extinction coefficient of the dyes were located in the range of
368.0–522.0 nm and 0.78 ꢂ 104 to 4.13 ꢂ 104 L molꢀ1 cmꢀ1 in
different solvents, respectively. The uorescence maxima and
Stokes shi of the dyes ranged from 460.6 to 596.6 nm and from
51.4 to 101.0 nm in different solvents, respectively, and dyes C1
and C3 showed a relatively high uorescence quantum yield.
The interaction between these dye molecules and six biological
molecules showed that there was larger enhancement of uo-
rescent quantum yield for dye C1 in the presence of DNA. The
investigation of the cytotoxicity and cell staining of the selected
dye C3 revealed virtually no toxicity at the dose and duration
applied and demonstrated that the dye could stain cytoplasm. It
is suggested that this dye is a potential uorescent reagent for
observing cells clearly, and for determining their characteristics
and developmental stages.
ˇ
Acknowledgements
22 L. Glavas-Obrovac, I. Piantanida, S. Marczi, L. Masic,
I. I. Timcheva and T. G. Deligeorgiev, Bioorg. Med. Chem.,
2009, 17, 4747–4755.
23 X. H. Zhang, L. Y. Wang, Z. X. Nan, S. H. Tan and Z. X. Zhang,
Dyes Pigm., 2008, 79, 205–209.
24 S. L. Wang and Y. T. Chang, J. Am. Chem. Soc., 2006, 128,
10380–10381.
We appreciate the nancial support for this research from a
grant from the Scientic and Technological Research and
Development Projects in Shaanxi Province (2012K07-06) and the
Special Science Research Foundation of Education Committee
(no. 11JK0558).
25 Q. Li, Y. Kim, J. Namm, A. Kulkarni, G. R. Rosania and
Y. T. Chang, Chem. Biol., 2006, 13, 615–623.
26 R. M. Abd El-Aal, A. I. M. Koraiem, Z. H. Khalil and
A. M. M. El-Kodey, Dyes Pigm., 2005, 66, 201–209.
27 T. G. Deligeorgiev, N. Gadjev, S. Kaloyanova, N. Lesev,
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