Development of 3-alkyl-6-methoxy-7-hydroxy-chromones (AMHCs) from natural isoflavones, a new class of fluorescent scaffolds for biological imaging

Article abstract of DOI:10.1039/c4cc06762b

Starting from 7-hydroxyisoflavones, we developed a new class of fluorescent scaffolds, 3-alkyl-6-methoxy-7-hydroxy-chromones (AMHCs, M_W ～ 205.19, λ_ab ～ 350 nm, λ_em ～ 450 nm) via a trial and error process. AMHCs have the advantages of being a small molecular moiety, having strong fluorescence in basic buffers, reasonable solubility and stability, non-toxicity, and are conveniently linked to pharmacophores. AMHCs were successfully used in fluorescence microscopy imaging of cells and tissues. This journal is

Full text of DOI:10.1039/c4cc06762b

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Development of 3-alkyl-6-methoxy-7-hydroxy-
chromones (AMHCs) from natural isoflavones, a
new class of fluorescent scaffolds for biological
imaging†
Cite this:DOI: 10.1039/c4cc06762b
Received 27th August 2014,
Accepted 21st November 2014
Jianzhuang Miao,‡a Huaqing Cui,‡*a Jing Jin,a Fangfang Lai,a Hui Wen,b
DOI: 10.1039/c4cc06762b
Xiang Zhang,b Gian Filippo Ruda,c Xiaoguang Chena and Dali Yin*a
www.rsc.org/chemcomm
Starting from 7-hydroxyisoflavones, we developed a new class
of fluorescent scaffolds, 3-alkyl-6-methoxy-7-hydroxy-chromones
(AMHCs, MW B 205.19, kab B 350 nm, kem B 450 nm) via a trial and
error process. AMHCs have the advantages of being a small mole-
cular moiety, having strong fluorescence in basic buffers, reasonable
solubility and stability, non-toxicity, and are conveniently linked to
pharmacophores. AMHCs were successfully used in fluorescence
microscopy imaging of cells and tissues.
Fluorescent dyes have been widely used in biological research
for analytical sensing and biological imaging.1–9 In spite of the
increasing demand, the library of conventional fluorophores only
contains a limited number of scaffolds, including naphthalimide,
styryl, xanthone, coumarin, dapoxyl, BODIPY, rhodol and tricarbo-
cyanine.3,4,7,8 Efforts to diversify the fluorophore library include
Fig. 1 Previous development of flavone 1 based fluorophore 3 and the
desired optimization of isoflavone 2 in this study.
both the de novo synthesis of new scaffolds and the optimization been further developed into a series of moderate to strong
of known scaffolds.8,10–17 However, due to the complex of photo- fluorophores in ethanol, such as 3 (F = 0.48 in 95% ethanol)
physical properties of fluorophores, it is difficult to predict the (Fig. 1). This optimisation has increased the fluorescence, and
emission wavelength or quantum yield of a fluorogenic scaffold, the size of the molecule was also increased.30,31
and most of these studies are performed empirically.8,10–19
To our knowledge, 7-hydroxyisoflavone 2 has not yet been
Natural products are important sources of new scaffolds for used as a lead for the development of new fluorophores.
fluorophores.1,20 Natural 7-hydroxyisoflavones and synthetic Nevertheless, 7-hydroxyisoflavone is able to exhibit fluores-
3-hydroxyflavones have been reported to have weak fluorescence cence, albeit weakly in a biological buffer, and has superior
in biological buffers. This limits their application in biological aqueous solubility compared to 3-hydroxyflavone (Fig. S3a
imaging (Fig. S1 and S2, ESI†).21–29 Efforts have been made to and b, ESI†). Thus, in this study we opted to optimize the
circumvent these problems. For example, 3-hydroxyflavone 1 has photophysical properties of isoflavones in order to develop a
new class of fluorescent dyes.
Considering the difficulty in theoretical prediction of optical
a State Key Laboratory of Bioactive Substances and Function of Natural Medicine,
properties,1 our optimisation was carried out by a trial and
Institute of Materia Medica, Peking Union Medical College and Chinese Academy
error process. We investigated the effect of various substituents
at different positions of the isoflavone core on its photophysical
of Medical Sciences, 1 Xiannongtan Street, Beijing, 100050, China.
E-mail: yindali@imm.ac.cn, hcui@imm.ac.cn
b Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation,
Institute of Materia Medica, Peking Union Medical College and Chinese Academy
of Medical Sciences, 1 Xiannongtan Street, Beijing, 100050, China
c TDI-SGC Nuffield Department of Medicine, University of Oxford, Roosevelt Drive,
Oxford, OX37FZ, UK
properties. Subsequently, favourable modifications were com-
bined to design new fluorophores. In addition, given the future
application for biological imaging, possible properties were
also designed into final fluorophores.
During our first round of optimisation, we developed a
specific library of isoflavone analogues via chemical synth-
esis.32 After fully evaluating the fluorescence properties of the
† Electronic supplementary information (ESI) available: Experimental procedures,
photophysical properties, and data of compounds. See DOI: 10.1039/c4cc06762b
‡ J.M. and H.C. contributed equally.
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Table 1 The photophysical properties of selected compounds
Compounda
emax
lab
lem
Fd
te
b
c
c
12 600
7300
311
288
334
468
0.05
1.72
419
454
0.21
0.21
5.24
2.11
Fig. 2 The key trend of structure fluorescence relationship of isoflavone
derivatives in 0.1 M Tris-HCl, pH 8.0.
12 700
isoflavones, a structure fluorescence relationship was observed
(Fig. 2 and Fig. S4–S8, ESI†):
ꢀ 7-Position: the isoflavones with 7-OH are often fluoro-
genic. Acetylation or methylation of 7-OH quenched the fluores-
cence of the isoflavone core (Fig. S4, ESI†).
16 400
16 300
345
346
447
445
0.48
0.41
4.49
4.31
ꢀ 6-Position: 6-OMe alone is able to improve the fluorescence
quantum yield (F = 0.21) of the isoflavone core. Other substituents
at this position (OH and CH3) do not have an effect (Fig. S5a
and b, ESI†). In addition, 6-OMe substitution marginally affects
the fluorescence quantum yield of 7-hydroxyisoflavone.
ꢀ 5-Position: 7-hydroxyisoflavones containing 5-hydroxyl are non-
a
b
Measurements were made in 0.1 M Tris-HCl buffer, pH 8.0. Unit
Mꢂ1 cmꢂ1
.
Unit nm. Determined with quinine sulfate (F = 0.54,
c
d
e
0.1 M H2SO4) as given in ref. 33. Unit ns. For details, please see ESI.
The first round of optimisation (Fig. 2) demonstrated that
fluorogenic. Previous studies elucidated that 5-hydroxylflavone 6-OMe alone activates the fluorescence of the 3-phenylchromone
undergoes the excited state intra-molecular proton transfer (ESIPT) core. Thus, a 6-OMe substitution was also incorporated into the
and results in the low fluorescence quantum yield (Fig. S6, ESI†).25–27 3-alkyl-7-hydroxychromone core to afford a new scaffold, 3-alkyl-
ꢀ 8-Position: substitution at the 8-position is not favourable 6-methoxy-7-hydroxy-chromone (AMHC). Intriguingly, AMHCs
to increase the fluorescence quantum yield of 7-hydroxyiso- exhibited a dramatically increased fluorescence quantum yield
flavone. In addition, 8-OH abolishes the fluorescence of of around 0.5 in 0.1 M Tris-HCl, pH 8.0, and reasonable extinc-
7-hydroxyisoflavone (Fig. S7, ESI†).
tion coefficients (e) in the range of (1.0–2.0) ꢁ 105 Mꢂ1 cmꢂ1
.
,
ꢀ 3-Position: for the 7-hydroxyisoflavone core, the 20-, 30-, and This results in a fluorophore brightness of B105 Mꢂ1 cmꢂ1
40-positions of the 3-phenyl ring were substituted with various which is sufficient to be used as fluorescent sensors (Table 1). In
groups to evaluate the effect on fluorescence. The results show addition, five of the representative fluorophores had their
that electron-donating 20-substituents can increase the fluores- fluorescence lifetimes measured (Table 1). For lifetime studies
cence quantum yield of 7-hydroxyisoflavone (CH3 4 OMe 4 OH) the excitation wavelength was set at lex = 375 nm, with all of the
(Fig. S8, ESI†).
tested compounds showing a lifetime in the range of 1.7–5.2 ns.
Next, we investigated the importance of the 3-phenyl group in the Our development has improved the fluorescence lifetime of
fluorescence of 7-hydroxyisoflavone. 3-Alkyl-7-hydroxy-chromones AMHCs compared to that of natural isoflavone 6, which makes
were synthesized to evaluate their fluorescence. Interestingly, AMHCs suitable as fluorescent probes in biological buffers.
3-methyl-7-hydroxychromone showed an increased fluorescence
The development process of AMHC fluorophores is pre-
quantum yield compared to 3-phenyl-7-hydroxychromone (Fig. S9a sented in Fig. 3. The fluorescence brightness was gradually
and b, ESI†). For comparison, we also synthesized and evaluated increased during optimisation. The commercial fluorescent
2-methyl-7-hydroxychromone, and found that this compound dye 7-amino-4-methylcoumarin (AMC) was chosen for com-
exhibits an identical fluorescence quantum yield (F = 0.21) to parison since AMC has a similar wavelength range of excita-
3-methyl-7-hydroxychromone (F = 0.21) (Table S2 and Fig. S10, tion and emission maximum to AMHCs. Interestingly, under a
ESI†). Thus, further optimisation was focused on 3-alkyl chro- monochrome camera (Fig. 3A), AMHC fluorescent dyes show
mone derivatives.
more intensive brightness than that of AMC tested under
3-Alkyl-7-hydroxychromone has increased fluorescence quantum identical conditions. Given that monochrome photography
yield and improved aqueous solubility. Interestingly, the 3-alkyl is commonly used in fluorescence microscopy,34,35 this bene-
chain serves as a natural spacer, and can be designed with a ficial property enhances the potential of AMHC sensors to be
terminal active group for attachment to a pharmacophore used in biological imaging. We also imaged the fluorescence
without seriously affecting the photophysical properties of the with a colour digital camera (Fig. 3B), and AMHC dyes show
fluorophore. However, so far the fluorescence quantum yield of similar colour intensity to AMC. The discrepancy between the
3-alkyl-7-hydroxychromone is only B0.20 in 0.1 M Tris-HCl brightness of AMHC and AMC under monochrome and colour
buffer, pH 8.0 (Table 1 and Table S2, ESI†), which is relatively cameras is likely caused by the different sensor systems of
poor for use as a fluorescent sensor in biological studies.
monochrome and colour cameras. Also, the optical behaviors
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from 1 to 12. Both 6 and 81 demonstrated pH dependent
fluorescence effects, but fluorophore 6 only showed moderately
high fluorescence intensity at the pH range of 9–11, with the
fluorescence dropping at pH 12. While, fluorophore 81 exhib-
ited moderate fluorescence intensity from pH 5 to 8, which will
enable it to be used in biological imaging. Moreover, 81
possesses exceptional fluorescence intensity above pH 9. This
demonstrates that AMHCs are a class of fluorophores with
strong fluorescence properties.
AMHCs were further evaluated for their suitability as bio-
logical reagents. Several AMHC analogues were screened for
cell toxicity and were not found to show any toxicity at 50 mM
(Table S4, ESI†). In addition, AMHCs are soluble in 0.1 M Tris-
HCl buffer, pH 8.0 at a concentration of 100 mM.
Fig. 3 Screening fluorescence intensity of selected compounds (100 mM)
in 0.1 M Tris-HCl, pH 8.0 in a microtiter plate. Yellow label, natural
products of isoflavones; red label, 3-phenyl-7-hydroxychromone analo-
gues; green label, 3-alkyl-7-hydroxychromone derivatives; purple label,
3-alkyl-6-methoxy-7-hydroxychromone derivatives; orange label: AMC.
For details of compounds, please see Table S1 (ESI†).
EdU (5-ethynyl-20-deoxyuridine) incorporates into DNA and
can be used to detect cell proliferation.39–41 A fluorescent dye
that contains an azide group can label EdU via click chemistry.
Therefore, we synthesized AMHC dye 83 (Table 1), which
contains an azide group at the end of the alkyl chain. HepG2
cells were incubated with 10 mM EdU, followed by the addition
of 5 mM 83 in a click chemistry reaction buffer, in order to label
EdU incorporated into DNA. During this experiment we
observed a strong blue fluorescence at the HepG2 cell nucleus
(Fig. 5A–C). It has previously been reported that the commercial
fluorescein-derived dyes Alexa488-azide and Alexa594-azide
(Molecular Probess) stain rat bone marrow cells in the absence
of EdU (false-positive staining).41 Therefore, the same rat bone
marrow cell staining experiment was performed using both
Alexa488-azide and 83 in the absence of EdU. We were able to
observe the same false staining of rat bone marrow cells by
Alexa488-azide, but not by AMHC dye 83 (Fig. S13, ESI†). This
shows an additional advantage of the AMHC scaffold dyes. In
addition, this false-positive staining case also enhances the
necessity to develop fluorophores with varied scaffolds.
AMHCs were also evaluated for their application in tissue
staining. Fluorescence imaging of tissue samples requires
of AMHCs and AMC are dependent on their different solvent
micro-environments.36–38
Natural 7-hydroxyisoflavones were reported to show a pH
dependent fluorescence effect.21–29 Thus, the fluorescence of
AMHCs was also evaluated at different pH values (Fig. 4).
Compounds 6 (natural isoflavone) and 81 (AMHC derivative)
were dissolved in methanol : water (1 : 1) with the pH ranging
Fig. 5 Application of AMHC dye to label and image EdU that was
incorporated into DNA in cultured cells (A–C) and mice tumor tissues
(D–F). (A) 83 detected EdU in cell nucleus of HepG2 cells, (B) HepG2 cell
imaged in bright field, (C) merged image of A and B. (D) 83 detected EdU in
the cell nucleus of tumor tissue, (E) tumor tissue imaged in bright field, (F)
merged image of D and E.
Fig. 4 Fluorescence emission of 6 and 81 show variation over different
pH values at the concentration of 10 mM (Ex = 350 nm). (A) Fluorescence
emission spectra and fluorescence intensity of 6 at pH ranging from 1 to
12; (B) fluorescence emission spectra and fluorescence intensity of 81 at
pH ranging from 1 to 12.
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