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Diketopyrrolopyrrole: brilliant red pigment

dye-based fluorescent probes and their applications
Cite this: Chem. Soc. Rev., 2015,
44, 58
Matinder Kaur and Dong Hoon Choi*

The development of fluorescent probes for the detection of biologically relevant species is a burgeoning
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topic in the field of supramolecular chemistry. A number of available dyes such as rhodamine, coumarin,
fluorescein, and cyanine have been employed in the design and synthesis of new fluorescent probes.
However, diketopyrrolopyrrole (DPP) and its derivatives have a distinguished role in supramolecular
chemistry for the design of fluorescent dyes. DPP dyes offer distinctive advantages relative to other
organic dyes, including high fluorescence quantum yields and good light and thermal stability. Significant
advancements have been made in the development of new fluorescent probes based on DPP in recent
years as a result of tireless research efforts by the chemistry scientific community. In this tutorial review,
we highlight the recent progress in the development of DPP-based fluorescent probes for the period
Received 18th July 2014 spanning 2009 to the present time and the applications of these probes to recognition of biologically
DOI: 10.1039/c4cs00248b relevant species including anions, cations, reactive oxygen species, thiols, gases and other miscellaneous
applications. This review is targeted toward providing the readers with deeper understanding for the future
[Link]/csr design of DPP-based fluorogenic probes for chemical and biological applications.

Key learning points

(1) Importance of diketopyrrolopyrrole (DPP) in supramolecular chemistry
(2) Biological roles of anions, cations, reactive oxygen species, and thiols
(3) Design strategies for DPP-based fluorescent probes
(4) Recognition mechanisms of DPP-based fluorescent probes
(5) Diverse applications of DPP-based small molecules and polymers

1. Introduction basically comprise an ion recognition unit (e.g., ionophore)

attached to a signaling unit (e.g., fluorophore) that converts
Biologically important species such as anions, cations, reactive molecular recognition into highly discriminative and easily
oxygen species (ROS), and thiols are essential for human health detected/measured optical signals that are associated with
and environment.1 An appropriate amount of these species is changes in the photophysical properties of the probes (Fig. 1).
crucial for the effective operation of biological systems as well These changes are associated with various signaling mechanisms
as environment and for maintaining the mandatory balance such as fluorescence enhancement/quenching and ratiometric
between them. However, these species are highly hazardous changes due to interaction of the probes with particular analytes
once their concentration exceeds a maximum limit, and there- (e.g., Lewis acid–base interaction, deprotonation, photoinduced
fore detection, quantitative determination, and removal of processes such as charge transfer, energy transfer, intermolecular
these species from environmental and biological systems are excited state proton transfer).
the main objectives of many studies. One of the most facile The binding of a specific analyte to a fluoroionophore (i.e.,
ways to recognize these species is to use fluorescent probes that fluorophore linked with an ionophore) can cause either
are capable of detecting these species in solution, in the solid enhancement (turn-on) or quenching (turn-off) of the fluores-
state, and in living cells with high sensitivity. Fluorogenic probes cence intensity, coupled with a red or blue shift of the emission
band or absorption band. Upon excitation, intramolecular
Department of Chemistry, Research Institute for Natural Sciences, Korea University,
charge transfer from a donor to an acceptor unit occurs within
5 Anam-dong, Sungbuk-Gu, 136-701 Seoul, Korea. E-mail: dhchoi8803@[Link]; the fluoroionophore. However, complexation of a metal ion
Fax: +82-2-925-4284; Tel: +82-2-3290-3140 with the donor or acceptor group results in significantly

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Fig. 1 Schematic representation of the photophysical response of a fluorescent probe to an analyte.

different photophysical responses. When the metal ion binds with strong acceptor units, (c) exhibit high fluorescence quantum
the donor group of the receptor, there is a consequent reduction of yields, and (d) possess exceptional thermal and photostability,
the conjugation length that decreases the electron-donating ability making them excellent building blocks for many applications.
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of the donor group, which results in a blue-shift in the absorption Owing to the above features, DPP and its derivatives have been
spectrum (Fig. 2a). On the other hand, when the metal ion interacts widely used as high-performance pigments (e.g., paints, plastics,
with the acceptor unit, there is an enhanced conjugative effect that and inks)3 and exhibit outstanding semiconducting properties
increases the electron-withdrawing nature of the acceptor group that have been primarily utilized in field-effect transistors and
and hence, generally induces a red-shift of the absorption or photovoltaic cells. These DPP-based materials and their various
emission spectrum (Fig. 2b). applications have been reviewed recently.4–7
Over the past decade, the design and synthesis of novel
fluorescent probes using various fluorogenic units such as coumarin,
2. Diketopyrrolopyrrole rhodamine, fluorescein, cyanine, boron-dipyrromethene (BODIPY),
Diketopyrrolopyrrole (DPP) is synthesized by the reaction of and DPP for the recognition of various biologically important species
aromatic nitrile with dialkyl succinate (Scheme 1).2 DPP has have been extensively reported.8 Rhodamine and fluorescein dyes
attracted considerable research interest over the last few years. exhibit high fluorescence quantum yields and good photostability
DPP has a planar structure and can induce strong intermolecular (Table 1). However, their biological applications are limited because
H-bonding and p–p stacking with neighboring molecules. The their absorption and emission maxima appear below 600 nm.
molecular frame of DPP has many reactive centers such as (i) the Similarly, although cyanine dyes are fluorophores of biological
aryl rings that undergo electrophilic and nucleophilic reactions interest, the poor photostability of these dyes has hampered their
and (ii) the bicyclic lactam chromophoric unit with three different applications. BODIPY derivatives have attracted much interest
functional groups: (1) –CQC– double bonds, (2) carbonyl, and because of their excellent absorption and emission spectra and high
(3) secondary amine (NH) groups that may potentially undergo molar absorption coefficient (Table 1). On the other hand, DPP has
structural modification for further derivatization (Scheme 1). earned distinction in the chemistry community because of its wide
The significant advantages of DPP derivatives are as follows: application as a fluorescent probe over other organic dyes because of
(a) they can undergo several synthetic modifications, (b) act as excellent photostability and good fluorescence quantum yields.

Matinder Kaur was born in Dong Hoon Choi was born in

Bareilly, India, in 1983. In 2012, Korea in 1960. He received his
she obtained her PhD in Organic PhD from department of
and Medicinal Chemistry under Macromolecular Science &
the supervision of Prof. Palwinder Engineering in the University of
Singh from the Department of Michigan, Ann Arbor, MI, USA.
Chemistry, Guru Nanak Dev After completing postdoctoral
University, Amritsar, India. She is studies at The State University
currently a postdoctoral fellow of New York, Buffalo, USA, he
with Prof. Dong Hoon Choi at joined as Senior Scientist at
Korea University. Her research Korea Institute of Science and
interests focus mainly on design Technology in 1992 and
Matinder Kaur and synthesis of OTFTs, OPVs Dong Hoon Choi transferred to Department of
and fluorescent probes for various Environment and Applied
analytes. Chemistry, Kyung Hee University in 1995 as a Professor. In 2005,
he moved to Korea University, where he is currently a Professor in
the department of chemistry.

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Fig. 2 Schematic illustration of the recognition response of a fluorogenic probe to an analyte.

the probes for various analytes such as anions, cations, reactive

oxygen species (ROS), thiols, pH, CO2, and H2; (b) the probes for
molecular imaging, (c) structurally modified DPP-based near-
infrared (NIR) dyes, and (d) DPP-based polymers as fluorescent
probes. Specific focus is placed on the research progress and
scholarly contributions made in recent years. Overall, this review
provides an outline of the design strategy for each class of DPP-
based fluorescent probes and their respective applications in the
Scheme 1 General synthetic route for DPP and its derivatives. format of a tutorial review.

To date, DPP-based high-performance materials have been well 3. DPP-based fluorescent probes for
developed,4–7 while the ability of DPP to recognize biologically various analytes
important species still remains relatively underdeveloped. However,
because of the high fluorescence quantum yields, exceptional 3.1 Probes for anions
photostability, and moderately high molar absorption coefficients Anions play significant roles in environmental, medicinal, chemical,
of DPP-containing fluorescent probes in both solution and and biological applications. Among the various important anions
membrane-bound forms, considerable effort has recently been (e.g., fluoride, chloride, cyanide, and phosphate), the fluoride (F)
dedicated to the development of such probes and investigation and cyanide (CN) ions are of considerable interest because of their
of their applications in supramolecular chemistry. Despite many well-known roles in biological systems. Despite the significant
emerging applications, a comprehensive review systematically benefits of F, excessively high or low concentrations have been
addressing DPP-based fluorescent probes has not yet been associated with gastric and kidney disorder, dental and skeletal
presented. In this tutorial review, we summarize the development fluorosis, urolithiasis in humans, and even death. Similarly, a small
of various DPP-based fluorescent probes and their applications amount of CN can cause death within a few minutes because of its
for tracing biologically and environmentally important species. strong preference for the active site of cytochrome c oxidase and its
The DPP-based fluorescent probes are broadly categorized as (a) ability to inhibit cellular respiration. Owing to the serious toxicity of

Table 1 Commonly used organic dyes and their photophysical properties

Absorption Fluorescence Photo Molar extinction Fluorescence

Organic dyes range (nm) emission range (nm) stability coefficient (M1 cm1) quantum yield
DPP 400–550 500–650 Very high B25 000 0.4–0.9
Coumarin 320–520 450–590 Good B20 000 B0.8
Rhodamine 470–528 510–610 High B116 000 B0.95
Fluorescein 400–550 460–700 High B80 000 B0.92
Cyanine 400–800 500–800 Moderate B100 000 0.12–0.67
BODIPY 500–645 506–760 Very high B80 000 B0.92

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F and CN ions, development of fluorogenic probes for detection spectra upon titration with TBAF in acetone and acetonitrile
of these ions has been widely pursued. Thus, in this section, we with a decrease in absorbance at B520 nm and formation of a
focus on the recent developments of DPP-based fluorescent probes new red-shifted band at B594 nm. In acetone, probe 4 exhibited
for F and CN ion detection. fluorogenic ratiometric behavior upon addition of F with a
3.1.1 Fluoride probes based on intermolecular proton transfer. decrease in emission intensity at 590 nm and formation of a new
Most fluorescent probes for F detection exploit the fact that F is emission band at 641 nm (Fig. 4a and b). However, in acetonitrile,
an electronegative atom and therefore can form strong hydrogen probe 4 exhibited a very weak fluorescence band at ca. 530 nm due
bonds with –NH groups or can easily deprotonate –NH protons of to aggregation-induced fluorescence quenching. With addition of
the designed receptor, which results in an evident change either in F ions, the fluorescence intensity was enhanced by ca. 40-fold at
the absorption or emission spectrum of the probe. 631 nm, which is attributed to deprotonation of the amide unit by
Hua et al. utilized the DPP moiety to synthesize fluorescent F, resulting in charge transfer (CT) from the deprotonated DPP
probes bearing bromo 1, phenyl 2, and fluorene 3 moieties for unit to the triphenylamine donor units (Fig. 4c and d). The F
the colorimetric and ratiometric detection of F (Fig. 3).9 The detection limit of probe 4 was 4.20 mM in acetone and 2.46 mM in
hydrogen atoms on the nitrogen of the amide units of these acetonitrile. Furthermore, the ratiometric absorption behavior and
probes 1–3 were used as a receptor for F sensing due to the turn-on fluorescence emission of probe 4 with F in acetonitrile
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strong H–F interactions. The absorption maxima of probes 1–3 has been employed to mimic a parallel double-INH logic gate in the
were observed at 477 nm, 485 nm, and 497 nm, respectively. presence of HSO4. Probe 4 demonstrated two-input–two-output
The absorbance decreased, accompanied by large bathochromic INH gate (parallel double-INH logic gate) with F (In1) and HSO4
shifts to 571 nm, 585 nm, and 594 nm, respectively, upon (In2) as two chemical inputs and ratiometric absorption and turn-
addition of tetrabutylammonium fluoride (TBAF), leading to on fluorescence emission as two outputs (Fig. 4e–g).
significant color changes. Furthermore, the emission spectra of A similar research was conducted by Yang et al. in the
probes 1–3 underwent a F induced red-shift from 532 to 613 nm synthesis of DPP-based one-photon and two-photon probes
for probe 1, from 545 to 630 nm for probe 2, and from 563 to 5–7 for detection of F ions (Fig. 3).11 These probes exhibited
635 nm for probe 3. ratiometric absorption and fluorescence behavior with F ions
The colorimetric and ratiometric changes were mainly due to the IPT between the amide moiety and F. In the
ascribed to the intermolecular proton transfer (IPT) between presence of F, ICT from the amide to the conjugated region
the hydrogen atom of the amide unit and F. Probe 3 can detect is increased, resulting in a bathochromic shift of the spectra.
F at concentrations in the range of 0–10 mM in the visible part Probes 5–7 exhibited practical two-photon absorption (TPA)
of the electromagnetic spectrum. Furthermore, to improve the cross-sections (d = 58–87 GM) that depend on the substituents
detection limit of F and enhance the intramolecular charge in the benzene rings of DPP.
transfer (ICT) from the amide unit of the DPP moiety to the In order to improve the TPA cross-section, they further
conjugated donor system, the new fluorescent probe 4 was synthesized the donor–p–acceptor–p–donor type DPP-based
synthesized by introducing two electron-donating substituents compound 8 and evaluated the F sensing properties using
(triphenylamine) at the DPP unit (Fig. 3).10 Recognition of F by one- and two-photon absorption and emission spectroscopy
probe 4 was studied in acetone and acetonitrile solvents, giving (Fig. 3).12 The red-shift of the absorption (from 538 to 608 nm)
rise to similar absorption changes but different fluorescence and emission bands (from 594 to 645 nm) of probe 8 in the
behavior. Probe 4 showed ratiometric behavior in absorption presence of F was attributed to deprotonation of the NH group

Fig. 3 Structures of DPP-based fluorescent probes 1–8 and mechanism of F detection.

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Fig. 4 (a) UV-Vis absorption and (b) fluorescence spectral changes of probe 4 upon the addition of F in acetone; (c) UV-Vis absorption and (d)
fluorescence spectral changes of probe 4 upon the addition of F in acetonitrile. (e) Ratiometric responses of absorption spectra (A594nm/A520nm) of 4
(10 mM) in the absence and presence of HSO4 and/or F in acetonitrile; (f) turn on responses of emission spectra (lem = 631 nm) of 4 (10 mM) in the
absence and presence of HSO4 and/or F in acetonitrile; and (g) schematic representation of a parallel double INH logic gate. Reproduced from ref. 10
with permission from Elsevier.

in DPP by the F ion. On the other hand, the TPA cross-section ions, accompanied by a color change from yellow green to green.
(d) of probe 8 increased (up to 1030 GM) because of the The binding behavior of probe 9 was proved by 19F and 1H NMR
incorporation of the triphenylamine groups. spectroscopy. The combined results of these experiments
These results revealed that structural modification of DPP showed the existence of two species 9a and 9b; finally, 9b was
with different donor units can change the sensitivity of probes found to be more stable than 9a. The interaction of probe 9 with
towards detection of F, and the modified-DPP probes can be F ions was based on the strong interactions of F ions with the
used for designing efficient optical probes for different analytes. sp2-hybridized boron center (Fig. 5). Probe 9 exhibits CT from
3.1.2 Fluoride probe based on fluoride–boron interaction. the fluorophore to the empty p-orbital of the boron atom;
Trivalent boron possesses inherent Lewis acidity and therefore however, after the addition of F ions, the CT was inhibited
forms strong covalent bonds with F. Trivalent boron is sp2 because of the occupied p-orbital at the boron atom.
hybridized with an empty p-orbital, and the interaction of 3.1.3 Fluoride probes based on cleavage of the Si–C bond.
boron with F occurs via donation of an electron pair on the The chemodosimeter approach involves an irreversible reaction
F anion into the empty p-orbital, resulting in a change in the between a dosimeter and a specific analyte that covalently binds to
structural geometry of the probe. the product or catalyzes the reaction. Thus, the reaction provides
Based on fluoride–boron interactions, Wang et al. synthesized significant changes in the absorption or emission spectra of the
a DPP-based colorimetric and ratiometric probe 9, bearing a probes. In the literature, a number of reports on chemodosimeters
pinacol boronate group via the Suzuki–Miyaura reaction for the for F that are based on the cleavage of the Si–O/Si–C bond due to
efficient detection of F ions (Fig. 5).13 A peak was observed at the great affinity of F for silicon (Si) are available.
535 nm in the emission spectrum of probe 9, and this peak Choi et al. utilized the chemodosimetric approach for recognizing
showed a five-fold enhancement of the emission intensity along F using DPP-based probes 10 and 11 with different alkyl chains
with a blue-shift from 535 nm to 527 nm after the addition of F bearing two trimethylsilyl groups (Fig. 6a).14 The design strategy

Fig. 5 Structure of fluorescent probe 9 and mechanism of F ion detection. Reproduced from ref. 13 with permission from Elsevier.

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exploited the high selectivity of F towards Si. The respective 12a and 12b, in solvents of different polarities (CDCl3 and DMSO),
absorption and emission maxima of probe 10 at 597 nm and at as elucidated using 1H NMR spectroscopy. Both isomers 12a and
617 nm were blue-shifted to 589 and 604 nm upon addition of F 12b selectively detected F ions via noncovalent interactions
(Fig. 6b and c). Probe 10 could be used for F detection not only between the amide NH protons of 2,6-dicarboxyamidopyridine
in solution but also in the solid state (e.g., test strips, glass films). macrocycle and F ions. These interactions result in the deproto-
The detection limit of probe 10 for F analysis was found to be nation of the amide cleft and induce a macrocycle shuttling
2  107 M. (Fig. 7b). The intensities of the absorption bands at 274, 525, and
3.1.4 Fluoride probes based on a preorganized rotaxane cavity. 564 nm of probe 12b in DMSO decreased, and new bands at
Lin et al. reported the synthesis of a DPP-stoppered-[2]rotaxane 257 and 457 nm appeared accompanied by a color change from
containing an orthogonal bifurcated pyridine–pyridine H-bonded pink to pale green (Fig. 7c). In contrast, the emission of probe 12b
motif, 12 (Fig. 7a).15 Probe 12 exists as two translational isomers, at 582 nm was quenched by the addition of F ions (Fig. 7d).
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Fig. 6 (a) Structures of fluorescent probes 10 and 11 and mechanism of F detection, (b) UV-Vis absorption, and (c) fluorescence spectral changes of
probe 10 upon addition of F.

Fig. 7 (a) Chemical structure of fluorescent probe 12, (b) representation of two translational isomers, 12a and 12b, in CDCl3 and DMSO and interaction
with F ions. (c) UV-visible absorption, and (d) fluorescence spectral changes of probe 12b upon the addition of F ions in DMSO. Reproduced from
ref. 15 with permission from American Chemical Society.

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The results indicate that the stimulated responses toward the physiological functions such as transmission of nerve impulses,
solvent and F ions were enhanced because of the presence of osmoregulation, metabolism, biomineralization, and signaling.
the preorganized cavity in the rotaxane unit of these probes. Optimal concentrations of these ions are essential for proper
3.1.5 Fluorescent probes for cyanide. Jang et al. reported a new growth and development, and any change in their concentrations
type of DPP-based fluorescent probe 13 for the selective detection of can be dangerous and may lead to various disorders in the body.
CN (Fig. 8).16 Addition of CN resulted in a decrease in absorbance Therefore, the development of optical probes for specific detection
at 442 nm, along with a corresponding increase at 550 nm, accom- of metal ions is a very important objective for protecting living
panied by a visible color change from green to red. Probe 13 systems and the environment from the toxic effects of metals.
exhibited the emission band at 512 nm which was quenched by 3.2.1 Probes for zinc. Zinc (Zn2+) is the first element of
addition of CN. The electron-withdrawing nature of the carbamate group 12 in the periodic table and the very plentiful transition
group makes the carbonyl carbon in the amide ring electron- metal ion in the human body. It is also an indispensable factor
deficient, and therefore CN attacks the carbonyl carbon because in various biological processes such as brain function, gene
of its high nucleophilicity. Probe 13 showed high selectivity for CN transcription, regulation of metalloenzymes, and immune
over other anions, indicating the specificity of the DPP units. function. Zinc plays a key role in the metabolism of RNA and
Thus, recent efforts in the development of DPP-based anion DNA, neural signal transmission, as well as gene expression.
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probes have provided new ideas for modification of the DPP core in Therefore, selective detection of Zn2+ is a highly desirable
the design and synthesis of new colorimetric and fluorescent anion pursuit in the fields of analytical chemistry and supramolecular
probes. The hydrogen atoms on the nitrogen of the amide units of chemistry.
DPP were utilized for the sensing of F and the electron-deficient Wang et al. synthesized fluorescence turn-on DPP-based
lactam ring of DPP was used for selective detection of CN ions. probe 1417 (Fig. 9a) bearing the N,N-di(pyridin-2-ylmethyl)-
Further introduction of electron-donating substituents onto the amine (DPA) group, and probe 1518 bearing the N,N-di(pyridin-
DPP moiety improves the ICT effect for better sensitivity towards 2-ylmethyl)ethane-1,2-diamine group (Fig. 10a) for Zn2+. Probe
F. Therefore, the present reports should prove useful for further 14 exhibited very weak red fluorescence at lem = 630 nm;
progress and modification of the sensing properties of DPP-based however, after complexation with Zn2+, a spectral blue-shift (Dl =
fluorescent probes. 70 nm) from 630 nm to 560 nm was observed with an enhance-
ment of the yellow fluorescence by 25-fold. Similar fluorescence
3.2 Probes for metal ions behavior was observed for probe 15 upon interaction with Zn2+. The
All living organisms require metal ions for their growth and large blue-shift was attributed to photo-induced electron-transfer
development given the significant role of metal ions in biological (PET) and ICT from the nitrogen atom to the DPP core through the
processes. Metal ions such as sodium, potassium, magnesium, conjugation pathway. On the other hand, probe 16 containing the
zinc, copper, manganese, iron, in particular, are vital for various acylated linker (Fig. 10b) exhibited no fluorescence enhancement

Fig. 8 Structure of fluorescent probe 13 and the mechanism of CN detection. Reproduced from ref. 16 with permission from John Wiley and Sons.

Fig. 9 (a) Structure of fluorescent probe 14 and the mechanism of binding with Zn2+ and (b) confocal fluorescence images of Zn2+ in HeLa cells: (top
(A1–A4)) incubated with a 10 mM solution of 14 for 30 min at 25 1C; (bottom (B1–B4)) incubated with a 10 mM of Zn2+ for 15 min. Bright-field transmission
images (A1 and B1) and double channel fluorescence images (A2 and B2: channels centered: 570  15 nm; A3 and B3: channels centered: 650  15 nm),
and overlap field (A4 and B4). Scale bar of HeLa cells 0 to 75 mM. Reproduced from ref. 17 with permission from Royal Society of Chemistry.

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Fig. 10 (a) Structure of probe 15 and mechanism of binding with Zn2+ and (b) structure of derivative 16. Reproduced from ref. 18 with permission from
Elsevier.

with Zn2+ under similar conditions because of the decrease in the addition of Hg2+, which was due to the conversion of the alkyne
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electron-donating capability of the conjugated N atom, which short- groups to carbonyl groups (Fig. 11b). On the other hand,
circuited the PET process. Probe 14 was the first DPP-based addition of Cu2+ also resulted in a decline of the absorption
fluorescent probe used for the successful detection of Zn2+ in HeLa intensity at 582 nm with an increase at 274 nm, 440 nm, and
cells using Leica TCSSP2 confocal fluorescence microscopy using 690 nm (Fig. 11b). The maximum emission of probe 17 at
both green and red channels (Fig. 9b). 603 nm was quenched by addition of Hg2+ and Cu2+ (Fig. 11c).
3.2.2 Probes for mercury and copper. Heavy and transition Thus, DPP-based fluorescent probes have emerged as promising
metal (HTM) ions have significant functions in many biological candidates not only for sensing anions, but also for the selective
systems and environmental processes. The fluorescent probes and sensitive detection of metal ions. Structural modification of the
for the recognition and the selective detection of HTM ions DPP core with specific metal binding units such as DPA, DEPA, and
attract much interest in supramolecular chemistry because of terminal alkynes generates DPP derivatives that lend themselves to
the importance of these ions. Among the HTM ions, fluorogenic fast, selective, and sensitive metal ion determination.
detection of copper (Cu2+) and mercury (Hg2+) is particularly
interesting because of their toxicity and vital role in biological
systems.19 3.3 Probes for reactive oxygen species and thiols
Choi et al. reported the DPP-based probe 17 with bis-alkyne 3.3.1 Probes for reactive oxygen species. Reactive oxygen
groups as a fluorescent chemodosimeter for the recognition of species (ROS) (e.g. hydrogen peroxide (H2O2), the hydroxyl
Hg2+ based on the Kucherov reaction (Fig. 11a).20 Probe 17 radical ( OH), peroxynitrite (ONOO), and hypochlorite (OCl))
exhibited absorption bands at 316 nm, 377 nm, 399 nm, are the byproducts of normal metabolism of oxygen and therefore
542 nm, and 582 nm, and addition of Hg2+ resulted in a display important roles in cell signaling and many biological
decrease in the intensity of the absorptions at 542 nm and processes in normal living cells. Among the various ROS, H2O2 is
582 nm, with a small increase at 341 nm and 422 nm. The band the most important species in living cells and is produced during the
at 582 nm underwent a bathochromic shift to 623 nm after activity of almost all oxidase enzymes. Imbalance in the production

Fig. 11 (a) Structure of fluorescent probe 17 and its interactions with Hg2+ and Cu2+; (b) UV-Vis absorption spectral changes; (c) fluorescence spectral
changes of probe 17 with Hg2+ and Cu2+.

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and consumption of H2O2 due to environmental stress or resulting in reduced p-conjugation. The recognition mechanism
genetic mutations results in its accumulation in the cellular was deduced by means of mass analysis, which demonstrated that
mitochondria, which is strongly related to serious diseases. along with Michael addition, the –CN group of the malonitrile
Therefore, it is essential to design and synthesize new fluorescent moieties reacted with thiols to form the 4,5-dihydrothiazole struc-
probes that not only recognize specific ROS, but can also detect the ture. It was found that probe 19 could be utilized as a ratiometric
intracellular overproduction of ROS in specific cells. probe for bio-imaging of intracellular thiols (Fig. 13b).
Choi et al. reported the DPP–tellurophene conjugate 18 Thus, this research indicates that DPP-based fluorescent
based on the redox properties of the tellurium atom for the probes are not strictly limited to the detection of anions and cations,
turn-on selective and sensitive fluorescence detection of H2O2 but are also functional for the determination of intracellular thiols,
over other ROS (Fig. 12).21 Probe 18 exhibited an absorption opening a new avenue for the design of DPP-based fluorescent
band at 610 nm that completely disappeared upon addition of probes for the recognition of intracellular thiols.
H2O2 accompanied by the appearance of a new small band at
982 nm. The fluorescence peak of probe 18 at ca. 565 nm was 3.4 Probes for pH, carbon dioxide and H2 gas
enhanced by titration with H2O2 with a spectral red-shift from Determination of pH is an extremely important issue because
565 nm to 579 nm. Moreover, the oxidized form of 18,
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pH plays a governing role in various physiological processes.

tellurophene-1-oxide, was reduced to regenerate the free probe The intracellular or extracellular pH sensitively affects many
18 in the presence of glutathione (GSH). Thus, probe 18 works cellular processes such as cell growth, various enzyme activities, and
as a reversible H2O2 probe that is responsive to the oxidative transport of ionic species through biological membranes.23
environment and could be reduced in the presence of GSH. The Alterations in the intracellular pH caused by metabolic processes
detection limits were determined to be 6.0 mM for H2O2 and such as metabolic acid production and membrane transport
8 mM for GSH. processes may lead to cellular dysfunctions. Therefore, determina-
Thus, combining the favorable features of DPP with tell- tion of extracellular/intracellular pH is very important for monitoring
urophene generates innovative optical probes that can detect the physiological and pathological status of organisms. Among
H2O2 with high selectivity and sensitivity. This approach should the various available methods for pH measurement, the use of
be useful for the further development of fluorescent probes for fluorescent probes is one of the most effective methods for pH
detecting ROS species. determination in solutions as well as in living cells. The impor-
3.3.2 Probes for sensing thiols. Thiols (e.g., cysteine, homo- tance of pH for both the environment and living systems is the
cysteine, and glutathione) are commonly used, highly active, reduced driving force for the design of new and efficient fluorescent
forms of sulfur found in biomolecules. A number of health problems probes for determination of the intracellular or extracellular pH.
are associated with thiol deficiency, including slow growth of 3.4.1 Probes for pH. Kanbara et al. reported that the DPP-
children, liver damage, and AIDS. On the other hand, high thiol based fluorescent probe 20 with morpholinyl moieties exhibited a
levels are responsible for the generation of several other undesired two-step fluorescence response against acid (Fig. 14).24 Titration of
syndromes. The detection of thiols by fluorescent probes proceeds probe 20 with trifluoroacetic acid (TFA) (0–1000 equiv.) induced a
by various mechanisms such as cyclization with aldehydes, cleavage decrease in the absorption at 373 nm and 522 nm and quenching
of sulfonamide and sulfonate esters, and Michael addition. There- of the emission spectra at B590 nm. With further addition of TFA
fore, a number of studies have focused on expanding the scope of (1250–5000 equiv.), a new fluorescence band was generated at
new fluorescent probes to the quantitative and selective detection of 540 nm. The fluorescence quenching might be attributed to ICT
thiols. because of the large dipole moment of the protonated species
Zhao et al. utilized the Michael addition mechanism in (20H+) relative to that of 20 and 202H+ with small dipole
constructing the DPP-based fluorescent probe 19 with a malo- moments. These experiments revealed that probe 20 undergoes
nitrile moiety (Fig. 13a).22 Probe 19 exhibited a blue-shift of the two-step protonation at the morpholinyl moieties. Probe 20 was
absorption bands and enhancement of the fluorescence emission further applied to the detection of acids in the gas phase as shown
after addition of cysteine (Cys). This is attributed to the Michael in Fig. 14b. Orange fluorescence could be observed on the silica gel
addition reaction of Cys to the ethylenic double bonds of the probe, sheet of probe 20; however, after exposure to formic acid, the

Fig. 12 Structure of probe 18 and its reaction with H2O2 and GSH.

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Fig. 13 (a) Structure of probe 19 and proposed mechanism of Cys detection; (b) differential interference contrast and fluorescence images of living
MDA-231 cells with probe 19; 1st row (i–iv): without probe 19, 2nd row (v–viii): MDA-231 cells incubated with 10 mM of probe 19, and 3rd row (xi–xii)
MDA-231 cells pretreated with 1 mM N-methylmaleimide and then incubated with 10 mM probe 19. Reproduced from ref. 22 with permission from
American Chemical Society.

Fig. 14 (a) Structure of fluorescent probe 20 and (b) photographs of a thin layer silica gel sheet showing the changes in the fluorescence of probe 20 in
the presence of formic acid and TFA vapor under UV light (l = 365 nm). Reproduced from ref. 24 with permission from Elsevier.

fluorescence disappeared. With further exposure to TFA vapor, an planar probe was intact. The probe beads in Eudragits RL100
intense yellow emission appeared. Based on the structure and polymer can be practically applied in microfluidic systems and
specific function of probe 20, new fluorescent probes could be also in fluorescence imaging and microscopy.
designed for application in materials chemistry. 3.4.2 Probes for CO2 gas. The same group reported the
Borisov et al. demonstrated the design and synthesis of the DPP-based pH sensitive indicator dyes 24–26 with sulfonamide
new pH indicators 21–23 (Fig. 15)25 based on deprotonation of groups for sensing CO2 (Fig. 16).26 These dyes showed strong
NH groups in the amide unit of DPP. These indicators could absorbance in the blue-green wavelength region and were
operate by means of two different mechanisms as shown in emissive in the neutral and the deprotonated forms. CO2
Fig. 15. Deprotonation of the phenolic groups in the indicators sensing both in the gaseous and liquid phase is of great
21 and 22 resulted in the PET effect, which was associated with importance in various fields such as environmental, oil, and
the fluorescence quenching in the pH range of 5.9–9.3 with no chemical industries. Electrochemical and infrared spectro-
alteration of the absorption spectra. At higher pH (9.7–11.6), scopic techniques are generally employed for CO2 sensing,
these indicators exhibit bathochromic shifts in the absorption but these processes are costly and time-consuming. Optical
spectra. On the other hand, the bathochromic shift in the chemoprobes for determination of the CO2 level are the most
absorption and emission spectra of the indicator 23 with a favorable analytical tools. These dyes, along with tetraoctylam-
morpholino group was ascribed to the deprotonation of the NH monium hydroxide, were embedded into ethyl cellulose to
groups in the amide unit of the DPP. By utilizing appropriate obtain plastic optical CO2 probes. The dyes were demonstrated
immobilizing polymer matrices, Hydromeds D4 (a commercially to be capable of working as colorimetric indicators for CO2,
available polyurethane-based hydrogel), poly(2-hydroxyethyl- exhibiting a blue color in the absence of CO2 and pink in the
methacrylate) in the form of planar sensors, and Eudragits presence of CO2 with a concomitant change in the fluorescence
RL100 (a positively charged acrylate polymer) in the form of color from deep red to orange. These indicators showed better
sensor nanobeads, these indicators worked as efficient pH probes CO2 sensing performance than the frequently used fluorescent
(e.g., planar or sensor beads) over a wide pH range from 5–12. The pH indicator, 8-hydroxypyrene-1,3,6-trisulfonate.
use of sensor beads was more effective than the use of planar 3.4.3 Probes for H2 gas. The development of H2 sensors is
sensors because of the larger surface area of the beads and higher of increasing importance because of its use as an energy carrier and
flexibility which can reveal instantaneous response whereas the as a chemical reactant. The development of new technologies based

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Fig. 15 Structures of DPP-based indicators 21–23 and two distinct mechanisms for pH-sensitivity.
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on H2 gas can overcome the complications of energy supply and revealed the usefulness of DPP derivatives containing a pyridyl
fuel reserve. However, in emerging technologies, it is highly unit as H2 sensors.
difficult to detect colorless, odorless, tasteless, and flammable
H2 gas leaks and accurately measure H2 gas during its produc-
tion, storage, and transportation. Thus, it is essential to develop 4. DPP-based fluorescent probes for
H2 sensors for safety monitoring. molecular imaging
Mizuguchi et al. reported a H2 gas sensor using DPP
derivative 27 containing pyridyl units (Fig. 16).27 The high Two-photon excitation fluorescence (TPEF) microscopy emerged
proton affinity of the N atoms of the pyridyl units significantly as the most promising technique for acquiring images of deep
affected the shade and electrical conductivities of compound 27. tissue (4500 mm) and is a non-invasive tool for living cell and
The H2 gas sensing behavior was evaluated in the vapor and tissue imaging. Recently, a number of reports have explored the
solution phases; the vapor phase showed a high tendency for two-photon excitation and two-photon absorption properties of
protonation, thus leading to substantial changes in H2 gas sensing. DPP-based conjugated molecules. The conjugation of the DPP
Furthermore, the effect of a high electric field on the dissociation of unit, having strong acceptor properties with different donor
H2 into protons in the presence of Pd or Pt was evaluated.28 It was units, was exploited in the development of efficient two-photon
observed that H2 dissociated exponentially with the electric field, probes. The DPP-based two-photon probes showed significant
which was further enhanced by the presence of a DPP moiety with a TPA cross-section values (B500–3000 GM), making them pro-
high affinity for protons. spectively useful for deep tissue imaging for disease detection.
Furthermore, Kratochvılova et al. reported the hydrogenation of Tian et al. synthesized the DPP-based hyperbranched polyyne
derivative 28, modulating its electronic properties such as ionization 29 with triphenylamine groups connected to the DPP unit and
potential, surface potential, and conductivity (Fig. 16).29 Upon the investigated its two-photon absorption properties (Fig. 17).30
interaction of the pyridyl units with protons, the conductivity of Upon excitation of 140 femtosecond laser pulses (l = 800 nm),
derivative 28 was significantly increased; however, the surface polymer 29 emitted strong frequency up-converted fluorescence
potential decreased, indicating its potential use as a H2 sensor. at 584 nm. Using the solution in tetrahydrofuran, the TPA cross-
Thus, it is crucial to develop optical probes for environmental section of polymer 29 was measured to be 579 GM per
and biological pH detection. By utilizing the strong absorbance repeating unit.
and fluorescence properties of the DPP core, efficient pH probes Yang et al. developed two donor–p–acceptor–p–donor type
have been developed. These pH probes provided new approaches DPP derivatives 30 and 31 with electron-donating diphenylamine
for the development of CO2 sensors. Further investigations and triphenylamine groups with TPA properties (Fig. 17).31

Fig. 16 Structures of DPP-based dyes 24–26 as CO2 indicators and DPP-based H2 sensing molecules 27, 28.

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These two derivatives had large TPA cross-sections (dmax = near-IR wavelength range, with maximum TPA cross-section
900–1200 GM) and emitted strong red two-photon excitation values of 2500–3000 GM at 940 nm.
fluorescence. These derivatives exhibited large bathochromic Bolze et al. reported the synthesis of DPP-based non-ionic
shifts because of enhancement of the ICT. Derivative 31 exhibited water-soluble TPEF microscopy dyes 33–39 (Fig. 18).33 The one-
weaker ICT than derivative 30, which indicates that weak photon photophysical properties of all of the synthesized dyes
electron donating benzene is not an effective conjugation in dichloromethane (CH2Cl2) and water gave rise to an absorption
bridge. Consequently, derivative 30 exhibited red emission at in the 470 nm region and intense green fluorescence at around
604 nm but with low quantum yield (f = 0.37) as compared to 530 nm. In the case of dye 37, the quantum yield decreased
derivative 31, which exhibited emission at 594 nm with a because of the remaining NH group, and the quantum yield of
slightly higher quantum yield (f = 0.46). These derivatives probe 37 dropped to 2% in water. The TPA cross-sections of
served as prototypes for the design of new DPP-based candidates probes 35 and 36 were very similar with a moderate effect of the
as two-photon fluorescent probes. DPP core substituent (150 GM at 830 nm). On the other hand,
Gryko et al. developed the new probe molecule 32 bearing a probes 38 and 39 with p-extended conjugated systems had
DPP bridge with two porphyrin units, which was synthesized by significantly increased TPA cross-sections (950 GM at 835 nm
coupling the porphyrin moiety to the DPP-alkyne partner for probe 38). These probes are highly photostable and were
Published on 04 september 2014. Downloaded on 24.10.2018 [Link].

(Fig. 17).32 Probe 32 displayed maximum fluorescence emission explored for their application in confocal and TPEF microscopic
at lmax = 710 nm and possesses large intrinsic TPA in the evaluation of HeLa cell cultures (Fig. 18).

Fig. 17 Structure of DPP-based TPA derivatives 29–32.

Fig. 18 (a) Structures of water soluble non-ionic DPP-based probes 33–39, two-photon excitation microscopy (TPEM) image of HeLa cells stained with
(106 M with 1% DMSO) (b) 35, (c) 36, (d) 38, and (e) 39. Reproduced from ref. 33 with permission from Elsevier.

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The structures of these fluorophores were further modified important advantages over the traditional dyes operating in the
for bio-conjugation with the amino group of biomolecules. The visible region. The NIR absorption of such dyes has been
same group reported the synthesis of DPP-based fluorescent primarily utilized in materials science; however, NIR fluores-
probes, such as monomers 40 and 41, and dimers 42 and 43 cence attracted considerable interest in cell biology because of
(Fig. 19).34 The authors utilized two approaches for the bio- the following reasons: (1) deep tissue penetration for cell
conjugation of these probes to the HIV Trans-Activator of recognition, (2) minimal autofluorescent background from
Transcription (Tat) protein. These bio-conjugates were used biological cells, (3) in vivo imaging, and (4) minimum photodamage
as two-photon fluorescent probes for imaging HeLa cells using to tissues. Various NIR dyes such as cyanines, squaraines, and
TPEF microscopy. In the first approach, the Tat (44-61) peptide BODIPYs have been developed; however, their applications are
was attached to the reactive end of the fluorophore 41 and in limited because of poor photostability or low fluorescence quantum
the second approach the Tat (44-61) peptide was attached to the yields. Therefore, the development of new NIR dyes with high
central DPP core 43 (Fig. 19). The Tat peptides 41-bAla–Tat and fluorescence quantum yields and good photostability provides
43-bAla–Tat tagged with the fluorophores were employed in avenues for various applications.
TPEF microscopic analysis of HeLa cell cultures (lex = 800 nm, Thus, the structural modifications of the DPP unit have been
laser power o5 mW) (Fig. 19c and d). These fluorescently carried out for the development of highly stable NIR dyes. The
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tagged Tat peptides are prospectively useful in microscopy for DPP core can be functionalized in many ways to provide suitable
living cells with low dose excitation (e.g. o1 mW for 43) and derivatives with desired properties. The carbonyl O atoms of DPP can
also in multicolor labeling of biomolecules utilizing optimized be replaced with different arylacetonitriles, S atoms, and aromatic
two-photon bio-conjugated fluorophores using two-photon amines.36 The reaction of carbonyl O atoms with arylacetonitriles is
excited microscopy (TPEM). interesting as it leads to p-expanded bathochromically shifted dyes
Thus, the development of two-photon probes is very attractive exhibiting absorption in the NIR wavelength region.
in light of their advantages such as deeper tissue penetration, Daltrozzo et al. modified the DPP core to synthesize cyanine-
and reduced photobleaching and photodamage during tissue type NIR absorbing dyes, pyrrolopyrrole cyanine (PPCy), con-
imaging. These reports are a source of valuable insights for the taining a pyrrolopyrrole moiety as the central component
construction of DPP-based two-photon probes with versatile (Fig. 20).37 The reaction of DPPs with heteroarylacetonitriles
applications. followed by the substitution of protons with BF2 or BPh2 group
rigidifies the chromophore to afford the corresponding NIR
dyes. These dyes exhibited fluorescence quantum yields of 0.59
5. Structurally modified DPP-based (lem = 773 nm) and 0.53 (lem = 831 nm) for 44a and 44a 0 ,
near-infrared (NIR) dyes respectively.
Using a similar strategy, a series of PPCys 45a–f (Fig. 20)38
Considerable efforts have been devoted to develop new near-infrared and bis(pyrrolopyrrole) cyanines 46–48 containing different
(NIR) dyes for applications in chemical biology35 and materials heterocyclic peripheral groups were synthesized (Fig. 21).39
science. Dyes with strong NIR absorption and emission offer Dyes 45a–f exhibited weak absorption in the visible spectral

Fig. 19 (a) and (b) Structures of DPP-based two photon fluorescent probes 40–43 and the structures of Tat (44-51) bio-conjugates 41-bAla–Tat and
43-bAla–Tat; (c) TPEM images of HeLa cells incubated in solutions of 41-bAla–Tat (i) 0.5 mM, (ii) 1 mM, (iii) 10 mM, and (d) TPEM image of HeLa cells
incubated in solutions of 43-bAla–Tat (iv) 1 mM. Reproduced from ref. 34 with permission from American Chemical Society.

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range, but strong and narrow absorptions in the NIR region Kobayashi et al. reported the synthesis of pyrrolopyrrole aza-
between 684 and 864 nm, high quantum yields, and good BODIPY analogs by the reaction of DPP with heteroaromatic
photostability. The photostability of dye 45e was compared to amines (pyridine; 50, benzothiazole; 51, and quinolone; 52) in
that of indocyanine green (ICG), which is used as a standard the presence of titanium tetrachloride (Fig. 22).41 In CHCl3,
for new NIR fluorophores. Dye 45e showed much better photo- these analogs exhibited red-shifted absorption bands at 638,
stability than ICG, indicating promising features for its appli- 655, and 671 nm and emission bands at 661, 676, and 692 nm
cation in cell biology. On the other hand, bis(pyrrolopyrrole) for 50, 51, and 52, respectively. These analogues exhibit high
cyanines 46–48 possess extended p-electron systems, thus fluorescence quantum yields of 40.8. These analogs may be
providing a very strong and narrow NIR absorption (in the useful in the field of molecular electronics and optoelectronics
range 815–941 nm) and no significant absorption in the visible and also in designing molecular probes for bioimaging.
spectral wavelength range. Dyes 47 and 48 are selective NIR Gryko et al. used a different strategy to expand the DPP core
absorbers, highly photochemical stable, and exhibited strong and synthesized a series of p-expanded S-shaped DPP-based
fluorescence, which could be successfully applied to many fluorescent dyes 53–58 (Fig. 23).42 The six-membered rings were
applications. formed by placing two vinylene moieties between the nitrogen
Furthermore, water-soluble PPCy dyes were synthesized as atoms and the aromatic substituents. The synthetic methodology
Published on 04 september 2014. Downloaded on 24.10.2018 [Link].

NIR fluorophores, 49a and 49b, and found to be more photo- involved the three step synthesis of DPP, followed by N-alkylation
stable than ICG (Fig. 22).40 Live cell imaging showed the easy and electrophilic substitution to aromatic rings. The final products
internalization of 49a and 49b into mammalian cells through were S-shaped, stable, p-expanded DPP analogues that were
endocytosis processes, demonstrating their advantages for obtained in good yields. This synthetic methodology could
imaging applications at the cellular level. be applied to the synthesis of various DPP-based dyes with

Fig. 20 Structures of DPP-based NIR dyes 44 and 45.

Fig. 21 Structures of DPP-based NIR dyes 46–48.

Fig. 22 Structures of NIR dye 49 and pyrrolopyrrole aza BODIPY derivatives 50–52.

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Fig. 23 (a) Structures of DPP-based fluorescent dyes 53–58, (b) structures of bithiophene functionalized DPP-based dyes 59–64 and (c) photographs of
the solutions of these dyes showing the decrease in fluorescence intensity based on the terminal groups. Reproduced from ref. 43 with permission from
American Chemical Society.

electron-rich or electron-neutral aromatic and heteroaromatic absorption and fluorescence spectra of dyes 65a–g were acquired
rings. The dyes displayed spectroscopic properties that differed in CH2Cl2; the absorptions and emissions were, respectively,
from that of the DPP core. The dye 57 was further used for labeling bathochromically shifted in the range of 451–518 nm and 528–
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mammalian cells indicating its potential for bio-applications. 642 nm. These dyes display an average Stoke’s shift of 70–80 nm,
The effect of substitution on the optical properties of except for 65c for which the Stoke’s shift was 124 nm. The optical
bithiophene-functionalized DPP-based dyes was explored by properties of pigments 66a–g were also recorded in DMSO,
Wurthner et al. who reported the synthesis of a series of DPP- where bathochromic shifts of the absorption spectra (in the
based dyes 59–64 with electron-donating groups (Me, SMe, OMe, range of 540–566 nm) and emission spectra (in the range of 550–
NMe2) and an electron-withdrawing group (CN) (Fig. 23).43 The 629 nm) were observed. These dyes are prospective candidates
effect of substituents on the electronic properties of the respective for use as molecular probes for bio-imaging.
DPP chromophores was evaluated using UV-Vis absorption and Wang et al. reported the synthesis of four symmetric DPP
fluorescence spectroscopy and cyclic voltammetry. All of the synthe- derivatives with substituents 67–70 (Fig. 24b).45 In THF, these
sized dyes exhibit a broad absorption band that covers the visible derivatives (67, 68, 69, and 70) exhibited absorption bands at
and NIR region (i.e., 450–800 nm) in CH2Cl2. The fluorescence 496 nm, 485 nm, 482 nm, and 470 nm. The green emission
spectra of these dyes covered the range of 637–753 nm. Among bands of these derivatives in THF were located at 575 nm,
these dyes, dye 63 displayed absorption at ca. 684 nm and emission 555 nm, 530 nm, and 527 nm. The effects of alkaline conditions
at 753 nm because of the incorporation of the strong electron on derivatives 68 and 69 were evaluated, illustrating remarkable
donating group (NMe2) into the dye, which resulted in planariza- changes in the photophysical properties. The UV-Vis absorption
tion of the bithiophenes through p-extended conjugation to the and emission spectra of derivative 68 were blue-shifted by
DPP electron acceptor. The fluorescence intensity based on the 20 nm in the presence of sodium hydroxide. On the other
substituents decreased in the order: H 4 Me 4 CN 4 SMe 4 hand, derivative 69 displayed a red-shift of the absorption with
OMe 4 NMe2 (Fig. 23c). The intriguing properties of these dyes no change of the emission because of the increased electron-
should motivate the synthesis of new candidate materials for donating ability of 69. These derivatives may be explored for the
organic photovoltaics. design and synthesis of new optoelectronic materials and may
Similarly, the effect of peripheral substituents around the also be used in bio-imaging because of their good solubility in
DPP core on the optical properties of dyes was evaluated by water derived from the oligoethyleneglycol chains.
Borsato et al. who synthesized a new class of soluble DPP-based Thus, the present research shows that structural modification
dyes with di-tert-butyl dicarbonate (t-Boc) groups 65a–g. After of DPP may be exploited to generate a series of functionalized
removal of the t-Boc groups by thermal treatment, the new DPP-based dyes with versatile optoelectronic properties that
insoluble pigments 66a–g were formed (Fig. 24a).44 The UV-Vis function as laser dyes, NIR dyes, and have prospective application

Fig. 24 (a) Structure of DPP-based dyes 65a–g, pigments 66a–g and (b) structures of symmetric DPP derivatives 67–70.

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in the synthesis of new DPP-based organic molecules for opto- accompanied by the formation of a new band at 625 nm that
electronic and biological application. corresponds to the deprotonated species. The color of the
solution changed from red to purple (Fig. 26b). Initial addition
of F resulted in fluorescence quenching at 570 nm, and
6. DPP-based polymers as fluorescent further addition of F resulted in the appearance of a new
probes emission peak at 656 nm, corresponding to the deprotonated
DPP species (Fig. 26c). The unalkylated imide groups in DPP
Recently, Lin et al. reported the synthesis of mechanically inter- function as emission traps for excited state transport, and F
locked polymeric architectures (MIPAs) possessing a 9-alkylidiene- disrupts the electronic energy transfer by acting as a quencher.
9H-fluorene monomer, DPP linked with a dumbbell unit, metalated Furthermore, polymer 75 was used for the fabrication of
[2]rotaxane, demetalated [2]rotaxane and simple alkyl chain- nanoparticles with diameters ranging from 100–200 nm that
tethering monomers 71–74 (Fig. 25a).46 In THF, polymer 73 were applied to live cell imaging.
exhibited absorption bands at 642 nm and 725 nm and an Confocal laser scanning microscopy was used to study the
emission band at 766 nm. After addition of TFA, the intensity of applicability of polymer 75 nanoparticles for real-time imaging
using KB tumor cells. Initially, no fluorescence signal was
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the absorption and emission bands decreased simultaneously.

The binding constant of polymer 73 with TFA was found to be detected; however, with elapse of the incubation time, red
1.22  105 M1. The complexation of TFA with the orthogonal fluorescence signals were observed (Fig. 26d). This scientific
H-bonded unit of polymer 73 was reversible in the presence of contribution is potentially useful for the design and synthesis
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and this on–off–on of DPP-based fluorescent polymers for diverse applications in
cycling process was reversible for up to four cycles. The rever- sensing and bio-imaging.
sible self-assembly process of polymer 73 was substantiated by He et al. reported three water soluble conjugated polyfluor-
SEM and TEM analysis. SEM images of polymer 73 illustrated ene polymers 76–78 in which the ratio of fluorene to DPP units
spherical vesicular type particles (Fig. 25b) which converted to varied (Fig. 27).48 The fluorene: DPP ratios of these polymers
distinctive flake and nanofiber structures after protonation were 95 : 5, 90 : 10, and 50 : 50 for 76, 77, and 78, respectively.
with TFA (Fig. 25c). The reversibility was achieved by treating Intramolecular energy transfer from the fluorene units to the
polymer 73–TFA with DBU to regenerate the spherical vesicles DPP units took place, and aqueous solution of the polymers
observed in the initial SEM images of polymer 73 (Fig. 25d). displayed red-shifted emissions. Polymer 77 has strong electro-
Polymer 73 underwent a controllable self-assembly process static interaction with Cy5-labeled ssDNA and the fluorescence
on the nanoscale, achieved by a reversible acid–base stimulated intensity of red Cy5 dye significantly increased through fluores-
molecular switch mechanism. The orthogonal H-bonded cence resonance energy transfer.
[2]rotaxane side chain unit in polymer 73 originally exists as Cao et al. demonstrated the synthesis of two cationic con-
metal-free vesicle-shaped particles and further morphs into jugated polyelectrolytes (CPEs) 79 and 80 having fluorene, DPP,
hexagonal nano-objects (e.g. sheets and fibers) in the presence and diacetylene moieties (Fig. 28a).49 The interaction of these
of acid (Fig. 25e). polymers with DNA was studied by UV-Vis absorption and
Recently, Hua et al. reported the synthesis of the conjugated fluorescence spectroscopy. The emission maximum of polyelectrolyte
polymer 75 with DPP (electron acceptor) and fluorene mono- 79 was present at 417 nm (originating from the fluorene ethynylene
mers (electron donor) (Fig. 26a)47 for the sensitive and selective segment). After the addition of DNA, the emission intensity at
detection of F. The UV-Vis absorption spectrum of polymer 75 417 nm decreases, accompanied by the appearance of a new
(7.5 mg mL1) in THF was characterized by a transition band at emission band at 431 nm (originating from the DPP unit). The
511 nm, the intensity of which decreased upon addition of F, interaction of positively charged polyelectrolytes with negatively

Fig. 25 (a) Structures of mechanically interlocked DPP-based polymers 71–74 and SEM images of (b) polymer 73, (c) 73 with TFA, and (d) 73–TFA with
DBU, (e) graphical representation of a possible hierarchical self-assembly process of polymer 73 on the nanoscale. Reproduced from ref. 46 with
permission from American Chemical Society.

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Fig. 26 (a) Structure of DPP-based conjugated polymer 75; (b) UV-Vis absorption; (c) fluorescence titration spectra of probe 75 with addition of F in
THF; (d) real-time fluorescence images of polymer 75 nanoparticles incubated in KB cells for: (i) 30 min, (ii) 60 min, (iii) 90 min, and (iv) 120 min.
Reproduced from ref. 47 with permission from Elsevier.

efficient quenching of the blue emission (from the fluorine

ethynylene-triphenylamine units) than the red emission (from the
DPP segments), accompanied by a color change from light blue to
light red. Hyperbranched polymer 82 showed better sensitivity for
DNA detection because of the presence of a large number of
branches in the polymer structure, which facilitated exciton migra-
tion and energy transfer than the linear polymer 81 (Fig. 29b).
Liu et al. reported the synthesis of anionic conjugated
polyelectrolytes 83 and 84 consisting of water soluble DPP
Fig. 27 Structures of water soluble conjugated polymers 76–78.
derivatives linked to phenylenevinylene (PV)/phenylene ethyny-
lene (PE) units (Fig. 30a).51 The absorption maxima of these
charged DNA results in complex formation as a result of polyelectrolytes were red-shifted in water (from 524 nm to
cooperative electrostatic attractions, which leads to aggregation 547 nm for 83 and from 507 nm to 522 nm for 84). Fluorescence
of the DPP-containing conjugated polyelectrolyte. Fluorescence quenching of polyelectrolyte 83, in contrast to 84, was observed
resonance energy transfer from the blue-emitting fluorene in aqueous solutions. This change is attributed to J-aggregation
ethynylene segments to the DPP units resulted in the observed in the polyelectrolytes. The hydrophobic main-chains in the
visible color change from blue to red allowing efficient detection polyelectrolytes interact strongly to form stacked layers and the
of DNA (Fig. 28b). hydrophilic sulfonium side chains are freely oriented to water
To improve the sensitivity of CPEs towards DNA, the water molecules. This J-aggregation results in a higher conjugative
soluble cationic polymers 81 and 82 with fluorene, DPP, triphenyl- effect, with facile transfer of excitons between the main chains,
amine, and diacetylene units in the main chain were synthesized for which might account for the fluorescence quenching in 83. The
studying the detection capability of DNA (Fig. 29a).50 Similar UV-Vis surfactochromicity of these polyelectrolytes was further evaluated
absorption spectral changes were observed as in the case of polymers in different solvents and in the presence of cationic surfactants or
79 and 80. Interaction of polymer 82 with CT DNA resulted in more polymer non-ionic surfactants. In the presence of oppositely

Fig. 28 (a) Structures of water soluble cationic polyelectrolytes 79 and 80, (b) pictorial representation of the analysis of optical changes caused by
complexation of 79 and 80 with CT DNA. Reproduced from ref. 49 with permission from John Wiley and Sons.

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Fig. 29 (a) Structures of water soluble cationic polymers 81 and 82; (b) pictorial representation of fluorescence amplification of (b) polymer 81 and (c)
polymer 82. Reproduced from ref. 50 with permission from Elsevier.

Fig. 30 (a) Structures of water soluble anionic conjugated polyelectrolytes 83 and 84; UV-Vis absorption and emission spectra of polyelectrolytes in
aqueous solutions with different concentrations of PVP: (b) polyelectrolyte 83, and (c) polyelectrolyte 84. Reproduced from ref. 51 with permission from
John Wiley and Sons.

charged surfactants, the homogeneous aggregates were broken Tremendous research effort has been expended in the develop-
and heterogeneous aggregates between the polyelectrolytes ment of DPP-related chemistry and for utilizing DPP derivatives
and the surfactant were formed, resulting in destruction of in the design of highly efficient materials for various applica-
the J-aggregation. tions. This manuscript summarizes the progress achieved from
This results in variation of the conjugation length of the 2009 to the present time in the design and development of DPP-
polymers and therefore lowering of the energy dissipation based fluorescent probes. As described herein, considerable
between the main chains, followed by fluorescence enhance- achievements have been accomplished by researchers in the
ment. The fluorescence of these polyelectrolytes could be exploration of DPP-based fluorescent probes and their applica-
enhanced by adding the specific non-ionic polymer surfactant tions in the detection of biologically important species (e.g.,
polyvinylpyrrolidone (PVP) (Fig. 30b and c). anions, cations, thiols, ROSs and gases).
The structures of different DPP-based fluorescent probes,
detection mechanisms, optical properties, and other miscellaneous
7. Conclusions applications were discussed, demonstrating that the application of
the DPP acceptor is not strictly limited to the creation of new
DPP is an emerging dye component in supramolecular chemistry organic materials for materials chemistry, but can also be exploited
that has a vast array of possibilities for functionalization. in the design of new derivatives for a plethora of applications.

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The applications of DPP-based small molecules and polymers 18 G. Zhang, S. Bi, L. Song, F. Wang, J. Yu and L. Wang, Dyes
in the development of two-photon probes, NIR dyes, and DNA Pigm., 2013, 99, 779–786.
detection were discussed. It is anticipated that this research 19 G. Aragay, J. Pons and A. Merkoci, Chem. Rev., 2011, 111,
area will become more active and productive and that the 3433–3458.
studies highlighted in this review will emphasize the great 20 M. Kaur and D. H. Choi, Sens. Actuators, B, 2014, 190,
potential of the DPP moiety to researchers. This is the first 542–548.
review focusing on DPP-based fluorescent probes, and it is 21 M. Kaur, D. S. Yang, K. Choi, M. J. Cho and D. H. Choi, Dyes
hoped that the information provided herein will provide new Pigm., 2014, 100, 118–126.
directives for the chemistry scientific community in the 22 L. Deng, W. Wu, H. Guo, J. Zhao, S. Ji, X. Zhang, X. Yuan and
exploration of novel ideas for structural modification of DPP C. Zhang, J. Org. Chem., 2011, 76, 9294–9304.
for the design and synthesis of new fluorescent probes to 23 M. Schaferling, Angew. Chem., Int. Ed., 2012, 51, 3532–3554.
extend the range of practical applications. 24 T. Yamagata, J. Kuwabara and T. Kanbara, Tetrahedron Lett.,
2010, 51, 1596–1599.
25 D. Aigner, B. Ungerbock, T. Mayr, R. Saf, I. Klimant and
Acknowledgements S. M. Borisov, J. Mater. Chem. C, 2013, 1, 5685–5693.
Published on 04 september 2014. Downloaded on 24.10.2018 [Link].

26 S. Schutting, S. M. Borisov and I. Klimant, Anal. Chem.,

This work was supported by the National Research Foundation
2013, 85, 3271–3279.
of Korea (NRF2012R1A2A1A01008797) and by Key Research
27 J. Mizuguchi, T. Imoda, H. Takahashi and H. Yamakami,
Institute Program through the National Research Foundation
Dyes Pigm., 2006, 68, 47–52.
of Korea (NRF) funded by the Ministry of Education, Science
28 H. Takahashi and J. Mizuguchia, J. Appl. Phys., 2006,
and Technology (NRF20100020209).
100, 034908.
29 O. Salyk, J. Vynuchal, I. Kratochvılova, T. Todorciuc,
J. Pavluch and P. Toman, Phys. Status Solidi A, 2010, 207,
References
2327–2333.
1 X. Li, X. Gao, W. Shi and H. Ma, Chem. Rev., 2014, 114, 30 Y. Jiang, Y. Wang, J. Hua, S. Qu, S. Qian and H. Tian,
590–659. J. Polym. Sci., Part A: Polym. Chem., 2009, 47, 4400–4408.
2 A. Iqbal and L. Cassar, US Pat., 4415685 A, 1983. 31 E. Q. Guo, P. H. Ren, Y. L. Zhang, H. C. Zhang and
3 Z. Hao and A. Iqbal, Chem. Soc. Rev., 1997, 26, 203–213. W. J. Yang, Chem. Commun., 2009, 5859–5861.
4 Y. Li, P. Sonar, L. Murphy and W. Hong, Energy Environ. Sci., 32 A. Nowak-Krol, M. Grzybowski, J. Romiszewski, M. Drobizhev,
2013, 6, 1684–1710. G. Wicks, M. Chotkowski, A. Rebane, E. Gorecka and
5 C. B. Nielsen, M. Turbiez and I. McCulloch, Adv. Mater., D. T. Gryko, Chem. Commun., 2013, 49, 8368–8370.
2013, 25, 1859–1880. 33 H. Ftouni, F. Bolze and J.-F. Nicoud, Dyes Pigm., 2013, 97,
6 D. Chandran and K.-S. Lee, Macromol. Res., 2013, 21, 77–83.
272–283. 34 H. Ftouni, F. Bolze, H. de Rocquigny and J.-F. Nicoud,
7 O. Wallquist, R. Lenz, in High Performance Pigments, ed. Bioconjugate Chem., 2013, 24, 942–950.
E. B. Faulkner and R. J. Schwartz, Wiley-VCH Verlag, 2009, 35 S. Luo, E. Zhang, Y. Su, T. Cheng and C. Shi, Biomaterials,
pp. 165–194. 2011, 32, 7127–7138.
8 L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. 36 R. Lenz and O. Wallquist, Surf. Coat. Int., Part B, 2002, 85,
Rev., 2013, 42, 622–661. 19–26.
9 Y. Qu, J. Hua and H. Tian, Org. Lett., 2010, 12, 3320–3323. 37 G. M. Fischer, A. P. Ehlers, A. Zumbusch and E. Daltrozzo,
10 Y. Qu, S. Qu, L. Yang, J. Hua and D. Qu, Sens. Actuators, B, Angew. Chem., Int. Ed., 2007, 46, 3750–3753.
2012, 173, 225–233. 38 G. M. Fischer, M. Isomaki-Krondahl, I. Gottker-Schnettmann,
11 C. Yang, M. Zheng, Y. Li, B. Zhang, J. Li, L. Bu, W. Liu, E. Daltrozzo and A. Zumbusch, Chem. – Eur. J., 2009, 15,
M. Sun, H. Zhang, Y. Tao, S. Xue and W. Yang, J. Mater. 4857–4864.
Chem. A, 2013, 1, 5172–5178. 39 G. M. Fischer, E. Daltrozzo and A. Zumbusch, Angew. Chem.,
12 Y. Li, M. Zheng, J. Wang, Y. Gao, B. Zhang and W. Yang, Int. Ed., 2011, 50, 1406–1409.
Dyes Pigm., 2014, 104, 97–101. 40 S. Wiktorowski, C. Rosazza, M. J. Winterhalder, E. Daltrozzo
13 G. Zhang, L. Wang, X. Cai, L. Zhang, J. Yu and A. Wang, Dyes and A. Zumbusch, Chem. Commun., 2014, 50, 4755–4758.
Pigm., 2013, 98, 232–237. 41 S. Shimizu, T. Iino, Y. Araki and N. Kobayashi, Chem.
14 M. Kaur, M. J. Cho and D. H. Choi, Dyes Pigm., 2014, 103, Commun., 2013, 49, 1621–1623.
154–160. 42 M. Grzybowski, E. Glodkowska-Mrowka, T. Stoklosa and
15 M. V. R. Raju and H.-C. Lin, Org. Lett., 2013, 15, 1274–1277. D. T. Gryko, Org. Lett., 2012, 14, 2670–2673.
16 Y.-H. Jeong, C.-H. Lee and W.-D. Jang, Chem. – Asian J., 2012, 43 H. Burckstummer, A. Weissenstein, D. Bialas and
7, 1562–1566. F. Wurthner, J. Org. Chem., 2011, 76, 2426–2432.
17 G. Zhang, H. Li, S. Bi, L. Song, Y. Lu, L. Zhang, J. Yu and 44 R. Beninatto, G. Borsato, O. De Lucchi, F. Fabris,
L. Wang, Analyst, 2013, 138, 6163–6170. V. Lucchini and E. Zendri, Dyes Pigm., 2013, 96, 679–685.

76 | Chem. Soc. Rev., 2015, 44, 58--77 This journal is © The Royal Society of Chemistry 2015
 View Article Online

Tutorial Review Chem Soc Rev

45 G. Zhang, L. Song, S. Bi, Y. Wu, J. Yu and L. Wang, Dyes 49 S. Lin, S. Liu, H. Zou, W. Zeng, L. Wang, R. Beuerman and
Pigm., 2014, 102, 100–106. D. Cao, J. Polym. Sci., Part A: Polym. Chem., 2011, 49,
46 M. V. R. Raju, P. Raghunath, M.-C. Lin and H.-C. Lin, 3882–3889.
Macromolecules, 2013, 46, 6731–6743. 50 S. Lin, S. Liu, F. Ye, L. Xu, W. Zeng, L. Wang, L. Li,
47 Y. Qu, Y. Wu, Y. Gao, S. Qu, L. Yang and J. Hua, Sens. R. Beuerman and D. Cao, Sens. Actuators, B, 2013, 182,
Actuators, B, 2014, 197, 13–19. 176–183.
48 F. He, L. Liu and L. Li, Adv. Funct. Mater., 2011, 21, 51 H. Shen, C. Kou, M. He, H. Yang and K. Liu, J. Polym. Sci.,
3143–3149. Part A: Polym. Chem., 2014, 52, 739–751.
Published on 04 september 2014. Downloaded on 24.10.2018 [Link].

This journal is © The Royal Society of Chemistry 2015 Chem. Soc. Rev., 2015, 44, 58--77 | 77