M.J.CONDENSED.MATER VOLUME 11, Number 2 July 2009
Single-Molecule Fluorophores as Environmental Nanoprobes
N. Liua, Z. Lua, H. Ougaddouma, H. Wanga, R. Webera, J. Williamsa, Z. Yanga,
R. Twiega Samuel J. Lordb
aKent State University, Department of Chemistry
Kent, Ohio USA 44242
bStanford University, Department of Chemistry
Stanford, California USA 94305
The DCDHF dyes are under development as a promising new class of single-molecule fluorophores. The utility of these
fluorophores is derived from a range of attributes including their synthetic accessibility, structural versatility and
photostability, all permitting a range of practical labelling applications. Their polarity- and viscosity-dependent emission,
required for their application as local environment nanoprobes, is of particular interest. A wide range of DCDHF
chromophores with different pi-systems have been prepared for control of absorption and emission characteristics. Structure-
function analysis has been performed in conjunction with theoretical evaluations of these chromophores.
Key words: Single molecule, DCDHF fluorophore, environmental sensitivity.
I. Introduction
Single-molecule detection techniques have
undergone intensive development for both spectroscopic
characterization and for imaging of individual fluorescent
molecules.1-8 Contemporary single-molecule techniques offer
several advantages in comparison with more conventional
imaging and spectroscopic methods. First, the observation of
individual molecules removes the ensemble-averaging
characteristics encountered in bulk experiments and permits
the extraction of extra information which, in turn, may reveal
otherwise hidden processes.9-13 For example, in a molecular
beacon study8, if some of the beacons exist in a closed state
while the remaining beacons exist in an open state then an
ensemble characterization will only reveal some average
properties of the fluorescence which are of little value.
However, a single-molecule imaging study of the same
beacon system, wherein the open and closed beacons are
observed individually, would permit the construction of a
frequency histogram for the actual distributions of both
closed and open beacon states. A second benefit of single-
molecule techniques involves removal of the need for
synchronization of many single molecules undergoing a time-
dependent process. Thus, single-molecule techniques could
provide a more detailed picture of some biological process so
that the exact mechanism could be elucidated.2 Third, and
more specifically, the observation of single molecules offers
the opportunity to collect information about the nanoscale
environment around an individual probe
molecule. Thus, a molecule may report on its unique local
environment by a change in its fluorescence intensity (related
to changes of the lifetime of the states from which
fluorescence occurs) and/or the energy of the fluorescent
photons (related to the changes in the relative energies of the
various electronic states involved). The environmental
influence might come from a variety of sources including the
surrounding viscosity and polarity and single-molecule
probes are particularly useful for the analysis of
inhomogeneities in such systems. A straightforward example
is found in the analysis of single-molecule chromophores in
glassy polymer matrices wherein information about the
distribution of local free volume is revealed instead of just the
average free volume (as is experimentally reflected in the
distributions of lifetimes of the exited state responsible for
fluorescence).14
Very generally, single-molecule imaging should be
very difficult to achieve since it demands the detection of
emission from an individual source above any background
signals in a system swamped with an overwhelming number
of host molecules as well as impurities in the focal volume
containing the molecule of interest. There are instrumental
challenges as well, including photon losses from filters and
optical components, scattered light at the incident wavelength
and detector dark current. Fortunately, the detection of single
molecules can be greatly simplified by utilization of
fluorescence and, as a result, more routine optical techniques
become relevant and sufficient. Here the photons resulting
from a fluorescence process are shifted to a longer
wavelength relative to the absorption (the Stokes shift) and
pump wavelength and are thus much more easily
discriminated against the background. In order to increase the
contrast of the fluorescence signal to background, one must
also strive to improve the photophysical properties of the
fluorophore so that a signal-to-noise ratio (SNR) for single-
molecule signal is sufficient to obtain enough fluorescence
information over a reasonable collection time.
Single-molecule techniques continue to evolve and
have become increasingly widespread and sophisticated. For
example, two-photon pumping can successfully be utilized on
the single-molecule level for fluorophores with sufficient
two-photon cross sections.15 Single-molecule detection is also
polarization sensitive and can even observe the orientations
of suitable optically anisotropic molecules relative to an
established experimental frame of reference.16 A particularly
exciting recent development involves new superresolution
techniques that may offer the opportunity to examine single
molecules in the sub diffraction limited regime.17,18
11 90 ©2009 The Moroccan Physical and Condensed Matter Society

91 N. Liu et al. 11
Fluorophores useful for applications in single-
molecule studies must have a large absorption cross section
so as to absorb excitation light efficiently, must have weak
bottlenecks into dark states (such as triplet states), should
have a high fluorescence quantum yield to emit fluorescence
efficiently (or, in special cases, a variable quantum yield
might be desirable), and finally, must have high
photostability so that sufficient emitted photons are collected
before the fluorophore ultimately photobleaches. At room
temperature, these requirements have already been fulfilled
by fluorescent labels based on laser dyes (such as
rhodamines, cyanines, oxazines, etc) as applied to many
biological applications and also by derivatives of some rigid
polynuclear aromatic hydrocarbons such as terrylene or
perylene. Other classes of fluorescent substances that have
proven useful for single-molecule imaging are derived from
naturally occurring proteins (GFP, the green fluorescent
proteins, as well as their engineered derivatives)19 and from
quantum dots.8 Even color center defects have been utilized
as “single-molecule” sources.20
II. Experimental results
As single-molecule techniques become more
sophisticated, fluorophores are now required that are not only
suitable for imaging the localization of the dye but are also
able to offer additional beneficial properties and reporting
function. Since an important goal of single-molecule
spectroscopy is to collect additional information from the
fluorescence emission, fluorophores sensitive to their local
environment may also serve as nanoscale reporters for
information about their immediate environment. Also,
fluorophores with good synthetic flexibility would allow
introduction of a wider range of functional groups, and thus
may be utilized in many different applications requiring
covalent attachment or other specific interactions. Many of
these demands are met by the DCDHF fluorophores.
Hereafter, we concentrate on a few of these requirements in
more detail. More specifically, we will concentrate on
representative structure variations, which influence the
quantum yield and wavelength of fluorescence and, in turn,
influence their nano-reporter function.
The general structure of a DCDHF dye is found in Fig
1. The individual chromophore (the electronic/photonic active
part) is found within the ellipse and is comprised of an R1,R2-
disubstituted amine donor, a π-link (some combination of
carbocyclic or heterocyclic aromatic rings and alkenes) and
the DCDHF (dicyanomethylene dihydrofuran) ring with three
acceptor cyano groups and additional R3, R4 groups. The
various combinations of the amine donor, the π-system and
the acceptor have primary influence on the electronic,
absorption and emission properties of the fluorophore. The
donor part of the molecule is relatively electron rich (the
nitrogen has a pair of electrons in an available lone pair) and
the acceptor part of the molecule is relatively electron
deficient (it contains heteroatoms with hybridizations and
configurations that attract and stabilize electron density). The
FG1 through FGn are additional functional groups attached to
the chromophore at various locations (in the R1, R2 donor
tails, on the π-core or in the R3 or R4 substitutents of the
acceptor). These FGs tend to serve two main roles. First, the
modifying FG will influence the lipophilicity of the
chromophore, i.e., where it will tend to distribute itself in an
environment with varying polarity. For example, the FG may
contain polar or even ionic sites (alcohols, carboxylic acids,
sulfonic acids, etc.), which tend to enhance miscibility of the
dye in an aqueous environment or the FG could be just simple
long aliphatic tails, which would enhance miscibility of the
dye in a nonpolar environment, as in a membrane.37 Second,
the FG may be reactive groups (maleimide, succinimide ester,
etc.) permitting covalent attachment of the DCDHF
fluorophore to biomolecules or other substrates. As an
additional level of complexity, the system may exist as a
monomer (m=1 and then with no need for an X linking entity)
or a dimer (m=2) with X as some linking structure which
organizes the individual chromophores spatially (control of
orientation and distance with implications on energy transfer
interactions of the individual chromophores). There are still
more complex systems, which are not
O
NC CN
NC
R3 R4
N
R1
R2
pi
FG1
FGn m
X

Fig. 1 The General Structure Features of a DCDHF
Chromophore.
adequately represented by the simple cartoon in Fig 1. One
such dimer system is a molecular beacon in which the “X”
component might be a very complicated biological system
such as a peptide or nucleic acid. Here the fluorophores
terminating such a system may be identical but often they
must be different and with their respective electronic
properties tuned for highly specific interactions (such as
FRET, Fluorescence Resonance Energy Transfer, etc.).
This DCDHF-type chromophores were first
synthesized for electro-optical applications (here they are
sometimes called TCF dyes)21 and have been subsequently
applied in a number of different fields including as
photorefractives and media for THZ generation.22-28 Many of
the DCDHF chromophores exhibit the interesting ancilliary
property of monolithic glass formation and it was in the
course of the studies of the photorefractive properties of these
chromophores that their attractive fluorescence properties
were first manifested. Subsequent studies to date have
revealed that this family of fluorophores can also serve as a
single-molecule imaging dye.14,30-32
A general synthetic route for DCDHF dyes is shown
in Fig. 2. For fluorophores with only a single phenyl ring in
the π-system (5), the synthesis can be accomplished via two
different methods. The α-ketol was synthesized by the

93 N. Liu et al. 11
that might contribute to a TICT (Twisted Intramolecular
Charge Transfer) state: the amine-aryl twist (α), the aryl-
dihydrofuran twist (β) and the dicyano-dihydrofuran twist (δ).
N
R2
R1
O
NC
NC
CNα β
δ
Γ 0
S1
hυk1 k2
A
B
Fig. 4: (A) Possible dihedral twists in a DCDHF fluorophore.
Note that the bond orders shown in the figure represent just
one limiting resonance form (at all of the the sites of rotation
indicated, the single bonds possess double bond character and
the double bonds have single bond character); (B) Proposed
energy level scheme and transitions to account for the
photophysics of DCDHF fluorophores (see reference 14 for
details).
The preliminary calculations indicated that the
amine-aryl rotation  has less influence on the energy of
excited state while the aryl-furan and dicyano-furan rotations
would lower the energy of the excited state. So, the following
mechanism (Fig. 4B) was proposed: the molecule is excited
by light from ground state (Γ0) into a Frank-Condon state S1
without a geometry change (Frank-Condon Rule). Given
sufficient time, the molecule can relax into one of the two
excited states through a change in δ or a change in β. From
either of these two excited states, the molecule can then relax
back to the ground state, reversibly changing the geometry
back to the original form (Γ0). So, there are two main
pathways to relax the excited state. And the calculations
indicate that the S1 state has lower energy than the S1 state
On the other hand, when the energy gap between S1δ
and S0δ becomes so small that internal conversion will take
the place of fluorescence emission (k2), it could result in a
fluorescence loss. The calculated energy gap also confirms
that the energy of fluorescence detected is more similar to the
energy gap between S1β and S0β. So, the observation that the
quantum yields of this family of fluorophores are polarity
dependent can be explained in this way: If the solvent favors
the nonradiative S1δ state, the fluorescence quantum yield of
the molecule will be quite low. Likewise, if the rotation of the
dicyano group is slowed or prevented in a particular
environment, the quantum yield will be larger. Thus enters
the role of more viscous solvents, which slow down this
rotation.
The absorption and emission properties of the DCDHF dyes
have been finessed to a significant degree. As an example of
the level of tuning that has transpired, consider the influence
of donor connectivity modification. Control of rotation about
“” is feasible but has not yet been accomplished and control
or rotation about “δ” may not be feasible by any reasonable
structure modification. While the preliminary calculations did
not identify a significant influence on the photophysical
properties for rotation at site “α” a contribution from this site
has been observed experimentally. Modifications to control
rotation about “” proved to be relatively straightforward
and we have prepared a series of molecules in which the
rotation here is essentially turned off by inclusion of the
amine donor in one or two rings.
The first series of styrene DCDHFs with zero, one or
two tetrahydroquinoline rings shown in Fig. 5 has been
synthesized. Basically it follows the same synthetic protocol
in Fig. 2. Different benzaldehydes (8 and 9) were made by
Vilsmeier reaction of julolidine and alkylated
tetrahydroquinolines. Together with commercially available
4-N,N-diethylaminobenzylaldehyde, they were condensed
with previously made intermediate 6 to give our desired
fluorophores (11 and 12).
Synthesis routes to the series of the phenyl DCDHF
with no, one or two tetrahydroquinoline rings are shown in
Fig. 6. Fluorophore 13 was made through the same
intermediate 4 in Fig 2. Fluorophore 18 and 19 were prepared
by bromination of 1-hexyl-1,2,3,4-tetrahydro-quinoline and
julolidine followed by lithiation and trapping with protected
cyano acetonhydrin 1 to make the respective 4-amino
substituted α-ketols. Condensation of the resulting ketols
with malononitrile gave the desired DCDHF dyes directly.
N DMF POCl3
O
CN
CN
CN
N
O
N
O
H
N
O
N
O
HN
DMF K2CO3
O
CN
NC
NC
N
N O
CN
NC
NC
N O
CN
NC
NC
N
10
11
12
DMF POCl3 N
O
5
5
1-bromohexane
5 5
9
8
H
H
H
H
6
Fig. 5: The synthesis of styrene DCDHF w/o
tetrahydroquinoline rings.

95 N. Liu et al. 11
HO
Br Burcherer reaction H2N
Br
N
Br
R2
R11)n-BuLi
2) 1NR2
R1
O
OH
CN
CNPy
N
R2
R1 O
NC NC
CN
20
Fig. 8: Synthesis route for DCDHF fluorophores with
naphthalene pi-system.
The synthesis of DCDHF fluorophores with
naphthalene rings starts with Bucherer reaction of 6-bromo-2-
napthol. The free amine is alkylated with different R1, R2
groups and the bromo end undergoes lithiation and attacks the
protected cyano acetohydrin (1) to give the desired α-
hydroxyketone, which, in turn, condenses with malononitrile
to obtain the target fluorophore 20. (Fig. 8) Compared with
DCDHF fluorophores with phenyl rings (DCDHF-6,
absorption at 486nm and emission at 505nm in toluene), this
naphthalene based fluorophore (20: R1= R2= n-hexyl), with
absorption at 547 nm and emission at 576 nm in toluene, is
above the excitation range of flavins but its emission could
overlap with that of flavins.
To further optimize the operational wavelengths, the
aromatic core needs to be pushed still further and so the
corresponding anthracene derivatives were examined. The
synthesis began with commercially available 2,6-
diaminoanthraquinone. However, all attempts to selectively
modify only one of the two amines were unsuccessful. (Fig.
9) An attempt to carry out a selective Sandmeyer reaction
(conversion of amine to bromide) on only one amine group
failed at least in part due to solubility problems, producing
mainly dibromide 24 and no monobromide 25 was

O
O
Br
BrO
O
NH2
H2N
NH2
H2N Br
Br
Br
H2N
21
22
+
23
24
O
O
NH2
Br
25
+
O
O
Br
Br 24
O
O
N(Hex)2
(Hex)2N 26
+
O
O
N(Hex)2
Br
27
O
O
NH2
Br
O
O
NH2
H2N
+
22
Zn, NH4OH
1 eq. t-BuONO
1 eq. CuBr2, CH3CN
dihexylamine, K2CO3,
DMSO, 125-140 C
CuI, NH4OH
1 eq. t-BuONO
1 eq. CuBr2, CH3CN
2.2 eq. t-BuONO
2.2 eq. CuBr2, CH3CN
Fig. 9: Unsuccessful approaches attempted for the
preparation of some asymmetric 2,6-disubstituted
anthraquinones.
isolated. We then reduced the anthraquinone to
diaminoanthracene (21) and tried to chemically differentiate
these two amines. Using the approach to convert only one of
the amino groups to a bromide by using one equivalent or less
of tert-butyl nitrite and cupric bromide resulted in only a
mixture of 22 and the starting material. Direct nucleophilic
aromatic substitution of 24 with a secondary amine usually
afforded an inseparable mixture. For example, reaction of 24
with dihexylamine in the presence of K2CO3 in DMSO
afforded a mixture containing less than 5% of the desired 2-
bromo-6-dihexylaminoanthraquinone (27) along with 2,6-
bisdihexylaminoanthraquinone (26). This mixture was too
difficult to separate for any practical application. Amination
of 24 with CuI catalyst and an excess of ammonium
hydroxide under pressure afforded only the starting amine
and no asymmetric product was obtained.

97 N. Liu et al. 11
the near infrared. Also beyond the scope of detailed
discussion here is the growing array of reactive functional
groups which have been successfully introduced into these
dyes: N-hydroxy succinimide ester was introduced into the
fluorophores to attach to different functionalized
ologonucleotides; maleimide was introduced into the
fluorophores to label different thiol containing peptides or
proteins; and AM (acetoxy methyl) protected APTRA (o-
aminophenol-N,N,O-triacetic acid group has been introduced
to detect different ions.
Table 2. Spectral parameters of fluorophores 13, 20 and 31 in
a representative range of liquid solvents and also in PMMA.
The pi-core homologation structure modification has pushed
the fluorescence of these dyes into the near infrared.
V. Conclusions
In summary, a group of novel single-molecule
imaging fluorophores, the DCDHF family, has been
discovered and successfully imaged at the single-molecule
level. Their polarity dependent absorption and emission
wavelengths, and viscosity dependent quantum yields have
provided additional benefits as polarity and viscosity
reporters for their local environment. A mechanism for this
dependence is proposed together with addition of
tetrahydroquinoline rings to the molecule to identify the
photophysical property related rotations at the amine donor
part of the molecule. The high synthetic flexibility allows
introduction of different functional groups into the
fluorophore to achieve different goals: addition of different
conjugation systems for optimal absorption and emission
wavelengths; addition of different bioactive functional groups
for different organelles labeling; and addition of different
functional groups to detect different ions.
Acknowledgements
This work has been supported by National Institutes
of Health grant Grant No. 1P20-HG003638, Department of
Energy Grant No. DE FG02-04ER63777 and the Ohio Board
of Regents. We also acknowledge contributions to the work
described here by earlier collaborators at Kent State
University and also collaborators at Stanford University who
are named in the subsequent references.
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acetone 0.0041 494 531
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