Modifications of DCDHF single molecule fluorophores to impart water solubility

Article abstract of DOI:10.1016/j.tetlet.2007.03.026

A series of dicyanomethylenedihydrofuran (DCDHF) fluorophores with different hydrophilic groups were synthesized and their photophysical properties and water solubilities were measured. Significant water solubility was achieved without compromising desirable photophysical properties, permitting applications of these fluorophores in biological systems.

Full text of DOI:10.1016/j.tetlet.2007.03.026

Tetrahedron Letters 48 (2007) 3471–3474
Modifications of DCDHF single molecule fluorophores to
impart water solubility
Hui Wang,a Zhikuan Lu,a Samuel J. Lord,b W. E. Moernerb and Robert J. Twiega,*
aDepartment of Chemistry, Kent State University, Kent, OH 44240, USA
bDepartment of Chemistry, Stanford University, Stanford, CA 94305-5080, USA
Received 9 February 2007; revised 4 March 2007; accepted 5 March 2007
Available online 12 March 2007
Abstract—A series of dicyanomethylenedihydrofuran (DCDHF) fluorophores with different hydrophilic groups were synthesized
and their photophysical properties and water solubilities were measured. Significant water solubility was achieved without compro-
mising desirable photophysical properties, permitting applications of these fluorophores in biological systems.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Dicyanomethylenedihydrofuran (DCDHF) chromo-
phores are useful not only as photorefractive materi-
als1–4 but also can be imaged at the single-molecule
level with additional beneficial properties such as viscos-
ity-dependent fluorescence, strong solvatochromism,
significant ground-state dipole moment and moderate
hyperpolarizability.5–11 From a synthetic point of view,
this family of fluorophores also has a high degree of flex-
ibility in all of the donor, acceptor and conjugated core
substructures (Fig. 1).
ble forms.12 To explore the water solubility potential of
these fluorophores, we modified the donor component
with a variety of hydrophilic groups (alcohol, carboxylic
acid, and sulfonic acid), and their water solubility and
photophysics in aqueous environments were determined.
Fluorophores 6 and 9 (alcohols) were first synthesized as
shown in Scheme 1. Intermediate 5 was made by
H C (CH )
a
3
2 5
HN
N
Most of the previously described DCDHF fluorophores
are soluble in organic solvents but are not soluble in
aqueous media. For example, DCDHF-61 (1: R1 =
R2 = n-hexyl, R3 = R4 = methyl; n = 0 and Ar =
phenyl) and DCDHF-2-V10 (2: R1 = R2 = ethyl; R3 =
R4 = methyl; n = 1 and Ar = phenyl) have less than
0.001 ppm water solubility. With alkyl substituents at
the various R positions, such molecules have an amphi-
philic motif and label cell membranes easily.11 In order
to prevent membrane binding and enable applications
in the cellular cytosol, it is useful to develop water solu-
HO
AcO
3
b
NC
NC
H C (CH )
3
2 5
H(H C)
2
CN
6
H
O
N
CN
N
CN
O
HO
O
HO
6
CN
4
5
c
NC
H
O
NC
N
N
CN
HO
(CH )
2 6
HO (CH )
2 6
O
8
9
b
NC
NC
CN
d
N
HN
O
(CH )
R1R2NAr
n
R3
AcO
2 6
7
R4
Scheme 1. Synthesis of DCDHF-V dyes with a single primary alcohol
group in the amine donor tail. Reagents and conditions: (a)
1-iodohexane, K2CO3, DMF and then Ac2O, Py; (b) POCl3, DMF
and HCl/methanol reflux; (c) Py, acetic acid; (d) 6-chloro-1-hexanol;
NaOH, KI, DMF and Ac2O, Py.
Figure 1. Generic structure of the DCDHF fluorophores.
*
Corresponding author. Tel.: +1 330 672 2791; fax: +1 330 672
3816; e-mail: rtwieg@lci.kent.edu
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.03.026

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H. Wang et al. / Tetrahedron Letters 48 (2007) 3471–3474
condensation of 3-hydroxy-3-methyl-2-butanone with
malononitrile.13 Alkylation of 2-(anilino)ethanol, protec-
tion of the alcohol group by esterification (3), Vilsmeier
reaction, and deprotection gave the key aldehyde precur-
sor 4, which was subsequently condensed with intermedi-
ate 5 to give fluorophore 6 with one free alcohol group
terminating the short alkyl chain. Alternatively, alkyl-
ation of N-ethylaniline with 6-chloro-1-hexanol, protec-
tion of the alcohol group by esterification (7), Vilsmeier
reaction and deprotection gives the key aldehyde precur-
sor 8, which was also condensed with intermediate 5 to
give the desired fluorophore 9 with one free alcohol group
terminating the longer chain. Measurements (Table 1)
show that both of these two mono-alcohol terminated
fluorophores still have very low water solubility (around
0.01 ppm) with fluorophore 6 slightly higher than fluoro-
phore 9 (Scheme 1).
without any alcohol group at all. Therefore, we designed
fluorophore 12 to increase water solubility by removing
the vinyl linkage. The synthesis of the key intermediate
10 has been reported before.1 Commercially available
2-(n-butylamino)ethanol 11 was chosen as a nucleophile
to prepare fluorophore 12.15 Measurements show a sol-
ubility of 0.40 ppm for 12, which is better than the dyes
with the vinyl linkage but still insufficient for our appli-
cations. Next, we decided to increase the number of the
free alcohols to improve water solubility and so fluoro-
phores 14 and 15 were subsequently synthesized. Gly-
cidol undergoes nucleophilic attack with n-butylamine
to give secondary amine 13, which then undergoes the
same nucleophilic aromatic substitution on 10 to give
fluorophore 14.15 The same reaction occurs between
N-methyl-D-glutamine and intermediate 10 to give mul-
tiply hydroxylated fluorophore 15.15 Measurements
(Table 1) show that the solubility increases almost 10
times with an increase of one free alcohol group, and
increases another 30 times with an increase of another
three free alcohol groups (two and five total alcohol
groups, respectively, in 14 and 15) (Scheme 2).
Although these two fluorophores show very low water
solubility, it is improved compared with fluorophores
Table 1. Photophysical properties and water solubility measurements
of different DCDHF (I) and DCDHF-V (II) chromophores
Thus far, we had been able to increase the water solu-
bility of the DCDHF fluorophores from less than
0.001 ppm to 150 ppm. Clearly, addition of hydroxyl
groups alone is not a particularly effective means to
enhance solubility so we switched our attention to other
functional groups beginning with carboxylic acid
groups. N-Methylbutyric acid and iminodiacetic acid
reacted with 10 to give fluorophores 16 and 17, respec-
tively.15 Fluorophore 16 with a single carboxylic acid
has only 3.80 ppm water solubility, which is comparable
to fluorophore 14 with two free alcohol groups. How-
ever, and to our surprise, fluorophore 17 with two
carboxylic acids was found to have 2830 ppm water
solubility, which is at least 20 times higher than that
compared with fluorophore 15, the one with five free
alcohol groups (Scheme 3).
kmax kem UF
emax
Solubility in
(water) (Lcmꢀ1 molꢀ1
)
water14 (ppm)
I
1
470a 546 0.12
89,900
15,100
44,200
64,700
37,300
31,500
79,300
65,800
57,300
42,700
<0.001
0.40
12 498
529 0.001
14 496a 531 0.002
1.40
15 493
16 500
17 489
19 500
21 496b
26 477
27 480
528 0.001
526 0.001
529 0.002
517 0.001
1.50 · 102
3.80
2.80 · 103
>2 · 104
>2 · 104
7.30 · 102
4.70 · 102
—
—
517 0.001
520 0.004
II
2
6
9
560a 642 0.002
535a 646 0.002
13,500
77,900
87,200
37,000
<0.001
ꢁ0.05
587b
—
—
ꢁ0.01
24 585
639 0.01
>2 · 104
a A small volume of ethanol stock solution added to the cuvette of
Although fluorophore 17 already has enhanced water
solubility, we still wanted to push further, and so a sul-
fonic acid group was introduced next into the system. In
this synthesis, 1,3-propanesultone underwent attack
from either n-butylamine or dodecylamine to give pre-
cursors 18 and 20, respectively, which underwent the
same reaction with aryl fluoride intermediate 10 to give
fluorophores 19 and 21.16 Measurements show that
water.
b Aggregation was observed in water.
NC
NC
HO
CN
NH
NH
N
O
11
12
HO
NC
HO
HO
NC
NC
NC
CN
CN
F
O
HO
HO
N
O
13
10
NC
NC
14
b
a
CN
N
O
O
O
OH
N
H
NC
OH
NC
O
HO
HO
16
H
C
3
CN
10
N
CH NHCH
2
OH
H
OH
OH
3
H
H
HO
H
C
O
NC
2
NC
H
HO
H
a
OH
H
OH
OH
HO
OH
CN
N
H
O
N
O
O
O
15
H
O
H
CH OH
2
HO
CH OH
17
2
Scheme 2. Synthesis of DCDHF dyes with alcohol functionalization in
the amine donor tail. (a) n-Butylamine, reflux; (b) pyridine.
Scheme 3. Synthesis of DCDHF dyes with carboxylic acid function-
alization in the amine donor tail. (a) Pyridine.

H. Wang et al. / Tetrahedron Letters 48 (2007) 3471–3474
3473
NC
NC
H N
2
CN
a
N
O
O
S
O
HO
O
HN
19
OH
3
S
10
O
NC
18
NC
b
H C (CH )
3
2 10
CN
(CH ) CH
2 10
N
O
HN
O
O
OH
S
S
O
O
HO
21
20
a
NH (CH ) CH
3
2
4 10
d
c
N
CHO
N
CHO
F
CHO
O
O
23
S
O
HO
22
HO
e
5
NC
NC
N
CN
O
O
O
S
O
HO
24
Scheme 4. DCDHF and DCDHF-V dyes functionalized with sulfonic acids. Reagents and conditions: (a) 1,3-propanesultone, reflux; (b) pyridine; (c)
DMF, K2CO3, 2-(methylamino)ethanol, 130 ꢁC; (d) NaH, DMF, 1,3-propanesultone; (e) pyridine, acetic acid.
these sulfonic acid substituted fluorophores have signif-
icant water solubilities (larger than 2 · 104 ppm). The
influence of this single sulfonic acid is also so large that
we decided to add the vinyl linkage back again so that
we could also assay the group of DCDHF-V fluoro-
phores containing the vinyl linkage. Fluorophore 24
was synthesized by nucleophilic aromatic substitution of
4-fluorobenzaldehyde with 2-(methylamino)ethanol.
The alcohol was then deprotonated and reacted with a
sultone to attach a sulfonic acid to the benzylaldehyde
substrate. Finally, the aldehyde was condensed with
intermediate 5 to provide the desired vinyl DCDHF-V
fluorophores functionalized with sulfonic acid (Scheme
4). Here again measurements indicate water solubility
larger than 2 · 104.
were not soluble in water at bulk concentrations, single
molecules were observed diffusing in the cell membrane
at the nanomolar regime.11 As such, this means that
while we increased the water solubility of this family
of fluorophores, the desirable spectroscopic properties
were essentially unchanged.
This outcome is also confirmed by independent viscosity
dependence experiments. Previously, in a mixed solvent
of glycerol and methanol, the quantum yield of fluoro-
phore 1 was found to grow with the increase of the glyc-
erol content.7 Here, two identical solutions, each of 17
and 24 in water, were prepared. For each pair, the
sample on the right was frozen while the other sample
remained at room temperature (Fig. 2). Under illumina-
tion with a UV lamp, the liquid sample (left of each pair)
shows limited fluorescence while the frozen sample
(right of each pair) is strongly emissive. Therefore, the
viscosity dependence of the emission is maintained after
the addition of dicarboxylic acid and sulfonic acid. This
suggests that our DCDHF fluorophores have the poten-
tial for application in a cellular environment beyond
lipid-analog membrane probes.
With these highly water soluble fluorophores available,
our concerns about whether or not the presence of these
hydrophilic groups will affect the photophysical proper-
ties of these fluorophores could be evaluated. To this
end, we compared the fluorophores bearing different
hydrophilic groups with their original low polarity ana-
logs 1 and 2. Table 1 shows that within groups I
(DCDHFs) and II (DCDHF-Vs) the absorption and
emission wavelengths are quite similar. The differences
for fluorophores 1, 2, 6 from their counterparts can be
attributed to the addition of ethanol to the aqueous
solution. A few other fluorophores in Table 1 (i.e., 17,
19, and 27) exhibit somewhat blue-shifted absorption
or emission relative to their counterparts. Given the
amphiphilic character of these molecules, the anomalous
photophysics may be due to the formation of aggregates
or micelles in water at the relatively high concentrations
required for bulk measurements. However, at the nano-
molar concentrations actually used for single-molecule
experiments, such effects are unlikely. For instance,
although the fluorophores used in our earlier studies
Figure 2. Fluorophores 17 and 24 dissolved in liquid water (vials l) and
in solid ice (vials s) under irradiation with a UV lamp (a filter was used
to block the scattered light). The fluorescence is significantly enhanced
in the ice samples responding to the dramatic enhancement in viscosity.

3474
H. Wang et al. / Tetrahedron Letters 48 (2007) 3471–3474
NC
NC
I
N
NC
NC
N
N
b
a
CN
CN
N
N
NH
10
O
O
26
25
c
CN
NC
CN
NC
NC
CN
N
N
N
N
27
O
O
Scheme 5. The preparation of water solubilized DCDHF dyes, which are functionalized with quaternary amines. Reagents and conditions: (a)
pyridine; (b) 1-iodomethane, CH2Cl2; (c) 1,3-diiodopropane, CH2Cl2.
5. Schuck, P. J.; Willets, K. A.; Fromm, D. P.; Twieg, R. J.;
Moerner, W. E. Chem. Phys. 2005, 318, 7–11.
6. Willets, K. A.; Callis, P. R.; Moerner, W. E. J. Phys.
Chem. B 2004, 108, 10465–10473.
All the examples of solubilized DCDHF fluorophores
described thus far are anionic. We have also briefly
examined some cationic derivatives. The secondary
amine in N,N,N0-trimethylethylenediamine reacts with
10 to give fluorophore 25, which next underwent alkyl-
ation at the trialkylamine end with methyl iodide to give
monocationic fluorophore 26. In a similar fashion,
alkylation of 25 with 1,3-diiodopropane gives dicationic
dimer fluorophore 27 (Scheme 5). Although 26 and 27
are not as water soluble (730 ppm, 470 ppm respectively)
as fluorophores 19, 21 and 24, they demonstrate still
another option to functionalize and solubilize this fam-
ily of DCDHF fluorophores.
7. Willets, K. A.; Nishimura, S. Y.; Schuck, P. J.; Twieg, R.
J.; Moerner, W. E. Acc. Chem. Res. 2005, 38, 549–556.
8. Willets, K. A.; Ostroverkhova, O.; He, M.; Twieg, R. J.;
Moerner, W. E. J. Am. Chem. Soc. 2003, 125, 1174–1175.
9. Willets, K. A.; Ostroverkhova, O.; Hess, S.; He, M.;
Twieg, R. J.; Moerner, W. E. Proc. SPIE 2003, 5222, 150–
157.
10. Wang, H.; Lu, Z.; Lord, S. J.; Willets, K. A.; Bunge, S.;
Moerner, W. E.; Twieg, R. J. Tetrahedron 2007, 63, 103–
114.
11. Nishimura, S. Y.; Lord, S. J.; Klein, L. O.; Willets, K. A.;
He, M.; Lu, Z. K.; Twieg, R. J.; Moerner, W. E. J. Phys.
Chem. B 2006, 110, 8151–8157.
12. Jose, J.; Burgess, K. J. Org. Chem. 2006, 71, 7835–7839.
13. Melikian, G.; Rouessac, F. P.; Alexandre, C. Synth.
Commun. 1995, 25, 3045–3051.
14. The water solubilities of the DCDHF chromophores were
determined from UV–vis data. A standard solution of
known concentration was prepared by dissolving about
1 mg of the compound in methanol/H2O (3/7, 10 ml) for
fluorophores 6, 9, 12, 14–16, or in methanol/H2O (2/8,
10 ml) for fluorophores 17, 19, 21, 24, 26, 27. The
saturated solutions were prepared by suspending about
2 mg or more fluorophores in H2O (10 ml) and then
stirring at 60 ꢁC for 2 h, followed by equilibration at room
temperature for 12 h. These samples were passed through
a syringe filter (2 lm). The absorption could be measured
from the UV–vis spectra and the absorption coefficients
could be calculated from standard solutions, which were
used to determine the concentration of the substrate in the
pure H2O sample. The results reported are averages of
duplicate runs. (Benbow, J. W.; Bernberg, E. L.; Korda,
A.; Mead J. R. Antimicrob. Agents Chemother., 1998, 42,
339-343).
15. General methods to obtain fluorophores 12, 14–17, 25: the
starting disubstituted amine was mixed with precursor 10
in dry pyridine. The reaction mixture was then stirred at
room temperature or heated to 40 ꢁC overnight depending
on the substrate. Pyridine was then distilled out under
vacuum and the residue was purified via flash
chromatography.
16. General methods to obtain fluorophores 19, 21: the
starting disubstituted amine was mixed with precursor 10
in anhydrous DMF with 2 equiv i-Pr2NEt. The reaction
mixture was then stirred at 40 ꢁC overnight. Hydrochloric
acid (10%) was added and the suspension was filtered to
give the product as a yellow solid.
In conclusion, we have synthesized a series of DCDHF
fluorophores with a range of functional groups and
modified their water solubility without compromising
their photophysical properties. These results suggest
that this class of fluorophores possess strong potential
for a broad range of bio-labeling applications. The
methods applied here to solubilize the DCDHF fluoro-
phores should also be applicable in other cases, where
water solubilization is required.
Acknowledgments
Support from DOE (DG-FG02-04ER63777), NIH
(1P20HG003638-01) and the Ohio Board of Regents is
acknowledged.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2007.03.026.
References and notes
1. Gubler, U.; He, M.; Wright, D.; Roh, Y.; Twieg, R.;
Moerner, W. E. Adv. Mater. 2002, 14, 313–317.
2. Ostroverkhova, O.; He, M.; Twieg, R. J.; Moerner, W. E.
ChemPhysChem 2003, 4, 732–744.
3. You, W.; Hou, Z. J.; Yu, L. P. Adv. Mater. 2004, 16, 356.
4. Ostroverkhova, O.; Moerner, W. E.; He, M.; Twieg, R. J.
Appl. Phys. Lett. 2003, 82, 3602–3604.