Water-Soluble Nile Blue Derivatives
FULL PAPER
Table 1. Spectroscopic properties of Nile Blue and its derivatives under
different conditions.[a]
Finally, Nile Blue derivative 2a was used to label ovalbu-
min by activation of the dicarboxylic acids on the dye by
using N-hydroxysuccinimide and N,N’-diisopropylcarbodii-
mide in DMF, followed by addition of this activated probe
to the protein in 0.1m aqueous NaHCO3 (pH 8.3). The dye/
protein ratio was calculated[25] to be 1.1 if five equivalents of
dye was used; this corresponds to a 22% labeling efficiency.
This sample was used to obtain the spectral data shown in
Figure 3a. The wavelengths for the absorption and fluores-
cence maxima for free 2a and the 2a–ovalbumin conjugate
were observed to be almost identical, but the fluorescence
intensity was much less.
One application of Nile Blue derivatives is to measure
protein concentrations; this is possible because the fluores-
cence intensities of Nile Blue derivatives tend to increase
with protein concentration.[3] However, one limitation of
this method is that the solubility of Nile Blue derivatives
can be problematic. Herein, Nile Blue derivative 2a was
mixed with increasing concentrations of ovalbumin in phos-
phate buffer at pH 6.8. The fluorescence data for this set of
experiments are shown in Figure 3b. The measurements
were performed at different pH values because in one case
a covalent interaction was formed by using a protocol at
pH 8.3, whereas the other was a simple addition at a more
standard pH. The fluorescence intensity of 2a increased
when the protein was added. These increases were small,
but the concentrations of ovalbumin were only varied be-
tween 3 and 12 mm, that is, small changes in protein concen-
tration that are hard to detect. Furthermore, unlike in some
previous works with lipophilic Nile Blue derivatives, use of
the water-soluble form 2a circumvented the need for any
detergent additives.
Dye labs [nm] e [mÀ1 cmÀ1
]
lem [nm] fwhm [nm] F[b]
Solvent
1a
1a
1a
1a
2a
2a
2a
2a
1b
1b
1b
2b
2b
2b
A[c]
B[c]
C[c]
628
630
631
630
629
630
629
631
629
628
630
632
631
630
635
633
637
14400
42400
51200
28400
58800
30300
64100
34500
33600
44400
21800
38100
14700
38000
4000
662
671
669
670
666
670
669
670
671
670
672
673
672
670
674
675
677
47
52
49
56
50
58
56
73
55
47
51
56
54
54
115
86
93
0.56 MeOH
0.14 PB
0.24 TX
0.02 BB
0.32 MeOH
0.10 PB
0.11 TX
0.02 BB
0.14 PB
0.23 TX
0.13 BB
0.13 PB
0.26 TX
0.08 BB
0.01 water
0.10 water
0.03 water
36000
11000
[a] PB: Phosphate buffer (pH 7.4), TX: 3% Triton X-100 in phosphate
buffer (pH 7.4), BB: borate buffer (pH 9.0). [b] Standard used for quan-
tum yield measurement: Nile Blue in MeOH (F=0.27), quantum yield
and extinction coefficient experiments (at 10À6 m) were repeated three
times. [c] Values obtained from ref. [12].
variations between the emission wavelengths in various buf-
fers indicates that changing the pH away from physiological
levels and adding lipophilic cosolvents have little effect on
these dyes. The sharpness of the fluorescent emissions are
expressed in terms of full width at half maximum peak
heights (fwhm; in which smaller is sharper). Dyes 1 and 2
emitted with sharper fluorescence peaks than Nile Blue or
the more water-soluble forms B and C (data shown in
Table 1 for these dyes is taken from the literature). Further-
more, in aqueous media the quantum yields for these emis-
sions for 1 and 2 were in all cases better than for Nile Blue
and its derivatives B and C.[12]
Conclusion
Figure 2 outlines experiments performed to explore the
aggregation of the dyes in aqueous media. Plots of the nor-
malized UV absorbance versus concentration reveal that the
The Nile Blue derivatives reported here have sharper fluo-
rescence emissions (fwhm=30 nm smaller), and improved
quantum yields in phosphate buffer at pH 7.4 relative to the
known water-soluble Nile Blue derivatives B and C. They
are formed by condensation reactions that do not require
additional acids or very harsh reaction conditions (DMF,
908C); this is in marked contrast to the syntheses of most
other Nile Blue derivatives. Preparative HPLC purification
of the products was not necessary; they were isolated by
using reverse-phase MPLC with acetonitrile/water as the
eluent. The phenolic OH functionalities of 1 and 2 almost
certainly increase the water solubilities of these compounds.
lmaxACHTUNGTRENNUNG(abs) for compound 1a at 4 mm occurs at 671 nm, with an
inflection on the blue side of the peak at approximately
600 nm (Figure 2a). This inflection point grew as the concen-
tration of the dye was increased; at 16 mm there are two dis-
tinct absorption maxima, and at higher concentrations, the
shorter wavelength absorption becomes dominant. Figure 2b
shows that at concentrations of up to 4.0 mm the absorbance
of 1a varies in a near-linear way with concentration. Above
that concentration, the absorbance deviates markedly from
linear concentration dependence. Overall, these data may
be interpreted to mean that the dye is aggregating at con-
centrations above around 4.0 mm. Similar analyses using UV
absorption indicate that 1a deviates from Beer–Lambert be-
havior above this concentration. Probably the dyes are
forming fluorescent J-aggregates at concentrations above
about 4.0 mm, rather than the nonfluorescent H-forms. Anal-
yses for dyes 1b, 2a, and 2b (Figure 2c–h) indicate very sim-
ilar behavior. Concentration versus absorbance studies indi-
cate that these materials tend to aggregate above 4.0 mm.
Alternatively, the phenolic group provides
a potential
avenue for further derivatization of the dyes (e.g., through
triflation and organometallic couplings, or for attachment of
a handle to enable 1 to be conjugated to proteins). Three
other groups that promote water solubility were included in
these studies: a sulfonic acid, dicarboxylic acids, and a tri-
ethylene glycol fragment. Despite this, the fluorescence
properties of the dyes, and presumably their aggregation
states at elevated concentrations, did not vary significantly.
Chem. Eur. J. 2009, 15, 418 – 423
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