A new solvatochromic fluorophore for exploring nonpolar environments created by biopolymers

Article abstract of DOI:10.1039/c0cc04917d

The fluorescence of a new aminocyanonaphthalene exhibits exquisite sensitivity to its environment and responds to a solvent change from water to hexane with greater than a 100-fold increase in intensity and 100 nm shift in λ_max.em. These properties should support many applications including the detection of abasic sites within duplex DNA as illustrated below.

Full text of DOI:10.1039/c0cc04917d

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COMMUNICATION
A new solvatochromic fluorophore for exploring nonpolar environments
created by biopolymersw
Abulfazl Fakhari M. and Steven E. Rokita*
Received 12th November 2010, Accepted 10th February 2011
DOI: 10.1039/c0cc04917d
The fluorescence of a new aminocyanonaphthalene exhibits
exquisite sensitivity to its environment and responds to a solvent
change from water to hexane with greater than a 100-fold
increase in intensity and 100 nm shift in kmax.em. These properties
should support many applications including the detection of abasic
sites within duplex DNA as illustrated below.
dependent 100-fold gain in emission intensity and 13%
change in Stokes shift.8 Likewise, certain aminonaphthyridine
derivatives selectively coordinate to unpaired nucleobases
within a duplex9 and support a sensitive fluorescence-based
method for detecting abasic sites in DNA.10 More recently, a
pyrazinecarboxamide has been reported for detecting abasic
sites containing an unpaired thymine as indicated by the more
than 2-fold decrease of its fluorescence.11
Solvatochromic probes have emerged as powerful tools for
identifying and characterizing microenvironments created by
macromolecules and molecular assemblies.1,2 Their use has
been particularly significant when characterizing interfaces
between regions of dramatically different polarity such as
those between bulk solution and surfaces of proteins and
nucleic acids. Investigators in this field have successfully
adapted classical fluorophores for convenient incorporation
into biopolymers of interest. For example, 4-N,N-dimethyl-
amino-1,8-naphthalimide3 and 6-propionyl-2-(N,N-dimethyl)-
aminonaphthalene (prodan)4 have been elaborated into amino
acids for direct coupling within peptides and proteins to
monitor ligand binding. Similarly, the dansyl group5 and
6-(dimethylamino)-2-acylnaphthalene (dan)6 have been attached
to nucleobases and incorporated into duplex DNA to examine
the polarity of the major and minor grooves. Progress has
additionally been reported on an alternative approach involving
nucleotide analogues that remain nearly isosteric yet are emissive
and sensitive to their local environment.2 A furan-conjugated
pyrimidine was generated for this purpose and responded with a
50 nm shift in lmax.em and greater than a 3-fold gain in emission
as the surrounding microscopic polarity increased from dioxane
to water.7
An abasic site was integral to our laboratory’s model for
studying excess electron transport in duplex DNA and
was based in part on the expectation that this site would
serve to bind and position diaminonaphthyl donors.12–14
Unfortunately, the emissive properties of these initial
compounds were not sufficiently sensitive to their environment
to report on their interaction with DNA.15 A new solvato-
chromic derivative based on naphthalene has now been
synthesized and used to examine its DNA association. This
derivative exhibits ideal characteristics for detecting abasic
sites in duplex DNA and should also find utility in studying
nonpolar surfaces of other macromolecules quite generally
since its fluorescence intensity increases by 140-fold when
transferred from an aqueous solution to a microenvironment
with a polarity equivalent to hexane. Its Stokes shift also
increases by more than 40% under equivalent conditions and
provides another complementary measure of binding and
polarity change with a very high dynamic range.
Typically, fluorophores with strong dipoles have the greatest
potential to exhibit significant dependence on solvent,1,2 and
consequently, 1,5-diaminonaphthalene was converted to a
strong dipolar derivative by enhancing the electron donating
ability of one amine and replacing the other amine with an
electron withdrawing group. Enhanced donor strength was
gained by monomethylation of the amine. Dimethylation would
ordinarily be expected to offer even greater electron donation,
but its resonance contribution is diminished in this system by an
out-of-plane twist required to accommodate the hydrogen at
the peri-position of the fused ring.16 Introduction of an electron
withdrawing group was first achieved by replacing the second
amino with a nitro group. However, preliminary studies on
1-methylamino-5-nitronaphthalene indicated that this fluoro-
phore expressed very weak emission and little dependence on
solvent.17 This may perhaps be due to a non-radiative
relaxation pathway available to the nitro group.18 Alternative
use of a cyano group proved more successful and was inspired
In a complementary approach, low molecular weight ligands
can be prepared with solvatochromic properties to interrogate
macromolecular surfaces, to monitor association with macro-
molecular receptors and even simply to detect the presence
of a specific receptor of interest. A bisbenzimide derivative
(Hoechst 33258) was one of the first fluorescent probes to
characterize the polarity of the minor groove through its solvent
Department of Chemistry and Biochemistry, University of Maryland,
College Park, MD 20742, USA. E-mail: rokita@umd.edu;
Fax: +1 301-405-9376; Tel: +1 301-405-1816
w Electronic supplementary information (ESI) available: Synthesis of
2, absorption spectra of 2 in each solvent, fluorescence quantum yield
measurement, fluorescence emission stability, titration of 2 with all
dsDNAs, and Kd analysis. See DOI: 10.1039/c0cc04917d
c
4222 Chem. Commun., 2011, 47, 4222–4224
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However, its absorptivity varies by as much as 50%. Most
importantly, the emission spectra reveal a very substantial
influence of solvent (Fig. 1). An aqueous solution of 2 emits
only weakly with a maximum at 575 nm whereas a hexane
solution of 2 emits over 100-fold more intensely at its maximum
of 476 nm (Table 1). Solvents of intermediate polarity display
intermediate effects, and both the quantum yield for emission
and the Stokes shift respond with near linearity to ET(30), a
measure of microscopic solvent polarity (Fig. 1).22,23 Proton
transfer does not appear to control the excited state properties
of 2 since there is no discontinuity in either fluorescence emission
or Stokes shift as a function of protic versus aprotic solvents
(Fig. 1).
Scheme 1 Synthesis of 2. Reagents and conditions: (a) FeBr3, Br2,
90 1C, 3 h, 60%; (b) Æ-BINAP, Pd(OAc)2, Cs2CO3, CH3NH2, toluene,
80 1C, 24 h, 69%.
Table 1 Photophysical parameters of 2 in the indicated solventsa
c
Solvent
lmax.abs (e)b
lmax.em
Dnꢀ
Ffd
H2O
MeOH
EtOH
n-BuOH
MeCN
EtOAc
DMSO
DMF
CHCl3
CH2Cl2
Toluene
Hexane
346 (3.62), 362 (3.76)
346 (3.24), 389 (4.26)
346 (3.00), 392 (4.00)
346 (3.20), 394 (4.28)
346 (3.10), 386 (4.24)
346 (3.20), 385 (4.34)
346 (3.22), 404 (4.48)
346 (3.10), 397 (4.36)
346 (2.58), 383 (3.50)
346 (2.94), 383 (4.02)
346 (3.44), 383 (4.54)
346 (3.40), 378 (4.34)
575
534
527
523
527
499
533
524
500
502
485
476
43.7
53.2
55.2
56.5
55.2
65.3
53.5
56.2
64.9
64.1
71.9
76.9
0.006
0.15
0.23
0.28
0.25
0.53
0.46
0.47
0.70
0.73
0.72
0.69
The large dipole created by the electron donating and
withdrawing substituents of 2 resulted in a large shift of
lmax.em ranging over 99 nm (from water to hexane). This
magnitude is nearly equivalent to the change in lmax.em of
130 nm for the widely applied prodan (from water to cyclo-
hexane)23,24 and greater than the change in lmax.em of 86 nm
for dansyl amide (from water to toluene) when comparing an
analogous change of 90 nm for 2 (from water to toluene).23
Furthermore, 2 is sufficiently stable photochemically to be
used routinely. Continuous irradiation of 2 in sodium
phosphate (10 mM) pH 7 and 100 mM NaCl at 346 nm for
1 h resulted in less than a 10% change in its fluorescence
emission (Fig. S3).21
a
l, e, nꢀ, and ET(30) are given in nm, mMÀ1cmÀ1, Â 103 cmÀ1, and
kcal molÀ1
independently by measuring absorbance of a series of solutions with
,
respectively. Extinction coefficients were obtained
b
c
d
known concentrations at lmax.abs
. lex = 346 nm. Ff was obtained
independently in each solvent using optically matched solutions of
anthracene in ethanol as a reference (Ff = 0.27).25 Errors are less than
10% for each individual value.
The striking solvatochromic properties of 2 provide a
sensitive indicator for testing the potential for the naphthyl-
based electron donors to bind within an abasic site.
Accordingly, the fluorescence of 2 under aqueous conditions
was monitored during titration with a series of oligonucleotide
duplexes containing abasic sites equivalent to those used
earlier in studies on electron transport in DNA in which the
unpaired base varied between C, G, A and T (Scheme 2).14
Since the environment within helical DNA is less polar than its
surrounding solvent, association between 2 and these duplexes
was predicted to increase fluorescence emission and induce a
hypsochromic shift of the lmax.em. Both results were clearly
evident by the 13- to 18-fold increase in emission and 42 nm
hypsochromic shift in lmax.em after addition of any of the
duplexes containing an abasic site (Fig. 2 and S4–S7).21
by the substantial effect of solvent on the fluorescent properties
of monocyanoanilines.19,20 1-Cyano-5-methylaminonaphthalene
(2) was synthesized in two steps by a Friedel–Crafts bromination
of commercially available 1-cyanonaphthalene followed by a
palladium-catalyzed cross coupling between the resulting 1-bromo-
5-cyanonaphthalene (1) and methylamine (Scheme 1).21
The photophysical properties of 2 were examined in a range of
solvents from water to hexane. Solvent polarity affects its light
absorption by a relatively modest 50% (Fig. S1).21 The long
wavelength lmax varies between 360 and 400 nm, and the short
wavelength lmax at 346 nm remains nearly constant (Table 1).
Fig. 1 Solvent dependent properties of 2 in aprotic (’) and protic (&) solvents. (A) Fluorescence emission of 2 (50 mM, lex of 346 nm)
was recorded under ambient conditions. (B) The quantum yield for emission and (C) the Stokes shift were compared to published values of ET(30), a
measure of microscopic solvent polarity.22 The quantum yields were calculated as illustrated in the Supplementary Information from multiple
independent determinations, and the Stokes shifts are averages of 3 independent measurements. Linear fits in (B) and (C) were generated by Origin 6.0.
c
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Z = C (Scheme 2). Only a slight increase in fluorescence emission
of less than 2-fold was observed in the presence of this canonical
DNA duplex at its maximum concentration of 40 mM (Fig. 2).
A corresponding hypsochromic shift in lmax.em of less than 10 nm
was more pronounced but still much smaller than that induced
by the presence of an abasic site.
Scheme 2 Nucleotide sequences of duplex DNA with abasic sites.
In summary, we have successfully created a novel solvato-
chromic fluorophore that should find application in many
studies examining the surface and structure of biopolymers.
The fluorescence is highly sensitive to solvent polarity and
provides a dynamic range of 100-fold for emission and 100 nm
for lmax.em. This probe was particularly useful to validate a
previously untested assumption that the naphthalene-based
electron donors are bound at an abasic site within duplex
DNA. Derivatives of 2 should also be easily prepared in the
future for conjugation to a broad array of low molecular
ligands and large macromolecules.
Financial support from the National Science Foundation
(CHE-0517498) is gratefully acknowledged.
Notes and references
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Fig. 2 The fluorescence of 2 responds selectively to the presence of
abasic sites in duplex DNA. Solutions of 2 (10 mM) in 10 mM sodium
phosphate pH 7 and 100 mM NaCl (measured ET(30) = 64.5 kcal molÀ1
)
10 K. Yoshimoto, S. Nishizawa, M. Minagawa and N. Teramae,
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were titrated with an oligonucleotide duplex (’, X = C, Z = G,
Scheme 2) or its derivatives containing an abasic alternatively opposite a
G (dsDNA(G), $), A (dsDNA(A), J), C (dsDNA(C), n), and T
(dsDNA(T), &). (A) The relative fluorescence emission (F/Fo) and
(B) lmax.em of 2 (lex 346 nm) were monitored as a function of total
DNA concentration.
13 T. Ito and S. E. Rokita, Angew. Chem., Int. Ed., 2004, 43,
1839–1842.
Additionally, the response of 2 to each of these duplexes was
saturable and followed an expected 1 : 1 binding isotherm.21
Fluorescence emission correlated well to this process and
yielded Kd values of 7–8 mM for X = pyrimidine and 5 mM
for X = purine. Interestingly, the lmax.em data did not
clearly distinguish between X = pyrimidine or purine but still
suggested Kd values between 3–10 mM. These affinities are also
similar to that measured for a neutral pyrazinecarboxamide
derivative (3.5 mM) used previously as a control when
developing reagents to identify specific abasic sites through
suppression of fluorescence rather than enhancement of
fluorescence as exhibited by 2.11
14 T. Ito and S. E. Rokita, J. Am. Chem. Soc., 2004, 126,
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17 N. P. Campbell, S. E. Rokita, Unpublished observations.
18 J. R. Lakowicz, in Principles of Fluorescence Spectroscopy,
Springer, 2006, pp. 1–21.
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21 See Supplementary Information.
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25 W. H. Melhuish, J. Phys. Chem., 1961, 65, 229–235.
Selectivity of 2 for an abasic site over the ubiquitous
alternatives of standard intercalation and groove binding was
demonstrated by further titration with the parent oligonucleotide
duplex lacking the abasic site (dsDNA) in which X = G and
c
4224 Chem. Commun., 2011, 47, 4222–4224
This journal is The Royal Society of Chemistry 2011