Attachable solvatochromic fluorophores and bioconjugation studies

Article abstract of DOI:10.1021/ol400292q

The synthesis and utility of attachable cyclopenta[b]naphthalene solvatochromic fluorophores related to Prodan are described. Two fluorophores were selected for functionalization and bioconjugation studies. The skeletons were chemically modified to include reactive functional groups and showed minimal alteration of the optical properties when compared to the parent dyes. The functionalized fluorophores were covalently attached to the carboxyl group of a fatty acid, and azido- and thiol-containing amino acids, demonstrating their potential for labeling biomolecules.

Full text of DOI:10.1021/ol400292q

ORGANIC
LETTERS
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Vol. XX, No. XX
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Attachable Solvatochromic Fluorophores
and Bioconjugation Studies
ꢀ
Erica Benedetti, Andrea B. E. Veliz, Melanie Charpenay, Laura S. Kocsis, and
Kay M. Brummond*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260,
United States
kbrummon@pitt.edu
Received January 31, 2013
ABSTRACT
The synthesis and utility of attachable cyclopenta[b]naphthalene solvatochromic fluorophores related to Prodan are described. Two fluorophores
were selected for functionalization and bioconjugation studies. The skeletons were chemically modified to include reactive functional groups and
showed minimal alteration of the optical properties when compared to the parent dyes. The functionalized fluorophores were covalently attached
to the carboxyl group of a fatty acid, and azido- and thiol-containing amino acids, demonstrating their potential for labeling biomolecules.
Solvatochromic fluorophores represent a fascinating
class of organic dyes particularly useful for the study of
complex biological systems.1 Their sensitivity to local
microenvironments, low cost, and ease of handling2 has
resulted in the recent applications of these compounds
to the field of proteomics and the study of protein folding
and proteinꢀprotein interactions.3 Solvatochromic fluo-
rophores are also employed both in vitro and in vivo as
sensitive indicators of lipid organization and dynamics in
cellular membranes.4 Prodan (1a) is an example of a com-
mercially available solvatochromic probe widely used for
labeling biomolecules and monitoring their structural trans-
formations or interactions (Figure 1).5 This naphthalene-
based dye exhibits desirable photophysical properties
such as changes in emission wavelength dependent upon
microenvironment polarity, high quantum yield, and
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10.1021/ol400292q
XXXX American Chemical Society

good photostability. Common derivatives of Prodan in-
clude the lipophilic Laurdan (1b), the thiol reactive Acry-
lodan (1c), and the amino acid containing Aladan (1d,
Figure 1).6 Several solvatochromic fluorophores are com-
mercially available, but development of probes with im-
proved photophysical and chemical properties for selective
binding and detection of biological targets remains a field
of active research.7
groups and/or biomolecules to the five membered ring,
as in 3a0, is also predicted to have little effect on the
photophysical properties because this ring is not conju-
gated with the chromophore. Regarding attachment sites
for fluorophore 2b, R1 on the appended aryl ring is syn-
thetically appealing. In addition, we have previously
shown that the aryl ring has little effect on the absorption
and emission maxima of the parent dye. We selected a
hydroxyl group as a reactive functionality for labeling
fluorophores 3a, 3a0, and 3b because of its synthetic
versatility, i.e. nucleophilic substitutions, oxidations, and
Mitsunobu reactions.9
We first set out to examine the functionalization of the
cyclopentane group in 3a0. A microwave-assisted intramo-
lecular DDDA reaction of styrenes 4a, 4b, and 4c afforded
cyclopenta[b]naphthalenes 5a, 5b, and 5c in 85%, 47%,
and 92% yield, respectively (Scheme 1). A low yield for the
conversion of 4b to 5b was attributed to the bulky tert-
butyl group. The resulting aryl chlorides 5a, 5b and 5c were
subjected to palladium-catalyzed cross-coupling amina-
tion conditions to isolate the protected fluorophores 6aꢀd
in 62%, 58%, 71%, and 35% yield, respectively. Reaction
conditions for the conversion of 5c to pyrrolidine 6d were
not optimized. The ketal groups of compounds 6aꢀc were
removed by treatment with 1 N HCl to afford diols
7aꢀc in 57%, 96%, and 52% yield, respectively. Tetra-n-
butylammonium fluoride (TBAF, 2 equiv) in THF was
used to deprotect the TBS groups of substrate 6d to afford
7c in quantitative yield.
Absorption and emission maxima of 6aꢀd and 7aꢀc
were measured in dichloromethane (DCM) revealing in-
teresting trends. Changes to the amine and ketone groups
influence the photophysical properties of these fluoro-
phores, as evidenced by the emission maxima for 6a
(566nm),6b (527nm),and6d (581 nm). However, variations
to the diol moiety had almost no effect on the optical
properties of these dyes. In fact, the ketal derivative 6a, the
TBS protected compound 6c, and the free diol 7a showed
almost identical fluorescence emission maxima (566, 564,
and 567 nm respectively) and only slight changes in the
absorption maxima were observed.
The diol group of 7aꢀc was considered for the fluo-
rescent labeling of carboxyl groups. To demonstrate this,
the fatty acid derivative 8 was obtained through a coupling
reaction of 7b with 10-undecenoic acid and dicyclohexyl
carbodiimide (DCC, Scheme 1). Despite the slightly infer-
ior photophysical properties of 7b when comparing it to 7a
and 7c, the tert-butyl group increases the lipophilicity of
this series of compounds and may serve to enhance its
potential as a membrane probe. The optical properties
of fluorophore 8 were found to be comparable to that of
substrate 7b with an absorption maximum of 324 nm,
an emission maximum of 531 nm, and a Stokes shift of
207 nm in DCM. This unusual fatty acid derivative 8 is
being examined for its potential to study membrane
structure.
Figure 1. Naphthalene-based solvatochromic fluorophores.
Recently, we reported a concise synthesis of fluorescent
dyes 2a and 2b (Figure 1). The synthesis employed a
dehydrogenative dehydro-DielsꢀAlder (DDDA) reaction
to obtain the keto-naphthalene core and a Buchwaldꢀ
Hartwig cross-coupling reaction to install the amine
group.8 These fluorophores were shown to absorb and
emit light at longer wavelengths and display larger Stokes
shifts in ethanol when compared to Prodan while exhibit-
ing similarly high quantum yields and good photo-
stability.8b Red-shifted absorption and emission spectra
are important because of the reduced phototoxicity in
biological systems. In addition, because many Prodan
analogs designed for bioconjugation use Prodan as a
starting material, we expect that a de novo synthesis will
afford fluorophores with enhanced biological relevance
and versatility.1f
To this end, fluorophore 2a has two functionalization
sites thatarereadily accessible, R1 andR2 (Figure1). Func-
tionalization of R1 in 3a with a variety of groups is possible
and should afford compounds with the same photophysi-
cal properties as parent 2a. Attachment of functional
(6) (a) Cohen, B. E.; McAnaney, T. B.; Park, E. S.; Jan, Y. N.; Boxer,
S. G.; Jan, L. Y. Science 2002, 296, 1700–1703. For other examples of
PRODAN derivatives, see: (b) Davis, B. N.; Abelt, C. J. Phys. Chem. A
2005, 109, 1295–1298. (c) Lu, Z.; Lord, S. J.; Wang, H.; Moerner, W. E.;
Twieg, R. J. J. Org. Chem. 2006, 71, 9651–9657. (d) Tainaka, K.;
Tanaka, K.; Ikeda, S.; Nishiza, K.-I.; Unzai, T.; Fujiwara, Y.; Saito,
I.; Okamoto, A. J. Am. Chem. Soc. 2007, 129, 4776–4784. (e) Jockusch,
S.; Zheng, Q.; He, G. S.; Pudavar, H. E.; Yee, D. J.; Balsenek, V.; Halim,
M.; Sames, D.; Prasad, P. N.; Turro, N. J. J. Phys. Chem. C 2007, 111,
8872–8877. (f) Kucherak, O. A.; Didier, P.; Mely, I.; Klymchenko, A. S.
J. Phys. Chem. Lett. 2010, 1, 616–620. (g) Abelt, C. J.; Sun, T.; Everett,
R. K. Photochem. Photobiol. Sci. 2011, 10, 618–622. (h) Lopez, N. A.;
Abelt, C. J. J. Photochem. Photobiol. A: Chem. 2012, 238, 35–40.
(7) The Molecular Probes Handbook, A Guide to Fluorescent Probes
and Labeling Technologies, 11th ed.; Life Technologies Incorporation:
2010.
(8) (a) Kocsis, L. S.; Benedetti, E.; Brummond, K. M. Org. Lett.
2012, 14, 4430–4433. (b) Benedetti, E.; Kocsis, L. S.; Brummond, K. M.
J. Am. Chem. Soc. 2012, 134, 12418–12421.
(9) Thermo Scientific Pierce Crosslinking Technical Handbook;
Thermo Fisher Scientific Incorporation: 2009.
B
Org. Lett., Vol. XX, No. XX, XXXX

Scheme 1. Synthesis of Fluorescent Diols 7aꢀc, Their
Scheme 2. Synthesis of Attachable Dye 13
Photophysical Properties, and Fatty Acid Labeling
a Absorption maximum (nm) in DCM. b Emission maximum (nm) in
DCM; c Stokes shift (nm) in DCM.
and subjected to microwave irradiation to produce fluo-
rescent dyes 15a and 15b in 30% and 40% yield for the
three steps (Scheme3). Thesecompounds displayedsimilar
optical properties when compared with dye 2b.
The versatility of the reactive fluorophore 15b was
demonstrated by its conversion into the maleimide deriva-
tive 17 employing a two-step protocol involving a
Mitsunobu reaction to form the protected maleimide 16. A
thermal retro-DielsꢀAlder reaction of 16 releases the thiol
reactive maleimide group of 17 in 75% yield (Scheme 4).
The fluorescent adduct 17 reacted in 10 min with N-Boc-L-
cysteine ethyl ester to afford conjugate18 as a (1:1) mixture
of diastereomers. Oxidation of 15b with Dess-Martin
periodinane (DMP) gave aldehyde 19 in 67% yield, which
was converted into alkyne 20 by treatment with the
BestmannꢀOhira reagent. Substrate 20 was successfully
employed in a copper-catalyzed click-reaction with N-tert-
butoxycarbonyl-L-β-azidoalanine methyl ester to yield the
covalently linked amino acid 21 (Scheme 4). The malei-
mide derivatives 16 and 17, the cysteine adduct 18, the
alkyne 20, and triazole 21 all displayed similar photophys-
ical properties to the parent dye 15b. Aldehyde 19 is the
only compound that exhibited a significantly red-
shifted fluorescence emission maximum when com-
pared to 15b (580 nm vs 537 nm). Finally, 15b was
converted into the fluorescent fatty acid analog 22
(Scheme 5). Derivative 22 maintained all the photo-
physical characteristics of its precursor 15b with an
absorption maximum in the visible region, a fluores-
cence emission maximum of 538 nm, and a Stokes shift
of 113 nm in DCM.
a Absorption maximum (nm) in DCM. b Emission maximum (nm) in
DCM. c Stokes shift (nm) in DCM
To widen the applicability and to show the versatility of
these dyes as labelsfor biological targets, ourefforts turned
to the labeling of 3a (Figure 1). This was accomplished by
reacting amide 9 and the lithium acetylide of alkyne 1010 to
produce the DDDA precursor 11 in 58% yield (Scheme 2).
Subjecting 11 to the DDDA reaction conditions followed
by a BuchwaldꢀHartwig cross-coupling reaction gener-
ated the TBS-protected fluorescent compound 12 in a 53%
overall yield. Deprotection of the TBS group with TBAF
afforded the attachable fluorophore 13 in 91% yield.
This convenient synthetic protocol allows for the pre-
paration of additional fluorophores with tethers of
varying lengths and conformationally mobility between
the carbonyl and the reactive hydroxyl group. With
regards to optical properties, the TBS-protected and
hydroxyl derivatives 12 and 13 showed absorption and
emission maxima in DCM comparable to those ob-
served for fluorophore 2a.
The final labeling strategy is depicted as 3b (Figure 1).
Analogs of fluorophore 2b are especially attractive because
this fluorophore displays an absorption maximum in the
visible region of the electromagnetic spectrum (425 nm).8b
In a manner entirely analogous to the preparation of other
DDDA precursors, 14a and 14b were prepared, isolated,
In conclusion, attachable cyclopenta[b]naphthalene sol-
vatochromic fluorophores structurally related to Prodan
were synthesized through the functionalization of fluo-
rescent compounds previously reported by our group.
(10) For more details on the synthesis of substrates 9 and 10, see
Supporting Information.
Org. Lett., Vol. XX, No. XX, XXXX
C

fluorophores. Utilizing the cyclopentane moiety of these
dyes, fluorescent diols were obtained. Probes incorporat-
ing conformationally mobile or rigid monohydroxyl-
functionalized linkers were alsoprepared. All fluorophores
maintained the photophysical properties of their parent
compounds showing enhanced solvatochromism when
compared to Prodan. Finally, fluorescent lipid analogs
and unnatural amino acid derivatives were prepared start-
ing from the newly synthesized dyes, demonstrating their
potential as versatile labels for biomolecules. We expect
thatthese fluorophoreswillbeofgeneral utilityinthe study
of lipid dynamics in cellular membranes and the detection
of protein-binding interactions.11 Futurestudies are direct-
ed toward expanding this chemistry-driven approach to
prepare fluorophores with enhanced chemical properties
such as multifunctionality and/or increased solubility in
buffered aqueous solutions.
Scheme 3. Synthesis of Dyes 15a and 15b
a Absorption maximum (nm) in DCM. b Emission maximum (nm) in
DCM. c Stokes shift (nm) in DCM.
Scheme 5. Synthesis of the Fluorescent Fatty Acid Analog 22
Scheme 4. Synthesis of the Unnatural Fluorescent Amino Acids
18 and 21
a Absorption maximum (nm) in DCM. b Emission maximum (nm) in
DCM. c Stokes shift (nm) in DCM.
Acknowledgment. We thank the National Science
Foundation (CHE0910597) and NIH (P50-GM067982)
for supporting this work.
Supporting Information Available. Detailed experimen-
tal procedures and characterization data for all new
compounds; fluorescent emission spectra for compounds
6aꢀd, 7aꢀc, 8, 12, 13, 15aꢀb, 16ꢀ22; solvatochromic
spectra for compounds 7b, 13, 15b, 21, and 22; quantum
yields and extinction coefficients for compounds 7b and
15b. This material is available free of charge via the
Internet at http://pubs.acs.org.
a Absorption maximum (nm) in DCM. b Emission maximum (nm) in
DCM. c Stokes shift (nm) in DCM.
(11) Barucha-Kraszewska, J.; Kraszewski, S.; Ramseyer, C. Lang-
muir 2013, 29, 1174–1182.
Three different sites for structural modification were con-
sidered to avoid altering the optical properties of the
The authors declare no competing financial interest.
D
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