Nonclassical SNAPFL analogue as a Cy5 resonance energy transfer partner

Article abstract of DOI:10.1021/ol801907b

(Graph Presented) We have synthesized a new SNAPFL analogue (1) that exhibits a large Stokes shift (78 nm) (abs. 542 nm, em. 620 nm) and a good quantum yield. Because of the large overlap between the emission spectrum of 1 and the absorption spectrum of C

Full text of DOI:10.1021/ol801907b

ORGANIC
LETTERS
2008
Vol. 10, No. 21
4931-4934
Nonclassical SNAPFL Analogue as a
Cy5 Resonance Energy Transfer Partner
Sung Hoon Kim, Jillian R. Gunther, and John A. Katzenellenbogen*
Department of Chemistry of UniVersity of Illinois, 600 South Mathews AVenue,
Urbana, Illinois 61801
jkatzene@uiuc.edu
Received August 27, 2008
ABSTRACT
We have synthesized a new SNAPFL analogue (1) that exhibits a large Stokes shift (78 nm) (abs. 542 nm, em. 620 nm) and a good quantum
yield. Because of the large overlap between the emission spectrum of 1 and the absorption spectrum of Cy5, 1 functions well as a fluorescence
donor to Cy5 and has been used in FRET-based experiments using estrogen receptor site-specifically labeled with Cy5 and a receptor ligand
conjugated to SNAPFL.
FRET is one of the most widely used spectroscopic tools
in drug discovery, elucidation of gene1 and protein function
(protein conformational change), examination of protein
interactions with ligands, genes, or other proteins, and use
in high-throughput assays.2-4 Fluorophores that are typically
used for FRET studies include those of the cyanine and Alexa
classes, tetramethylrhodamine (TMR), and fluorescein; each
of these, however, has a Stokes shift less than 30 nm and a
broad excitation and emission range (approximately 150 nm).
Consequently, many possible FRET pairs that provide ample
overlap of the donor emission and acceptor excitation spectra
cannot be optimally used because the broad acceptor excita-
tion spectrum (150 nm) results in the direct excitation of
the acceptor and subsequent undesired increase in acceptor
emission. This necessitates a more complicated calculation
to obtain an accurate determination of the energy transfer
and opens the results to systematic errors due to experimental
variability. To minimize this direct excitation of the acceptor,
a donor with an excitation spectrum sufficiently separated
from the acceptor excitation can be chosen, but oftentimes
this results in poor FRET overlap between the donor emission
and acceptor excitation. To improve this situation, we have
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10.1021/ol801907b CCC: $40.75
Published on Web 10/09/2008
2008 American Chemical Society

sought a new dye system having a large Stokes shift that
would allow efficient energy transfer between the donor and
acceptor with minimal direct excition of the acceptor
fluorophore; this would result in a more direct and accurate
measurement of the energy transfer between the fluorophores.
Scheme 1. Synthetic Scheme To Prepare for
Benz[c]xanthen-7-one (10)
hydroxyls in compound 9 were protected as tert-butyldim-
ethylsilyl ethers by reacting this bisphenol with TBDMSCl
and imidazole in DMF at rt. Xanthene moiety 10 was coupled
with Grignard reagent 13, generated from 4-iodo-3-methyl-
benzoic acid according to a modified Knochel method,8
followed by treatment with an aqueous 1 N HCl solution to
provide SNAPFL analog 1 in 64% yield (Scheme 2).
Upon searching through a variety of organic dyes to find
a better FRET pair for the Cy5 dye (ex. 653 nm, em. 665
nm), we discovered the unsymmetrical SNAPFL (Semi
Naphthalene Fluorescein) fluorophore, which exhibits a large
Stokes shift and has a good emission overlap with the
excitation spectrum of Cy5. Commercial SNAPFL is pro-
vided as a mixture of the 5- and 6-carboxylic acid isomers.
Recently, both Urano5 and Peterson6 reported that synthesis
of a novel type of fluorescein with a methyl at the 2 position
of the phenyl ring instead of a carboxy afforded a fluorophore
with a quantum yield and photophysical properties very
similar to fluorescein itself. Therefore, we designed a new
synthetic route to prepare the unsymmetrical SNAPFL analog
1 as a single isomer; our replacement of the carboxyl in
SNAPFL with a methyl group in 1 also eliminated a charged
group that can sometimes be troublesome in biological
applications that require cell uptake.
Scheme 2. Synthesis of SNAPFL Analogue (1) and
Cyclofenil-Conjugated SNAPFL (2)
Here, we describe the synthesis of a new SNAPFL
analogue dye 1 ((3,10-dihydroxybenz[c]xantene-7-yl)-4-
carboxy-2-methylbenzene), characterization of its physical
properties, conjugation of 1 with a nonsteroidal estrogen
receptor ligand, and investigation of its potential as a FRET
partner for Cy5 using a biological model system for ligand-
protein interactions.
To synthesize SNAPFL analogue 1 (3,10-dihydroxyben-
z[c]xantene-7-yl-(4-carboxy-2-methylbenzene), the precursor
10 (3,10-di[(tert-butyl-dimethyl)silyloxy]benz[c]xanthen-7-
one) was prepared as shown (Scheme 1). 2-Formyl-1,6-
dimethoxy-naphthalene 6 was synthesized according to
previously described methods.7 Addition of 2,4-dimethox-
yphenyl magnesium bromide to aldehyde 6 afforded alcohol
7 in 96% yield, and subsequent oxidation with MnO2 (3
equiv) in methylene chloride gave ketone 8 quantitatively.
Xanthene moiety 9 was obtained quantitatively by the
simultaneous demethylation and cyclization of 8 with pyri-
dinium hydrogen chloride salt over 180 °C. Both phenolic
This fluorophore, intended for use as a FRET partner with
Cy5, was conjugated with monoaminoethyl cyclofenil 14,9,10
a nonsteroidal antagonist of the estrogen receptor (ER), which
is a ligand-modulated transcription factor. After cyclofenil
conjugation with SNAPFL, the λmax for emission shifted from
620 to 624 nm, and the emission intensity decreased 15%
compared to SNAPFL. Interestingly, upon binding of the
ligand-SNAPFL conjugate to unlabeled ER, the emission
intensity was almost fully recovered with essentially no
change in emission wavelength (λmax 624 nm).
The binding affinity of compound 2 for both estrogen
receptor subtypes, ERR and ERꢀ, was evaluated using a
radiometric binding assay whereby test compounds compete
with a tritiated ligand estradiol for binding to the receptor.
(5) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano,
T. J. Am. Chem. Soc. 2005, 127, 4888.
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Hahm, J, B.; Kulp, K.; Hochberg, R. B.; Zhou, H.; Katzenellenbogen, J. A.;
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Biol. 2008, 4, 241
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Org. Lett., Vol. 10, No. 21, 2008

Binding affinities for compound 2, expressed relative to that
of estradiol set at 100%, were determined to be 3.3 ( 0.5%
for ERR and 28 ( 3.2% for ERꢀ.11 These percentages
correspond to a Ki of approximately 6 nM for the tethered
compound binding to ERR, based on a Kd of 0.2 nM for
estradiol binding to ERR.
The relative quantum yield (SI) (Φ ) 0.21) was measured
using rhodamine B as a standard in phosphate buffer at pH
9.0, according to previously described protocols.12 The pKa
value was calculated by measuring the absorbance of 1 with
varying pH. As shown in Figure 1A, the pKa of 1 (7.5) is a
full log unit less acidic than fluorescein (6.5).6,13
Figure 2. Schematic of SNAPFL-Cy5 FRET. Cy5-labeled ER (left)
binds cyclofenil-SNAPFL (2) and FRET is observed (middle).
When excess estradiol out-competes the ligand-fluorophore (right),
FRET signal is decreased.
Figure 1. Characterization of compound 1. (A) Plot of pH versus
compound 1 absorbance allowed calculation of the pKa of com-
pound 1 (7.5). Different pH sample solutions were prepared using
a 0.1 M NaH2PO4/Na2HPO4 buffer and diluting the stock dye
solution 1:10 into buffer (final dye concentration ) 10 µM). (B)
Absorption spectrum of 1 (blue broken line) and Cy5 (red broken
line) and emission spectrum of 1 (blue solid line) and Cy5 (red
solid line). Extinction coefficients for λ542 at pH 9, 8, 7, 6, and 5
are 58300, 46500, 26800, 18000, and 16500 cm-1 M-1, respectively.
Figure 3. FRET experiment between 2 and Cy5-ERR. Emission
spectrum of 2 alone (4 nM in pH 8.5 Tris buffer, black line), Cy5-
ERR alone (1 nM in pH 8.5 Tris buffer, blue line), preincubated
Cy5-ERR and 2 (1 nM ERR and 4 nM 2 in the same buffer, green
line), and preincubated 1 nM Cy5-ERR and 4 nM 2 in the presence
of excess 1 µM E2 (red line). The excitation was fixed at 520 nm.
To evaluate the possible use of this new fluorophore in
FRET experiments, we site-specifially labeled the ligand
binding domain of ERR at cysteine 417 with a thiol-reactive
Cy5 conjugate.14 Based on the quantum yield of SNAPFL,
the Forster radius for Cy5 at pH 9.0, estimated by the formula
R0 ) [(8.8 × 1023)κ2n-4QYDJ(λ)]1/6 Å,15 was R0 ) 62.5 Å.
As shown in Figure 1B, compound 1 has a large Stokes
shift (78 nm), and its emission nicely overlaps with the
excitation spectrum of Cy5 around 620 nm. To mimimize
any direct excitation of Cy5, we excited the cyclofenil
conjugate 2 at 520 nm instead of at its λmax, 542 nm.
Incubation of ERR-Cy5 with 2 for 1 h at 4 °C (1 nM ERR,
4 nM compound 2, 50 mM Tris buffer, pH 8.5) resulted in
a 70% increase in Cy5 emission intensity, with a concomitant
decrease in the emission intensity of 2 by 23% (see Figure
2 (schematic) and Figure 3 (data), green line vs black and
blue lines). When 2 was blocked from binding to the estrogen
receptor by addition of competing ligand estradiol at 1 µM,
energy transfer between the dyes disappeared, as expected
(Figure 3, red line vs green line). For this experiment, a 1 h
incubation usually decreased the FRET signal by 80%; we
extended the incubation to 8 h to ensure complete ligand
exchange. The loss of FRET is evident by the decrease in
acceptor (Cy5) emission intensity at 665 nm and the increase
in donor (SNAPFL) emission intensity at 624 nm. The
fluorescence remaining after estradiol has been added (red
curve) represents not FRET but background fluorescence
from Cy5 and SNAPFL in the absence of FRET; it is,
essentially, the sum of the blue curve and the black curve,
respectively.
The wavelength used to excite the SNAPFL fluorophore
2, 520 nm, results in very little direct excitation of the Cy5
FRET partner. In fact, this bleed-through energy resulting
from direct excitation comprises only 3% of the total intensity
of Cy5 emission at 649 nm (when excited through a FRET
transfer) and is therefore negligible for most experiments.
In contrast, other commonly used fluorophores having
smaller Stokes shifts must be excited at longer wavelengths,
resulting in a higher percentage of Cy5 emission due to direct
excitation instead of FRET transfer. For example, the
following commonly used FRET partners for Cy5 produce
varying degrees of this direct excitation: TMR (ex. 543 nm,
(12) Anstead, G. M.; Hwang, K.-J.; Katzenellenbogen, J. A. Photochem.
Photobiol. 1993, 57, 616.
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J. A. Biochemistry 1997, 36, 14897.
(13) Olmstead, J., III. J. Phys. Chem. 1979, 83, 2581.
Org. Lett., Vol. 10, No. 21, 2008
4933

em. 567 nm; 7% bleed-through at 543 nm), Alexa Fluor 568
(ex. 578, em. 603 nm; 18% bleed-through at 578 nm), and
Alexa Fluor 594 (ex. 590 nm, em. 618 nm; 30% bleed-
through at 590 nm), Rhodamine RedX (ex. 572 nm, em. 592
nm; 20% bleed-through at 572 nm) and Cy3 (ex. 553 nm,
em. 565 nm, 9% bleed-through at 553 nm) (see Figure 2 in
the Supporting Information). The SNAPFL fluorophore
analogue described here, in contrast to many of the above-
mentioned commonly used fluorophores in this spectral
range, is excited at a shorter wavelength, which minimizes
direct Cy5 excitation in FRET experiments and, therefore
provides a truer measure of the energy transfer between two
molecules.
valuable new fluorophore and a potential FRET partner for
the well-known fluorescent dye Cy5.
Acknowledgment. We are grateful for support of this
research by a grant from the National Institutes of Health
(PHS R37 DK15556). Jillian R. Gunther was supported by
a David Robertson Fellowship and the National Institutes
of Health (NRSA 1 F30 ES016484-01 and NRSA 5 T32
GM070421). We thank Kathryn Carlson for performing the
radiometric binding assays.
Supporting Information Available: Procedures for the
synthesis of SNAPFL (1) and spectrometric characterization
of intermediates (5-10), 2, and 1. This material is available
free of charge via the Internet at http://pubs.acs.org.
This new SNAPFL dye, which is characterized by a large
Stokes shift and a pH-dependent absorbance spectrum, is a
(14) Tamrazi, A.; Katzenellenbogen, J. A. Methods Enzymol. 2003, 364,
37.
(15) Fo¨rster, Th. Ann. Phys. 1948, 2, 55.
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Org. Lett., Vol. 10, No. 21, 2008