Photophysics and biological applications of the environment-sensitive fluorophore 6-N,N-dimethylamino-2,3-naphthalimide

Article abstract of DOI:10.1021/ja0449168

We have synthesized a new environment-sensitive fluorophore, 6-N,N-dimethylamino-2,3-naphthalimide (6DMN). This chromophore exhibits valuable fluorescent properties as a biological probe with emission in the 500-600 nm range and a marked response to changes in the environment polarity. The 6DMN fluorescence is red-shifted in polar protic environments, with the maximum emission intensity shifting more than 100 nm from 491 nm in toluene to 592 nm in water. Additionally, the fluorescence quantum yield decreases more than 100-fold from chloroform (Φ = 0.225) to water (Φ = 0.002). The scope and applications of the 6DMN probe are expanded with the synthesis of an Fmoc-protected amino acid derivative (5), which contains the fluorophore. This unnatural amino acid has been introduced into several peptides, demonstrating that it can be manipulated under standard solid-phase peptide synthesis conditions. Peptides incorporating the new residue can be implemented for monitoring protein-protein interactions as exemplified in studies with Src homology 2 (SH2) phosphotyrosine binding domains. The designed peptides exhibit a significant increase in the quantum yield of the long wavelength fluorescence emission band (596 nm) upon binding to selected SH2 domains (e.g., Crk SH2, AbI SH2, and PI3K SH2). The peptides can be used as ratiometric sensors, since the short wavelength band (460 nm) was found almost invariable throughout the titrations.

Full text of DOI:10.1021/ja0449168

Published on Web 01/04/2005
Photophysics and Biological Applications of the
Environment-Sensitive Fluorophore
6-N,N-Dimethylamino-2,3-naphthalimide
M. Eugenio Va´zquez, Juan B. Blanco,† and B. Imperiali*
Contribution from the Department of Chemistry and Department of Biology,
Massachusetts Institute of Technology, 77 Mass AVenue, Cambridge, Massachusetts 02139
Received August 23, 2004; E-mail: imper@mit.edu
Abstract: We have synthesized a new environment-sensitive fluorophore, 6-N,N-dimethylamino-2,3-
naphthalimide (6DMN). This chromophore exhibits valuable fluorescent properties as a biological probe
with emission in the 500-600 nm range and a marked response to changes in the environment polarity.
The 6DMN fluorescence is red-shifted in polar protic environments, with the maximum emission intensity
shifting more than 100 nm from 491 nm in toluene to 592 nm in water. Additionally, the fluorescence quantum
yield decreases more than 100-fold from chloroform (Φ ) 0.225) to water (Φ ) 0.002). The scope and
applications of the 6DMN probe are expanded with the synthesis of an Fmoc-protected amino acid derivative
(5), which contains the fluorophore. This unnatural amino acid has been introduced into several peptides,
demonstrating that it can be manipulated under standard solid-phase peptide synthesis conditions. Peptides
incorporating the new residue can be implemented for monitoring protein-protein interactions as exemplified
in studies with Src homology 2 (SH2) phosphotyrosine binding domains. The designed peptides exhibit a
significant increase in the quantum yield of the long wavelength fluorescence emission band (596 nm)
upon binding to selected SH2 domains (e.g., Crk SH2, Abl SH2, and PI3K SH2). The peptides can be
used as ratiometric sensors, since the short wavelength band (460 nm) was found almost invariable
throughout the titrations.
Introduction
fluorophores, the development of new extrinsic fluorophores
remains an essential element for the design of new fluorescent
probes.
Environment-sensitive fluorophores are a special class of
Fluorescence spectroscopy has become one of the most
valuable tools for the development of new probes for biochemi-
cal research,1 being extensively used for monitoring ions,2 small
molecules,3 and biological processes,4 such as protein folding,5
protein-protein interactions,6 and phosphorylation events.7
While many fluorescence applications rely on the use of intrinsic
chromophores that have spectroscopic behavior that is dependent
on the physicochemical properties of the surrounding environ-
ment.8 Particularly useful are the solvatochromic fluorophores
that display sensitivity to the polarity of the local environment,
such as 2-propionyl-6-dimethylaminonaphthalene (PRODAN),9
4-dimethylamino phthalimide (4-DMAP),10 and 4-amino-1,8-
naphthalimide derivatives11 (Chart 1). These molecules generally
exhibit a low quantum yield in aqueous solution, but become
highly fluorescent in nonpolar solvents or when bound to
hydrophobic sites in proteins or membranes.
Introduced more than 20 years ago,9a PRODAN and deriva-
tives still constitute the most widely used environment-sensitive
fluorophores, regardless of certain limitations mainly resulting
from the relatively intense fluorescence even in aqueous
† Departamento de Qu´ımica Orga´nica y Unidad Asociada al CSIC.
Universidad de Santiago de Compostela, 15782 Santiago de Compostela,
Spain.
(1) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. Mol. Cell Biol.
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Fabrizzi, L.; Licchelli, M.; Rabaioli, G.; Taglietti, A. Coord. Chem. ReV.
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9
1300
J. AM. CHEM. SOC. 2005, 127, 1300-1306
10.1021/ja0449168 CCC: $30.25 © 2005 American Chemical Society

Environment-Sensitive Fluorophore 6DMN
A R T I C L E S
environments. Herein we present the synthesis and preliminary
photophysical characterization of a new environment-sensitive
fluorophore 6-N,N-dimethylamino-2,3-naphthalimide (6DMN),
together with its application for the development of new probes
for biological interactions using a new amino acid residue for
Fmoc-based solid-phase peptide synthesis (Fmoc-Dap(6DMN)-
OH, 5 (Chart 2)). The 6DMN fluorophore combines some of
the advantageous fluorescence properties of PRODAN with the
extreme sensitivity to the local polarity exhibited by the
4-aminophthalimide family of environment-sensitive fluoro-
phores.10 The 6DMN fluorophore may also find important
technological applications analogous to those of 4-amino-1,8-
naphthalimides in new materials such as liquid crystal displays12
or light-emitting diodes.13
Therefore, SH2 domains facilitate phosphorylation-dependent
protein-protein interactions that result in signal propagation
within the cell.19 Examination of the structures of phosphopep-
tides bound to SH2 domains reveals that the interaction of the
phosphopeptides with the SH2 domains takes place on the
surface of the protein, and the phosphopeptides remain largely
exposed to solvent even when bound to the proteins.20 Thus,
initial experiments using DANA as fluorescent reporter did not
reveal a significant increase in fluorescence upon binding.
Development of fluorescent sensors for specific SH2 domains
would allow for a deeper understanding of their function within
complex cellular networks and aid in the screening of small-
molecule inhibitors that could be used to modulate SH2 activity
and, ultimately, serve as pharmaceutical agents.21
Results and Discussion
Chart 1. Examples of Environment-Sensitive Fluorophores
PRODAN (1), 4-DMAP (2), and 4-Amino-1,8-naphthalimide (3)
Synthesis of 6DMN Fluorophore and the Amino Acid
Building Block (5). Synthesis of the 6-N,N-dimethylamino-2,3-
naphthalene derivatives 4 and 5 required access to the key
anhydride intermediate 11 (Scheme 1). The first step involved
protection of the commercially available 2-nitrobenzaldehyde,
6, by refluxing in toluene with acid catalysis and continuous
removal of water using a Dean-Stark apparatus.22 The resultant
nitroarene 7 was then coupled with chloromethyl phenyl sulfone
via a vicarious nucleophilic substitution.23 The reaction pro-
ceeded in acceptable yield, affording the mixture of isomers at
positions 2 and 4, in a 1:1 ratio. The desired 2-(2-phenylsufo-
nylmethyl-5-nitrophenyl)-1,3-dioxolane isomer was separated
by silica gel chromatography and, after deprotection of the
aldehyde, afforded intermediate 8 in good yield. The naphthalene
moiety was then assembled by reaction of 8 in a multistep
process. Addition of the nucleophilic sulfone anion to the
electrophilic diethyl maleate, followed by addition of the
resulting anion to the electrophilic aldehyde and subsequent
elimination of phenylsulfinic acid and water, gave the desired
Chart 2. 6-Dimethylamino-2,3-naphthalimide 6DMN Derivatives
Studied: Model Compound 4 (6DMN-GlyOMe), Amino Acid
Building Block 5 (Fmoc-Dap(6DMN)-OH)
Previously, we reported that solvatochromic amino acids
could be used to probe phosphorylation-dependent peptide-
protein interactions.14 In the previous study, the DANA amino
acid (which included the PRODAN fluorophore) was used to
signal binding by a phosphoserine peptide to 14-3-3, which
is a protein involved in cell cycle control.15 Another common
phosphopeptide binding motif in nature is the Src homology 2
(SH2) domain. SH2 domains are small protein modules (ap-
proximately 100 amino acids) that are involved in tyrosine
kinase signaling networks.16 SH2 domains specifically recognize
short phosphotyrosine-containing peptide sequences in target
proteins through interactions with the phosphotyrosine side chain
and a short sequence of C-terminal residues (three to five amino
acids).17 The specificity of the interaction of the peptide for
any of the different SH2 variants is largely determined by the
nature of those residues adjacent to the phosphotyrosine.17b,18
Scheme 1. Synthesis of 6-N,N-Dimethylamino Naphthalic
Anhydride Intermediate 11a
a (a) HOCH2CH2OH, p-TsOH, toluene, reflux, overnight, 100%; (b)
ClCH2SO2Ph, KOH, DMSO, 23 °C, overnight, 69%; (c) AcOH/water 4:1,
reflux, 5 h, 100%; (d) diethyl maleate, 18-crown-6, K2CO3, CH3CN, 23 °C
then reflux, 3 h, 74%; (e) formalin, 10% Pd/C, H2 (1 atm) room temperature,
4 h, 84%; (f) KOH aq 40%, reflux, 4h; (g) 4, vacuum, 4 h, 77%.
(12) Grabchev, I.; Moneva, I.; Bojinov, V.; Guittonneau, S. J. Mater. Chem.
2000, 10, 1291-1296.
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VCH: Weinheim, Germany, New York, 2001.
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Kuriyan, J.; Cowburn, D. Annu. ReV. Biophys. Biomol. Struct. 1997, 26,
259-288.
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D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14053-14058. (b) Pauptit, R.
A.; Dennis, C. A.; Derbyshire, D. J.; Breeze, A. L.; Weston, S. A.; Rowsell,
S.; Murshudov, G. N. Acta Crystallogr. 2001, D57, 1397-1404. (c)
Waksman, G.; Shoelson, S. E.; Pant, N.; Cowburn, D.; Kuriyan, J. Cell
1993, 72, 779-790.
(21) Shakespeare, W. C. Curr. Opin. Chem. Biol. 2001, 5, 409-415.
(22) Babler, J. H.; Malek, N. C.; Coghlan, M. J. J. Org. Chem. 1978, 43, 1821-
1823.
(23) Makosza, M.; Golinski J. Org. Chem. 1984, 49, 1488-1494.
(13) Cacialli, F.; Friend, R. H.; Bouche, C.-M.; Le Barny, P.; Facoetti, H.; Soyer,
F.; Robin, P. J. Appl. Phys. 1998, 83, 2343-2356.
(14) Va´zquez, M. E.; Nitz, M.; Stehn, J.; Yaffe, M. B.; Imperiali, B. J. Am.
Chem. Soc. 2003, 125, 10150-10151.
(15) Muslin, A. J.; Tanner, J. W.; Allen, P. M.; Shaw, A. S. Cell 1996, 84,
889-897.
(16) (a) Pawson, T. Cell 2004, 116, 191-203. (b) Pawson, T.; Gish, G. D. Cell
1992, 71, 359-362. (c) Moran, M. F.; Koch, C. A.; Anderson, D.; Ellis,
C.; England, L.; Martin, G. S.; Pawson, T. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 8622-8626. (d) Koch, C. A.; Anderson, D.; Moran, M. F.; Ellis,
C.; Pawson, T. Science 1991, 252, 668-674.
(17) (a) Lee, C.-H.; Kominos, D.; Jacques, S.; Margolis, B.; Schlessinger, J.;
Shoelson, S. E.; Kuriyan, J. Structure 1994, 2, 423-438. (b) Bradshaw, J.
M.; Waksman, G. AdV. Protein Chem. 2002, 61, 161-210.
9
J. AM. CHEM. SOC. VOL. 127, NO. 4, 2005 1301

A R T I C L E S
Va´zquez et al.
naphthalene derivative 9.24 Reductive amination of intermediate
9 with formalin and hydrogen in the presence of Pd/C afforded
the desired diethyl 6-N,N-dimethylamino-2,3-naphthalene di-
carboxylate 10 in excellent yield. Treatment of 10 with aqueous
potassium hydroxide yielded the corresponding diacid, which
was dehydrated by sublimation under reduced pressure to afford
the desired anhydride 11.
For the assembly of the amino acid, commercially available
Fmoc-Dap(Boc)-OH (12) was allyl-protected (Scheme 2). The
side-chain amine of the fully protected intermediate 13 was
selectively deprotected by treatment with trifluoroacetic acid
(TFA) to afford the free amine 14, which was coupled without
further purification to the naphthalic anhydride 11. As expected
from previous studies in the group,25 the first coupling reaction
of the free amine 14 with the anhydride proceeded smoothly.
However, the ring closure reaction and formation of the
corresponding 6DMN fluorophore took place only after activa-
tion of the free acid using a 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate/N-hydroxybenzotria-
zole (HBTU/HOBt) mixture. This procedure afforded the desired
protected amino acid Fmoc-Dap(6DMN)-OAllyl (15) under mild
conditions. Allyl deprotection of the fully protected building
block 15 afforded the desired building block 5 in good yield.
For the synthesis of the model compound 4, glycine ester
hydrochloride was reacted in a similar way with anhydride 11.
Scheme 2. Coupling of Anhydride 11, Synthesis of 5a
Figure 1. (a) UV absorption spectra of 4 (40 µM) in methanol. (b)
Fluorescence emission spectra of 4 in different solvents. Labels show
maximum emission wavelengths for selected solvents (toluene, chloroform,
and methanol).
to those found in the unsubstituted parent compound.26 The
absorption spectrum was found to be relatively insensitive to
changes in the polarity of the environment, with only a small
red shift of the long wavelength absorption band from 372 nm
in 1,4-dioxane to 388 nm in water (Figure 1a).
In general, we have observed that 6DMN shows fluorescence
properties parallel to those exhibited by the closely related
fluorophore 4-DMAP, showing, however, a far higher emission
intensity (Figure 1b). The maximum excitation wavelength of
6DMN is at 375 nm, and the fluorescence emission spectra are
strongly dependent on the polarity of the environment: the
maximum emission wavelength is red-shifted almost 100 nm
from 491 nm in toluene to 589 nm in methanol, and the
fluorescence quantum yield decreases more than 100 times from
chloroform (Φ ) 0.225) to water (Φ ) 0.002).
In contrast to the behavior of the parent compounds (2,3-
naphthalimides), which hardly show any fluorescence response
to changes in the polarity of the solvents,26 6DMN shows
outstanding solvatochromic fluorescence properties. The most
remarkable effect is the quenching observed in protic solvents
that results in a strong reduction of the fluorescence quantum
yield and a significant red shift of the emission band to the
vicinity of 590 nm. Polar aprotic environments (acetonitrile,
N,N-dimethyl formamide, acetone, tetrahydrofuran) also induce
an increase in the quantum yield, as well as a marked blue shift.
Apolar aprotic solvents such as chloroform, dichloromethane,
toluene, or 1,4-dioxane cause the largest blue shift and increase
in the quantum yield relative to polar protic solvents. For
example, in toluene the maximum emission intensity is at 491
nm, and in water it is at 592 nm (see Table 1).
a (a) 1. Cs2CO3, MeOH, 23 °C, 10 min, 2. Allyl bromide, 23 °C, 78%;
(b) TFA, CH2Cl2, 0 °C, 30 min, 23 °C, 2h; (c) 1. 11, DIEA, DMF, 23 °C,
15 min, 2. HBTU, HOBt, 23 °C, overnight, >95%; (d) Pd(PPh3)4, phenyl
silane, CH2Cl2, 23 °C, 30 min, >95%.
Photophysics of 6DMN: UV Spectroscopy, Steady-State
Emission Spectra, Quantum Yield, and Solvatochroism. The
6DMN fluorophore is closely related to the 4-amino-1,8-
naphthalimides; however, as has been already established for
unsubstituted aromatic dicarboximides, the photophysical prop-
erties of these systems have a strong dependence on the position
of the dicarboximide group on the naphthalene system, and
therefore the new system required detailed characterization.26
The most significant feature in the UV spectrum is the intense
absorption band at 378 nm. This band is not present in the
unsubstituted 2,3-naphthalimide, while the other two bands at
shorter wavelengths (282 and 237 nm) are qualitatively similar
(24) Tyrala, A.; Makosza, M. Synthesis 1994, 3, 264-266.
(25) Va´zquez, M. E.; Rothman, D. M.; Imperiali, B. Org. Biomol. Chem. 2004,
2, 1965-1966.
(26) Wintgens, V.; Valat, P.; Kossanyi, J.; Biczok, L.; Demeter, A.; Be´rces, T.
J. Chem. Soc., Faraday Trans. 1994, 90, 411-421.
9
1302 J. AM. CHEM. SOC. VOL. 127, NO. 4, 2005

Environment-Sensitive Fluorophore 6DMN
A R T I C L E S
Table 1. Photophysical Properties of 4 in Different Solvents
absorbance
maximum
(nm)
emission
maximum
(nm)
quantum
yield
solvent
type
solvent
(Φ)
watera
methanol
2-propanol
ethanol
388
382
385
379
592
589
589
584
0.002
0.012
0.018
0.027
protic
acetonitrile
DMF
acetone
tetrahydrofuran
1,4-dioxane
380
380
375
373
372
549
545
532
510
498
0.135
0.155
0.148
0.147
0.220
polar
aprotic
dichloromethane
chloroform
toluene
379
380
373
517
509
491
0.210
0.225
0.208
apolar
aprotic
a Because of solubility limitations, the quantum yield in water was
calculated using peptide Abl-bp (see Table 2).
It is known that the solvent sensitivity to polarity can be
analyzed in terms of difference in the dipole moments in the
ground and excited states, and it has been found that the most
sensitive fluorophores are those with the largest changes in the
dipole moment. This can be estimated from a Lippert-Mataga
plot, which is essentially a plot of the Stokes shift of the
fluorescence emission versus the solvent polarity.27,28 The
difference in the maximum absorption and emission wave-
lengths, expressed in wavenumbers (∆νj), is fitted to the
following equation:
Figure 2. (a) Plot of the Stokes shift vs the solvent polarity measured as
the orientation polarizability ∆×c4, derived from fluorescence spectra of 4
in a series of 1,4-dioxane/acetonitrile mixtures. ∆×c4 is obtained for each
mixture using ꢀ and n values calculated using the molar fractions as
follows: ꢀ ) øacetonitrile × 38.8 + ødiox × 2.218; n ) øacetonitrile × 1.3442 +
ødiox × 1.4224; øacetonitrile + ødiox ) 1. (b) Lippert plot of 4 in different
solvents. The line corresponds to the best linear fit to the data, excluding
the values obtained for protic solvents (methanol, 2-propanol and ethanol,
b).
2
ꢀ - 1
2ꢀ + 1
n2 - 1
2n2 + 1
νjA - νjF ) ∆νj )
-
(µe - µg)2
3
(
)
hca0
solvent effects for protic solvents; as can be seen in Figure 2b,
in most solvents the Stokes shift is proportional to the orientation
polarizability. However, protic solvents induce a disproportion-
ately large Stokes shift. This phenomenon has also been
observed for other environment-sensitive fluorophores and has
been explained as the result of emission from two different
excited states: one with higher dipole moment that is stabilized
in polar environments and another less polar excited state
responsible for the emission in less polar media.32
SH2 Binding Peptides: Design and Synthesis. Upon
completion of the building block synthesis, 5 was incorporated
into peptides using standard Fmoc solid-phase peptide synthesis
(SPPS). The peptide targets were designed for use in evaluating
the potential applicability of 6DMN-based peptidic sensors in
monitoring phosphorylation-dependent peptide-protein interac-
tions in the context of SH2 domains. The phosphotyrosine
peptide-binding SH2 domains were selected as targets for their
high biological significance and the unique challenges presented
for probe design.19,33 SH2 domains interact with phosphorylated
2∆f
)
3∆µ2
(1)
hca0
where µe - µg is the difference between the dipole moments of
the excited and the ground states, respectively (∆µ), c is the
velocity of light, h is Plank’s constant, and a0 is the radius of
the Onsager cavity around the fluorophore. The parameters ꢀ
and n are the solvent dielectric constant and refraction index,
respectively, which are grouped in the term ∆f, known as
orientation polarizability. The Onsager radius was calculated
from the optimized structure obtained with a DFT minimization
using the Gaussian program29 (B3LYP functional using 6-31G-
(d) orbital base). The Onsager radius (4.19 Å) was taken as
half of the average distance between nitrogen of the amine donor
and the two carbonyl oxygens, which corresponds to the longest
distance across the molecule where charge separation can take
place.30 As can be seen in Figure 2a, the Stokes shift of a series
of mixtures of 1,4-dioxane/acetonitrile changes linearly in
response to the solvent polarity, which correlates with an
increase in the dipole moment (∆µ) of 5.5 D. This value is
similar to reported values for other environment-sensitive
fluorophores.30,31 Lippert plots also give evidence of specific
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R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.;
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A R T I C L E S
Va´zquez et al.
tyrosine-containing sequences at peptide-protein or protein-
protein interfaces. With phosphotyrosine-dependent protein-
protein interactions, the essential phosphoamino acid contacts
are commonly complemented by other simultaneous protein-
protein interactions that greatly enhance selectivity.17,34 In
general, SH2 domains are highly conserved, and the largest
energetic contribution to their interaction with peptides or
proteins comes from the contacts made with the phosphoty-
rosine. It is therefore challenging to achieve tight and selective
binding to a specific SH2 domain with small molecules or
peptides.35 Additionally, analysis of the available structures of
phosphopeptides bound to SH2 domains reveals that the peptides
are docked onto the protein surface, rather than sequestered in
a deep pocket, as is the case with the phosphoserine peptide-
binding proteins such as 14-3-3.36 The shallow surface for
interaction exposes most of the peptide side chains to the bulk
aqueous environment, thus making it more difficult to use
polarity-based (environment-sensitive) sensors as probes for the
interaction, since the polarity of the microenvironment sur-
rounding the fluorophore does not necessarily change upon
binding to the SH2 domain.
expressed in bacteria as GST fusion proteins for purification
purposes (GST-Crk SH2, GST-Abl SH2, and GST-PI3K SH2).
It has been shown previously that the GST fusion does not affect
the affinity of SH2 domains for phosphotyrosine-containing
peptides.39
Binding to SH2 Domains: Influence of N-Terminal
Backbone Flexibility on the Fluorescent Signal. Peptides Crk-
bp and Crk-bp2 were designed to evaluate the influence of
conformational flexibility on the fluorescence emission upon
SH2 binding. Crk-bp and Crk-bp2 share the same C-terminal
sequence recognized by Crk SH2 (pTyr-Asp-His-Pro), but they
differ in the N-terminal amino acid residue between the
phosphotyrosine and the fluorescent Dap(6DMN). Crk-bp
contains a glutamine found in many Crk binding sites.37 In Crk-
bp2, the glutamine is substituted for a glycine. Introduction of
glycine next to the reporting amino acid residue was proposed
to allow greater flexibility and potentially enhance interactions
between the protein and the 6DMN side chain, inducing an
increased fluorescent signal upon binding to the target SH2.
Such an interaction would be conformationally inaccessible with
the R-substituted amino acids.
Peptides for SH2 binding were designed on the basis of
previous studies with peptide libraries that identified the
optimized amino acid sequence for binding to different members
of the SH2 domain family.37 The binding sequences with the
highest scores from the library targeting Abl SH2 and Crk SH2
domains were incorporated into peptides, along with the reporter
amino acid, 5, inserted at the (+2) position relative to the
phosphotyrosine residue38 (Table 2).
The fluorescence spectrum of the peptide Crk-bp shows two
emission maxima: a band at 470 nm, similar to the fluorescence
emission observed with model compound 4 in very hydrophobic
solvents, and another band at 620 nm, similar to that observed
for 4 in polar protic solvents such as methanol or water.
Incubation of Crk-bp with the target SH2 domain, GST-Crk
SH2, results in an approximately 5-fold enhancement of the long
wavelength emission band, which is blue-shifted 55 nm from
620 to 565 nm (Figure 3a). Incubation of the glycine-containing
peptide, Crk-bp2, with GST-Crk SH2 induces a similar blue
shift on the long wave emission band, but a much larger increase
in the emission intensity (11-fold, Figure 3b). Binding constants
with GST-Crk SH2 are 4.8 ( 2 µM and 2.4 ( 1 µM,
respectively. The larger fluorescence increase observed for Crk-
bp2 may be the result of the higher flexibility of the peptide
that allows for better docking of the fluorescent side chain in a
hydrophobic environment created by the protein. Incubation of
Crk-bp2 with other SH2 domains (GST-Abl SH2 or GST-PI3K
SH2), to assess the selectivity of the peptides, induces only a
modest increase in the fluorescence emission intensity of the
long wavelength band (Figure 3c) and also shows reduced
affinities for these other nontarget SH2 domains (binding
constants 20 ( 2 µM for GST-Abl-SH2 and 40 ( 6 µM for
GST-PI3K SH2).
A similar phenomenon in the fluorescence emission (increase
of quantum yield of the long wavelength emission band) has
been previously observed for the related fluorophore p-(N,N-
diethylamino)benzoic acid in aqueous â-cyclodextrin solutions.
This has been attributed to burial of the donor moiety (an amine)
in the hydrophobic cyclodextrin cavity, with exposure of the
acceptor group into bulk water.40 The observed increase in
fluorescence emission upon protein binding has been associated
with a destabilization of the nonemissive twisted-intramolecular
charge-transfer state as a direct consequence of the reduced
polarity of the fluorophore environment,41 although a more
comprehensive study would be needed to obtain a definitive
conclusion about the mechanism.
Table 2. Peptide Sequences and Corresponding SH2 Domain
Targeteda
target
peptide SH2
peptide sequence
Crk-bp Crk Ac-Glu-Dap(6DMN)-Gln-pTyr-Asp-His-Pro-Asn-Ile-(CONH2)
Crk-bp2 Crk Ac-Glu-Dap(6DMN)-Gly-pTyr-Asp-His-Pro-Asn-Ile-(CONH2)
Abl-bp Abl Ac-Glu-Dap(6DMN)-Gly-pTyr-Glu-Asn-Val-Gln-Ser-(CONH2)
Abl-bp2 Abl Ac-Glu-Dap(6DMN)-pTyr-Glu-Asn-Val-Gln-Ser-(CONH2)
a pTyr refers to phosphotyrosine. All peptides are capped as N-terminal
acetyl and C-terminal amide derivatives. The SH2 recognition sequence is
noted in bold.
Peptides including the fluorescent environment-sensitive
amino acid were synthesized using standard Fmoc-SPPS,
cleaved/deprotected from the solid support, and purified by
reverse-phase high-performance liquid chromatography (HPLC).
The new 6DMN side chain proved resistant to the standard
mildly basic amino acid coupling conditions (0.12 M diisopro-
pylethylamine), the Fmoc deprotection conditions (20% pip-
eridine), and the acidic resin cleavage and deprotection cocktail
(95% TFA).
The designed peptides were examined for changes in fluo-
rescence upon binding to the respective SH2 domains. The SH2
domains (Crk SH2, Abl SH2, and C-terminal PI3K SH2) were
(34) Pawson, T.; Raina, M.; Nash, P. FEBS Lett. 2002, 513, 2-10.
(35) Ladbury, J. E.; Arold, S. Chem. Biol. 2000, 7, R3-R8.
(36) Rittinger, K.; Budman, J.; Xu, J.; Volinia, S.; Cantley, L. C.; Smerdon, S.
J.; Gamblin, S. J.; Yaffe, M. B. Mol. Cell 1999, 4, 153-166.
(37) Songyang, Z.; Shoelson, S. E.; Chaudhuri, M.; Gish, G.; Pawson, T.; Haser,
W. G.; King, F.; Roberts, T.; Ratnofsky, S.; Lechleider, R. J.; Neel, B. G.;
Birge, R. B.; Fajardo, J. E.; Chou, M. M.; Hanafusa, H.; Schaffhausen, B.;
Cantley, L. C. Cell 1993, 72, 767-778.
(38) In previous studies in our group using DANA as fluorescent reporter
(unpublished results), substitution at the (+2) position was shown to induce
the biggest change in fluorescence emission intensity and wavelength.
(39) Ladbury, J. E.; Lemmon, M. A.; Zhou, M.; Green, J.; Botfield, M. C.;
Schlessinger, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3199-3203.
9
1304 J. AM. CHEM. SOC. VOL. 127, NO. 4, 2005

Environment-Sensitive Fluorophore 6DMN
A R T I C L E S
Figure 3. Fluorescence titrations of peptides (a) Crk-bp and (b) Crk-bp2 (20 µM in PBS buffer, pH 7.5) with GST-Crk SH2. Insets show plots of the
fluorescence emission intensity with the best fitting binding curves. Spectra were corrected for the dilution upon addition of the protein solution. (c) Relative
fluorescence emission intensities at 565 nm for (1) peptides Crk-bp and Crk-bp2 in buffer, (2) peptide Crk-bp saturated with GST-Crk SH2, (3) peptide
Crk-bp2 saturated with GST-Crk SH2, (4) peptide Crk-bp2 saturated with GST-Abl SH2, and (5) peptide Crk-bp2 saturated with GST-PI3K SH2.
Figure 4. Fluorescence titrations of peptide Abl-bp (20 µM in PBS buffer, pH 7.5) with (a) GST-Abl SH2 or with (b) GST-PI3K SH2. Insets show plots
of the fluorescence emission intensity at 599 nm, with the best fitting binding curves. Spectra were corrected for the dilution upon addition of the protein
solution. (c) Relative fluorescence emission intensities at 590 nm for (1) peptide Abl-bp, (2) peptide Abl-bp saturated with GST-Abl SH2, (3) peptide
Abl-bp saturated with GST-PI3K SH2, and (4) peptide Abl-bp saturated with GST-Crk SH2.
Binding to SH2 Domains: Selectivity of SH2 Binding.
Peptide Abl-bp also showed dual fluorescence emission bands
at 470 and 610 nm. When peptide Abl-bp was incubated with
increasing amounts of GST-Crk SH2 there was no significant
change in the fluorescence emission spectra (Figure 4c).
However, there was a marked increase in the emission intensity
of the long wavelength band induced by GST-Abl SH2 (almost
4-fold), and even more significantly by GST-PI3K SH2 (almost
10-fold), even though the affinity of Abl-bp for PI3K SH2 is
lower than that for Abl SH2 (Figure 4). Binding constants for
GST-Crk SH2, GST-Abl SH2, and GST-PI3K SH2 are 30 (
8, 13 ( 2, and 42 ( 11 µM, respectively.
Finally, peptide Abl-bp2, with the fluorescent reporting amino
acid directly adjacent to the phosphotyrosine, was synthesized
to investigate if the interaction between the 6DMN side chain
and the phosphotyrosine could be enhanced. The fluorescence
emission spectra of this peptide show the dual fluorescence
behavior found in Abl-bp and demonstrate higher selectivity
for binding to Abl SH2 against any of the other SH2 domains.
The binding constants for GST-Abl SH2, GST-Crk SH2, and
GST-PI3K SH2 are 12 ( 3, 400 ( 93, and 100 ( 32 µM,
respectively. However, the induced increase in emission intensity
in the long wavelength band is clearly smaller than that for Abl-
bp (Figure 5). Note that the Abl-bp2 signal increase is higher
with GST-Abl SH2 than GST-PI3K SH2.
The larger fluorescence increase with GST-PI3K SH2 as
compared to GST-Abl SH2 can be attributed to the different
sets of specific interactions of each SH2 domain with the bound
peptide. Although there is no structural information on Abl-
peptide complexes, it has been proposed that there is an
accessory interaction between an arginine side chain of the Abl
SH2 domain with the backbone carbonyl of Gly (+1) and the
side chain of residue (+2) of the bound peptide. The microen-
vironment of those peptide positions should be largely affected
by the presence of the charged arginine in Abl SH2, which is
likely more polar than the same position in PI3K SH2, for which
no such interaction has been proposed.42
In summary, studies with the Crk-binding peptides show that
it is feasible to design a good fluorescent probe for the Crk-
SH2 domain. Crk-bp2 binds to Crk-SH2 with an affinity of
2.4 µM and undergoes an 11-fold change in fluorescence
emission intensity at 565 nm. Studies with the related Crk-bp
peptide show that the flexible amino acid between the reporter
amino acid and the phosphotyrosine in Crk-bp2 is important
for achieving the maximum fluorescent response with the Crk-
SH2. Studies with the Abl-bp peptide, which is targeted at the
Abl-SH2 domain, show good affinity (13 µM) for the targeted
SH2 domain (Abl-SH2) together with a 4-fold signal increase.
However, in this case the fluorescence increase with the
alternative SH2 domain, PI3K-SH2, is larger, although the
affinity for this domain is reduced. In this case, if the fluorescent
amino acid is inserted directly adjacent to the phosphotyrosine,
as in Abl-bp2, the fluorescent change is minimal and the affinity
greatly reduced. These results suggest that some flexibility
(40) Kim, Y. H.; Cho, D. W.; Yoon, M.; Kim, D. J. Phys. Chem. 1996, 100,
15670-15676.
(41) Nag, A.; Dutta, R.; Chattopadhyay, N.; Bhattacharyya, K. Chem. Phys.
Lett. 1989, 157, 83-86.
(42) Zvelebil, M. J. J. M.; Panayotou, G.; Linacre, J.; Waterfield, M. D. Protein
Eng. 1995, 8, 527-533.
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J. AM. CHEM. SOC. VOL. 127, NO. 4, 2005 1305

A R T I C L E S
Va´zquez et al.
Figure 5. Fluorescence titrations of peptide Abl-bp2 (20 µM in PBS buffer, pH 7.5) with (a) GST-Abl SH2 and with (b) GST-PI3K SH2. Insets show plots
of the fluorescence emission intensity at 599 nm, with the best fitting binding curves. Spectra were corrected for the dilution upon addition of the protein
solution. (c) Relative fluorescence emission intensities at 599 nm for (1) peptide Abl-bp2, (2) peptide Abl-bp2 saturated with GST-Abl SH2, (3) peptide
Abl-bp2 saturated with GST-PI3K SH2, and (4) peptide Abl-bp2 saturated with GST-Crk SH2.
between the signaling module and the phosphotyrosine is
important to enable adoption of the conformation that simul-
taneously affords a fluorescence increase and accommodates
good binding. In the case of the Abl-SH2 targeted peptides, we
have observed large changes in the emission intensity with a
nontarget SH2 domain (Abl-bp fluorescence increase with GST-
PI3K SH2) with the current design. However, the modular nature
of the probe, together with the ease with which further
refinement of the peptide sequence can be processed through
combinatorial methods, suggests that it should be possible to
optimize the binding and reporting properties of each probe to
effectively target specific SH2 domains. Thus, results compa-
rable to those observed with Crk-bp2 and the Crk-SH2 should
be attainable. Taken together, these results show the potential
applications of this new fluorophore for the development of
fluorescent peptidic sensors for biological interactions. In the
future, we also anticipate that larger fluorescence changes and
higher specificities can be obtained if the 6DMN-based fluo-
rescent amino acid is incorporated into full-length proteins via
protein semi-synthesis or unnatural amino acid mutagenesis,
since in this case shielding from water should be maximized in
the protein-protein interface.
was synthesized to explore the application of 6DMN to probe
biological interactions. This amino acid can be easily incorpo-
rated into peptides using standard Fmoc-SPPS methods and was
used to synthesize peptides that selectively bind SH2 phospho-
tyrosine binding domains (Crk SH2 and Abl SH2). The results
show that 6DMN fluorescence emission intensity significantly
increases upon binding of the designed peptides to specific SH2
domains.
Acknowledgment. This research was supported by the Cell
Migration Consortium (GM64346) and the National Science
Foundation (CHE-0414243). We also acknowledge the support
of the International Human Frontier Science Program Organiza-
tion for the award of a postdoctoral fellowship to E.V. and the
Spanish Ministry of Science and Technology for the graduate
fellowship to J.B.B.C. We also wish to thank Prof. Timothy
M. Swager (MIT) and Prof. A. Samanta (University of Hy-
derabad) for their helpful advice and Prof. Martin Schwartz
(University of Virginia) for the generous gift of the clones
expressing SH2 domain-GST conjugates.
Supporting Information Available: Experimental procedures
and spectroscopic data for the synthesis of 6DMN-GlyOMe (4)
and Fmoc-Dap(6DMN)-OH (5). Detailed peptide synthesis
protocols. Quantum yield determination of 4. GST fusion SH2
domains expression and purification. Fluorescence titration
binding assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
Conclusions
A new environment-sensitive fluorophore, 6DMN, has been
synthesized. 6DMN displays excellent fluorescent properties for
biochemical studies: long excitation (375 nm) and emission
wavelengths (460-590 nm) and also a large quantum yield and
Stokes shift. Additionally, a new amino acid building block
containing this fluorescent group, Fmoc-Dap(6DMN)-OH (5),
JA0449168
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1306 J. AM. CHEM. SOC. VOL. 127, NO. 4, 2005