Signal-amplifying resonance energy transfer: A dynamic multichromophore array for allosteric switching

Article abstract of DOI:10.1002/anie.200701410

(Figure Presented) Cooperative structural folding and unfolding of a dynamic multichromophore array provides an unusual signal amplification as part of a resonance energy transfer from the central donor unit to peripheral acceptor ensembles. The stepwise

Full text of DOI:10.1002/anie.200701410

Angewandte
Chemie
DOI: 10.1002/anie.200701410
Allosteric Switches
Signal-Amplifying Resonance Energy Transfer: A Dynamic
Multichromophore Array for Allosteric Switching**
Justin A. Riddle, Xuan Jiang, John Huffman, and Dongwhan Lee*
Cooperativity is an important operation mode for regulatory
proteins with multiple binding sites.[1–3] The interdependence
of stepwise binding events in such multi-subunit receptors is
manifested by a sigmoidal binding isotherm, in which a sharp
change in the response curve relates the “on” and “off” states
that are separated by a narrow concentration window
(Figure 1a). Extreme cooperativity in homotropic allosterism
implicates orientation-dependent D–A coupling as a viable
mechanism for signal amplification.[10]
We recently showed that the photophysical properties of a
two-dimensional (2D) electronic conjugation can be rever-
sibly modified by structural folding and unfolding motions.[11a]
The molecular C3 symmetry of this first-generation com-
pound 1 (Figure 2a) intrinsically suggested cooperativity in
the stepwise assembly and disassembly of its cyclic hydrogen-
bonding network as part of the conformational switching, but
direct experimental evidences for this intriguing phenomenon
could not be attained. To probe the solution dynamics of this
emerging family of switchable 2D fluorophores,[11e] we turned
our attention to the RET of multichromophore arrays.[10,12]
Our molecular design, as shown in Figure 2c, consists of
three A units placed around a common D unit to cause RET
from the donor excited state (D*A3) to an ensemble of
acceptor excited states (D(A3)*). Unlike typical RET sys-
tems, in which the acceptor emission is modified by changing
the D···A distance (while all other parameters are treated as
constants),[10] this dynamic system operates by structural
folding and unfolding, a procedure that reversibly tunes the
donor quantum yield (FD) to directly impact emission from
the acceptor units (FA) at essentially fixed D···A distances.
This seemingly straightforward and arguably incremental
structural modification has highly unexpected photophysical
consequences, as described below.
results in
a binary-function-like response curve that
approaches that of genuine on–off switching (Figure 1b)
and is difficult to reproduce with non-cooperative host–guest
systems displaying
a simple hyperbolic behavior (Fig-
ure 1c).[4–6] Undoubtedly, artificial systems with self-regula-
tory properties remain an engaging challenge.[3,5,7]
Despite elegant demonstrations of positive allosterism
with selected synthetic receptors,[5,8] a genuine switching
cycle, shown as reversible turn-on and turn-off scans in
Figure 1a, is yet to be realized.[9] Herein, we describe a
dynamic multichromophore array (DA3), which transduces
allosteric conformational changes of the energy-donor unit
(D) into amplified fluorescence emission from the energy-
acceptor units (A). The characteristic sigmoidal response
function and signal buffering of this array convincingly
demonstrate cooperativity in both the turn-on and turn-off
switching cycles. Notably, the unusual nonlinear behavior
observed in its resonance energy transfer (RET) process
For efficient RET with the tris(N-salicylideneaniline)[11]
energy donor, a boron–dipyrrin (BODIPY)[13] derived energy
acceptor was employed to prepare compound 2 in a highly
convergent four-step synthetic sequence from readily avail-
able starting materials (Scheme 1).[14] The characteristic
ꢀ
ꢀ
proton NMR signals of Nenamine H (13.58 ppm) and Cvinyl
H
ꢀ
(9.95 ppm) and the highly deshielded O H (5.08 ppm) proton
resonance signal of compound 2 match closely with the signals
ꢀ
ꢀ
of structurally characterized analogues that have O H···O
H···Oketone hydrogen-bonding networks supporting the
C3 symmetric molecular core (Figure 2).[11a,b] Variable-tem-
Figure 1. Response curves describing the cooperative binding of a
ligand L to a receptor Swith: a) n=6 and b) n=100 binding sites. A
non-cooperative binding isotherm is shown in (c). These plots were
constructed from the Hill equation with arbitrary binding constants
(for comparative purposes).[4]
1
perature H NMR studies of compounds 1 and 2 furnished
comparable DdOꢀH/DT plots (see Figure S1 in the Supporting
Information),[14] which were consistent with the conforma-
tional switching depicted in Figure 2c.
At 258C, compound 2 displays electronic transitions in
CH2Cl2, which are associated with local chromophores
resembling 1 and 3 (see Figure S2 in the Supporting Informa-
tion).[14] The essentially orthogonal geometric relationship
between the BODIPY plane and the aniline ring (Scheme 1)
prevents direct electronic conjugation, thus minimizing the
interchromophore interactions in the ground state. With
[*] J. A. Riddle, X. Jiang, Dr. J. Huffman, Prof. Dr. D. Lee
Department of Chemistry, Indiana University
Bloomington, IN 47405 (USA)
Fax: (+1)812-855-8300
E-mail: dongwhan@indiana.edu
Homepage: http://mypage.iu.edu/~dongwhan/home.html
[**] This work was supported by Indiana University, the National Science
Foundation (CAREER CHE 0547251), and the American Chemical
Society Petroleum Research Fund (42791-G3).
ꢀ
restricted torsional motions around the Caniline CBODIPY bonds,
which are enforced by the BODIPY methyl groups pointing
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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quantitative RET (see Figure S2 in the Sup-
porting Information).[14] Notably, this RET
cascade resulted in an approximately fivefold
enhancement of the overall emission quantum
yield relative to that of the donor-only com-
pound 1. This enhanced dynamic range
prompted us to use the emission from remotely
located energy acceptors to probe structural
folding and unfolding of the energy donor
(Figure 2c).
In CH2Cl2, the fluoride ion, added as
nBu4NF, elicits a structural unfolding of the
energy donor (compound 1), which can be
readily monitored by following the decrease in
the fluorescence quantum yield (FD).[11a]
Under similar conditions, compound 2 under-
goes an expected quenching of the energy-
acceptor emission, but with an unexpected
signal amplification towards the Fꢀ ion. As
shown in Figure 3, compound 1 exhibited a
decrease of about 40% in its emission intensity
(FD) upon treatment with 6 equiv of Fꢀ ions. In
contrast, the fluorescence from the RET
ꢀ
Figure 2. a) Chemical structures of compounds 1–3 with a close-up view of the Ohydroxy
acceptor in compound
2 was essentially
H···Ohydroxy H···Ocarbonyl hydrogen-bonding network (shown as capped-stick model).[11a,b]
b) Energy-minimized (PM3) structure of compound 2 in which the tris(N-salicylideneani-
line) and BODIPY units are color-coded with blue and red, respectively. c) Schematic
representation of conformational switching between the emissive “folded” (top) and the
nonemissive “unfolded” (bottom) states of compound 2 (D=energy donor; A=energy
acceptor).
ꢀ
quenched (> 90% decrease in FA) under
similar conditions. Control experiment with
compound
3 ruled out direct interaction
between BODIPY and the Fꢀ ion in this
process (Figure 3b).
The apparent 1:6 binding stoichiometry
between compound 2 and the Fꢀ ion (see
Figure 3b) defined a narrow concentration window to probe
the details of this molecular-recognition event. As shown in
Figure 4a, the fluorescence intensity of compound 2 could be
reversibly switched by the stepwise addition and removal of
Fꢀ species.[16] Notably, the turn-off and turn-on “scans” trace
essentially superimposable sigmoidal isotherms reminiscent
of that in Figure 1a. The rapid rise/drop in the response
function, bracketed by signal-buffering zones (dI/d[Fꢀ] = 0)
on both sides, prevents undesired turn-on/off by noise-level
Scheme 1. Synthetic route to compound 2.[15] a) Ag2SO4, I2, EtOH,
room temperature (RT); b) 3-ethyl-2,4-dimethylpyrrole (2.1 equiv),
CH2Cl2, RT; c) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
(1.1 equiv), CH2Cl2, RT; d) Et3N (10 equiv), BF3·Et2O (15 equiv),
CH2Cl2, ~; e) HCCHCH2OH, [PdCl2(PPh3)2] (2 mol%), CuI (3 mol%),
iPr2NH/THF, RT; f) 1,3,5-triformylphloroglucinol, EtOH, ~.
Figure 3. a) Emission spectra of compounds 1 and 2 normalized to
the absorbance at the excitation wavelength (340 nm) 1) prior to and
2) after addition of Fꢀ ions (6 equiv). b) Changes in the fluorescence
intensity of compounds 1–3 as a function of the amount of Fꢀ. For
direct comparison, emission data have been normalized to the same
value of integrated peak area at [Fꢀ]=0. lex =340 nm for compounds 1
and 2 and 480 nm for 3. [1]=1.0 mm, [2]=1.0 mm, and [3]=3.0 mm in
CH2Cl2 (at 298 K). IFl. =fluorescence intensity.
toward the aniline rings, the multichromophore array 2 with
well-defined spatial relationships between the energy-donor
and -acceptor units is established.
Upon excitation at 380 nm, compound 2 emits an intense
yellow BODIPY fluorescence signal at lmax,em = 546 nm with a
quantum yield of 20%. The complete disappearance of the
donor emission centered at 457 nm indicates an essentially
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Key to the observation of this remarkable allosteric
switching is the highly unusual signal-amplification
behavior that we observed as part of the RET.
Specifically, the normalized response profile shown in
Figure 3b deviates significantly from the simple pro-
portional relationship between the donor and acceptor
emissions that would normally be anticipated from an
RET with fixed D–A distance (RDA), donor lifetime
(tD), and spectral overlap integral (J), as described by
Equation (1). The J(l) term expresses the degree of
ꢀ
ꢁ
k2
tDR6DA
kRET / FD
JðlÞ
ð1Þ
spectral overlap between the donor emission and the
acceptor absorption, which remains constant in the
absence of spectral shifts (see Figure 3a).[10] Prelimi-
nary lifetime measurements showed that the tD value
also remains essentially independent of the fluoride
concentration [Fꢀ], which is characteristic of a static
quenching mechanism. Therefore, the only remaining
term that can be responsible for the experimentally
observed nonlinearity is the orientation factor k2, which
varies depending on the relative orientations of the
electronic transition moments associated with D and A
(0 ꢁ k2 ꢁ 4, see Figure 5a).[10,18]
Figure 4. a) Sigmoidal fluorescence response curves obtained by titration of
compound 2 with nBu4NF and subsequent back-titration with Me3SiCl
([2]=1.0 mm in CH2Cl2 at 298 K). b) On–off switching cycles of compound 2
monitored by changes in the fluorescence intensity upon successive addition
and removal of Fꢀ ions (6 equiv), as described in (a). Corrections were made
for dilution effects.
As a consequence of the restricted rotations around
ꢀ
the Caniline CBODIPY bonds (Figure 2), the pairwise
interactions between electronic transition moments
residing on the tris(N-salicylideneaniline) and
input, which is a critical requirement for genuine switching
devices.[5–7] This switching cycle can be repeated over ten
times without significant loss of reversibility (Figure 4b).
For a given n value, multiple isomeric 2·(Fꢀ)n species can
exist which differ in the positional distribution of the fluoride
ions among the n binding sites. The absence of hysteresis in
the forward and backward scans shown in Figure 4a impli-
cates a rapid establishment of equilibrium between these
positional isomers to produce a population-averaged fluores-
cence signal that is independent of the reaction pathways
followed to reach the same fluoride inventory, either by
addition (turn-off scan) or by removal (turn-on scan) of Fꢀ
ions. The 1H NMR spectrum of a mixture comprising
compound 2 and nBu4NF (6.5 equiv) in CD2Cl2 revealed the
disappearance of the wing-tip hydroxy-proton resonances
along with a significant broadening of the signals from
adjacent methylene groups; this observation is consistent
BODIPY planes in compound 2 are no longer subjected to
conventional models of rapidly tumbling RET pairs in which
k2 is simply treated as an orientation-averaged constant
(Figure 5c).[10] In addition, it is reasonable to conceive that
the correlated tilting motions of the three peripheral aryl
rings, which are directly attached to the BODIPY groups,
could assist in defining conformation-dependent finite trajec-
tories of k2 (Figure 5b). The validity of this proposal needs to
be tested against elaborate models that account for the
probability distribution of k2 as a function of structural folding
and unfolding and for the collective effects of the D–A
ꢀ
with a rapid chemical exchange of the O H protons via a
hydrogen-bonding interaction with the Fꢀ ions.[17]
The self-accelerated reactivity of compound 2 can equally
well be explained by invoking either the two-state concerted-
switching model (MWC model) or the sequential conforma-
tional transition model (KNF model) of allosterism (see
Schemes S1 and S2 in the Supporting Information).[1,2,14] The
progressive structural unfolding of compound 2 should
present an increasing number of OH groups, the interde-
pendence of which is already preprogrammed in their
symmetric arrangements in a tight hydrogen-bonded network
(Figure 2a).
Figure 5. The orientation factor k2 depends on the three parameters
qD, qA, and f, which relate mD (the emission transition moment of the
donor) and mA (the absorption transition moment of the acceptor), as
defined in (a).[10] For D–A pairs that belong to a freely tumbling RET
ensemble, such as that shown in (c), k2 becomes an orientation-
averaged constant of 2/3.[10] For a system comprising spatially well-
defined D–A pairs, such as in (b), a finite distribution of the k2 value
is anticipated.
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Communications
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(Figure 3).[18–20] Efforts are currently underway to understand
and exploit this previously underutilized orientational aspect
of RET as a viable amplification mechanism for signal
transduction.
In summary, signal amplification in RET has enabled us to
demonstrate the genuine allosteric switching of a dynamic
multichromophore array, which operates in a perfectly
reversible manner in response to a stepwise addition and
removal of chemical input at a mm concentration level. The
underlying molecular mechanism of the unique amplification
behavior observed both in the recognition and the energy-
transfer steps holds significant implications for molecular
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[9] One challenge is to devise ingenious schemes to systematically
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lenge arises from the typically weak chemical forces involved in
noncovalent interactions, which require a large excess of ligand
L to drive the formation of S·Ln species from a multisite receptor
S with n binding sites. To complete the switching cycle depicted
in Figure 1a, this excessive amount of L needs to be quantita-
tively removed to regenerate free S. This is an operationally
nontrivial task and has not been demonstrated before.
[10] a) J. R. Lakowicz, Principles of Fluorescence Spectroscopy,
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Experimental Section
The synthesis and characterization of compounds 1–3 (including
crystallographic data)[14] are described in the Supporting Information.
Received: April 2, 2007
Published online: August 6, 2007
Keywords: cooperative phenomena ·
.
FRET (fluorescence resonant energy transfer) · hydrogen bonds ·
molecular devices · signal amplification
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[6] A sharp structural transition of macromolecules often involves
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[15] CCDC-642093 contains the supplementary crystallographic data
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request/cif. See also the Supporting Information.
[16] The stoichiometric reaction between Me3SiCl and Fꢀ affords
Me3SiF and Clꢀ, neither of which reacts with the receptor
molecule.
[17] The 1:1 binding between a Fꢀ ion and the OH group (schemati-
cally drawn in Figure 4) is consistent with the experimentally
observed 1:6 stoichiometry between compound 2 and nBu4NF,
although alternative geometries can also be considered. See:
a) G. A. Jeffrey, An Introduction to Hydrogen Bonding, Oxford
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[20] Additionally, one could also consider the orientation-dependent
self quenching of BODIPY as another RET pathway leading to
signal amplification. This mechanism is currently under inves-
tigation.
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