Allosteric Control of Photofoldamers for Selecting between Anion Regulation and Double-to-Single Helix Switching

Article abstract of DOI:10.1021/jacs.8b10538

Allosteric regulation of protein structure and function is a hallmark of biology. The structures of protein-like abiological foldamers have been subject to allosteric control, however, regulation of their function is rare. We report this behavior using a

Full text of DOI:10.1021/jacs.8b10538

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Article
Allosteric Control of Photofoldamers for Selecting between
Anion Regulation and Double-to-Single Helix Switching
Fred C Parks, Yun Liu, Sibali Debnath, Sydney R Stutsman, Krishnan Raghavachari, and Amar H Flood
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 22 Nov 2018
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Allosteric Control of Photofoldamers for Selecting between
Anion Regulation and Double-to-Single Helix Switching
Fred C. Parks,† Yun Liu,†,‡ Sibali Debnath,† Sydney R. Stutsman,† Krishnan Raghavachari,† and
Amar H. Flood*,†
†Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, IN 47405, USA
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Abstract
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Allosteric regulation of protein structure and function is a hallmark of biology. The structures of
protein-like abiological foldamers have been subject to allosteric control, however, regulation of
their function is rare. We report this behavior using a photoactive foldamer following the
discovery that small and large anions select between single and double helical structures,
respectively. Correspondingly, these anions activate different functions in the photofoldamer;
small anions turn on photoregulation of anion concentrations while large anions turn on
chiroptical switching of quaternary structure. For this demonstration, we used an aryl-triazole
based photofoldamer in which the light-driven trans-cis isomerization of azobenzenes alters
intrastrand - contacts while the triazoles define the allosteric anion-binding site. Binding to
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–
–
–
–
eleven anions of increasing size was quantified (Cl–, Br–, NO2 , I–, NO3 , SCN–, BF4 , ClO4 ,
–
–
–
ReO4 , PF6 , SbF6 ). Contrary to expectations that single helices will expand to accommodate
–
larger and larger guests, this behavior only occurs for smaller anions (Cl– – NO3 ; < 45 Å3)
–
beyond which the larger anions form double helices (SCN– – SbF6 ; > 45 Å3). With small anions,
the single helix regulates anion concentrations when the azobenzenes are photoswitched. The
binding of large anions favors a chiral double helix and activates light-driven switching into
racemic single helices thereby modulating the quaternary structure and chiroptical activity. This
work shows how complex multifunctional outcomes emerge when allosteric changes in structure
are expressed in intrinsically functional foldamers.
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INTRODUCTION
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Foldamers offer a powerful abiological platform for investigating the structures and
functions common to biology.1 Recent examples include structural emulation of DNA,2
ribosomal synthesis,3 and the hydrophobic collapse of proteins.4 Allostery is a common
biological character where the activity of an enzyme is altered by a change in its structure upon
binding an allosteric effector.5-8 Mimicry of this classic behavior has been expressed in
supramolecular catalysis involving transition metals,9-14 and has inspired modulations of
molecular recognition where the binding of one guest (the effector) activates binding of a
different guest.15-21 With foldamers, allostery has inspired incorporation of other functions. In
one case, the helix sense of transmembrane foldamers is photoswitched to alter
enantiocatalysis.22 In another, photoswitching of the foldamer between helix and random coil
states produced an affinity swing to alter the solution concentration of chloride guests.23 Nature
also expresses other roles for which pyruvate kinase (PYK) is exemplary of a multifunctional
protein.24-25 It can exist in multiple quaternary forms (tetramers,25-26 dimers,27-28 and monomers24,
29) based on a variety of allosteric effectors24-29 with each form having different functions. With
fructose-1,6-bisphosphate25, 29 bound, the tetramer is activated to catalyze formation of pyruvate.
With triiodo-L-thyonine (T3) binding as an effector, it drives formation of an active PYK
monomer that homotropically regulates the concentration of T3 in cells.25-26, 29 The dimer is
different again and phosphorylates proteins upon binding tyrosine phosphopeptide.27-28 Different
quaternary structures have different functions when activated by different allosteric effectors. We
were surprised to find similar behavior emerge in a photofoldamer that derives multifunctional
behavior from the allosteric binding of anions of different sizes (Figure 1).
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This work was originally undertaken to investigate how the affinity swings of a
photofoldamer are influenced by the size of the anionic guest. Based on prior anion-binding
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foldamers,30-31 we expected the helix to expand to accommodate larger guests to lower the
affinity swing. Instead, and for the first time with aromatic foldamers, we found that guest size
controls access to a single or double helix. We also found the different helices had different
light-driven functions. Based on anion size, therefore, we activated either homotropic regulation
of anion concentrations in solution, a behavior similar to PYK’s T3 regulation, or photoswitching
between chiral double helices and racemic single helices. For this reason, we realized that these
differences were similar to the complex multifunctional allostery displayed by PYK.
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Anion Size
< 45 Å3
Function 1
Function 2
Chiral
Double Helix
Binding
> 45 Å3
N
N
3 π-π
contacts
430
nm
365
nm
Vis
UV
Vis
UV
N N
Photo
Activity
Racemic
Single Helix
Release
–
–
= Cl–, Br–, NO2 , I–, NO3
–
–
–
–
–
= SCN–, BF4 , ClO4 , ReO4 , PF6 , SbF6
Figure 1. Guest size dictates selection of single or double helices and the function of the helices upon light
switching of the azobenzene end groups. Under the working conditions, the single helix switches between
trans (F1tt) and cis (F1cc) to drive cycles of anion binding and release whereas large anions allow switching
between double and single helices.
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We know that foldamers can respond to various stimuli to alter their structure. Folding
into single helices can be driven using low temperature,23, 32 poor solvents4, 23, 32-36 and guest
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23, 32-41
binding.4,
Light-responsive photofoldamers4,
incorporate a covalently linked
photoswitch,42-47 like azobenzene.33 Reversible activation of the photoswitch changes the
conformational distribution of the foldamer to favor or disfavor the helix over the random coil. In
prior work we coupled this helix-coil switching to the binding and release of anions,23 an
outcome gaining interest with photo-switchable receptors.37, 48-57 Furthermore, even though we
have observed a double helix with one photofoldamer,4 the switching efficiency was poor.
Structural access to double helices instead of single helices requires additional
considerations. Specific interactions between the intertwining strands can enhance double helix
formation, such as with the coordination of transition metals.58-60 External driving forces can be
used, like solvophobic61-62 and hydrophobic effects,4, 63 in which double helices bury more of the
total  surface than is possible in a single helix.4 These solvent-driven effects benefit from
structures with longer chains.61 Torsion angles64 can also be tuned to better accommodate the
larger pitch present in the structures of double helices.65 Finally, double helices can form around
almost any guest (alkali66-67 and transition-metal cations,58, 60, 68 and anions4, 69-71). Returning to
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–
the role of guest size, anionic guests spanning a wide range of sizes (Cl– to PF6 ranges 20 to 73
Å3) have been studied with many foldamer classes, e.g., aryl-triazoles4, 23, 30-32, 37, 72-76 indoles,77-81
aliphatics,82 and triazole-carboxamides.83 However, the size of the bound guest has never been
observed as a design feature for driving double helix formation despite studies directed at
varying anion size.
Switchable foldamers that can interconvert reversibly between single and double helices
have only been observed in three instances. Lehn used a Ag+-templated double helix that formed
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a single helix upon addition of cryptand, and was switched using acid/base addition to control
Ag+-cryptand binding.84 Yashima used an activator-inhibitor85 concept to unwind an
oligoresorcinol double helix by addition of cyclodextrin and then re-wind upon addition of
adamantane. Yamaguchi used thermally responsive oligomers to either denature86 or form87
double helices at elevated temperatures. Foldamers that switch reversibly between quaternary
states using light as a non-destructive and non-invasive stimulus are not known.
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Herein, we describe the unexpected selection of single and double helices from
photofoldamers by using guest size, and the ability to use these guests as allosteric effectors to
change the light-driven function of the photofoldamer (Figure 1). We demonstrate this finding
with a flexible aryl-triazole foldamer F1 (Scheme 1). Therein, the allosteric site is composed of
triazole-based binding pocket and where the azobenzenes serve as functional sites. We selected
11 anions spanning volumes from 20 to 83 Å3 and representing an array of geometries including
–
–
–
–
–
spherical (Cl–, Br–, I–) or pseudospherical (BF4 , ClO4 , ReO4 , PF6 , SbF6 ), as well as linear
–
–
(SCN–), bent (NO2 ), and trigonal planar (NO3 ). In contrast to prior works by Jiang31 and
Craig30 to bind foldamers with a wide range of anions, we first characterized the solvent-driven
folding as a means to identify the best solvent for preparing a pre-folded initial state; acetonitrile
in this case. Using a combination of NMR, circular dichroism (CD), and UV-Vis titrations, we
characterize all binding constants for the helix and photoswitched random-coil states to identify
how the function of the photofoldamer changes with anion size; we initially only expected to see
changes in the affinity swing. Instead, these studies reveal that smaller anions (<45 Å3, Cl– to
–
–
NO3 ) form single helices while larger anions (>45 Å3, SCN– to SbF6 ) form double helices with
varying degrees of positive cooperativity88 ( = 4 K2:1  K1:1 = 50 – 10,000). Thus, we realized
that small anions can activate the photofoldamer to change the free anion concentrations
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(Function 1, Figure 1) as monitored using solution conductivity. This behavior was part of our
original design.48, 52-57, 89 With the larger anions, however, we activate a new chiroptical function
in which the quaternary structure can be switched between chiral double helices and racemic
single helices (Function 2, Figure 1) as seen by CD spectroscopy. This novel demonstration
ultimately relies upon the use of an intrinsically functional photofoldamer in conjunction with
the regulation of structure following the binding of allosteric effectors.
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Scheme 1. Synthesis of foldamer F1 via the arm-body approach.a,b,c
b
aTetra-n-butylammonium fluoride, THF, acetic acid, 0 °C. CuI, 1,8-diazabicyclo[5.4.0]undec-7-ene, toluene,
60 °C. cTrimethylsilylacetylene, CuI, Pd(PPh3)2Cl2, triethylamine, THF, 60 °C.
RESULTS AND DISCUSSION
Molecular Design. The primary sequence of aryl and triazole residues in the C2-symmetric
photofoldamer F1 (Scheme 1) is derived from prior designs.23, 32 The current design is a
configurational isomer in which the orientation of the triazoles is inverted. The east-west
phenylenes are C-linked to the two neighboring triazoles rather than being N-linked, which
enabled synthetic scalability. Azobenzene moieties provide an external means to control folding
preferences using light.23, 32 With both azobenzenes in their thermodynamically preferred trans
(t) conformations in isomer F1tt, three π-π contacts (Figure 1) enhance the stability of the helical
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conformation.23 Once folded into a helix, the binding pocket is pre-organized for anion binding.
Photoisomerization to the cis states lowers the π contact area to lower the helix-coil transition
and thus reduce the foldamer’s pre-organization and anion affinity. Finally, the chiral group on
the central phenylene imparts handedness to the helix allowing CD spectroscopy to diagnose the
extent of folding.
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Foldamer Synthesis. Foldamer F1 was synthesized in a straightforward manner using a
body-arm approach (Scheme 1 and §S2). The Body was synthesized by installing the azide by
the diazonium displacement of the amines on 3,5-diaminobenzoic acid with sodium azide,90-91
followed by amide coupling with a chiral leucine derivative.23 The Arm was synthesized by
copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)92-93 of 3-azidoazobenzene 2 with tri-
substituted phenylene94
1
followed by thermal Sonogashira95-96 reaction with
trimethylsilylacetylene. Subsequent deprotection of the Arm and CuAAC with the Body in a 2:1
ratio afforded foldamer F1 as a combination of isomers. Isolation of the trans-trans isomer (F1tt)
from cis-trans and cis-cis was accomplished by thermal isomerization of the azobenzenes in the
dark. Compound identity was verified using 1D and 2D NMR spectroscopy (§S3), and high-
resolution electrospray ionization mass spectrometry (HR-ESI-MS).
Photoisomerization of the azobenzenes from trans to cis using UV light (Figure 1)
changes the folding propensity. The isomerization is not perfect with 20:80 ratios being typical.97
Consequently, a total of three isomers are produced23, 32 in the UV-based photostationary state
(PSS) of the foldamer F1UV-PSS: trans-trans (tt), cis-trans (ct), and cis-cis (cc). Each cis isomer
results in removal of one of the three π-π contacts, shifting stability to the random coil23 to lower
the anion affinity of F1UV-PSS
.
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Pre-organization of Foldamer F1. The helical folding of F1tt was examined as a
function of solvent quality98 using CD spectroscopy (Figure 2a) to identify the ideal solvent to
drive folding. The magnitude of the CD signal relates to both (Figure 2b) folding of the random
coil into a helix and the preference of one twist sense over the other. Solvent quality was varied
from least to most polar as constrained by compound solubility. The ET(30) scale99 of polarity is
generally applicable to solvophobic processes involving  systems.100 We see (Figure 2a) that in
solvents of modest polarity, such as THF, CHCl3, and CH2Cl2 (ET(30) = 37 – 45), there was no
CD response. Starting from CH2Cl2, increases in the amount of MeCN from 20% to 100%
(ET(30) = 42 – 46) generated a continual increase in the CD signal (Figure 2a). This trend is
consistent with solvophobic driving forces stabilizing the helix.61, 101-102 Use of protic solvent
mixtures by introduction of water, however, led to a decrease in the CD response likely arising
from equalization between P and M helical preferences rather than from unfolding. To
distinguish between these two options we measured the melting temperature and found an
increase from Tm = 32 ± 2°C (Fig. S10) in pure MeCN to Tm = 40 ± 2°C (Fig. S11) upon the
introduction of 10% H2O. The higher melting temperature is consistent with water producing
greater P-M equalization. Pure acetonitrile was chosen for subsequent studies to avoid the
ambiguities of mixed solvents while maximizing the helical excess of the foldamer.
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We also verified that the photoisomerized form of the foldamer, F1UV-PSS, is less able to
support a helix than F1tt. The CD response of F1UV-PSS (Fig. S83) was reduced even in pure
acetonitrile. The residual peak at 270 nm of modest intensity was attributed to residual trans-
trans form (F1tt , 16%) in the photostationary state. This CD response is consistent with a loss in
the stability of the helix for the cis-cis and cis-trans isomers.
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(a)
(b)
random coil
folding
0
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-13
42
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50
M helix
P helix
ET(30)
(d)
(c)
Equivalents
TBACl
5
1
100
80
60
40
20
0
1:1
0

F1tt
-13
0
270
330
0
1
5
Wavelength (nm)
Equivalents of TBACl
(f)
(e)
100
80
60
40
20
0
0
2:1
1:1
80
40

Equivalents
TBAClO4
20
5
F1tt
0
-13
0 5
20
40
80
270
330
Wavelength (nm)
Equivalents of TBAClO4
Figure 2. (a) Molar ellipticity (θ, mdeg) values at 270 nm of foldamer F1tt (189 μM) in various solvent
mixtures versus solvent polarity (ET(30), kcal / mol). ET(30) values of mixed solvents were determined with
mole fractions. (b) Equilibrium between unfolded coil conformation and the chiral folded helices of F1tt. (c)
CD titration of the F1tt with TBACl (F1tt, 150 μM, CH3CN, 0.05 cm, 298 K) and (d) the corresponding
speciation curve (log K1:1 = 4.1). Analogous (e) CD titration and (f) speciation model with TBAClO4 (log K1:1
= 2.6, log β2 = 7.3).
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Structural Allostery: Selection of Single and Double Helices. The selection of single or
double helices using anionic guests of different sizes was identified from 1H NMR (§S6) and CD
(§S5) titrations. Smaller anions (<45 Å3) favor single helices where binding of chloride is
1
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exemplary. H NMR studies were conducted in dichloromethane (CD2Cl2) to resolve peaks and
replicated in 20% acetonitrile in dichloromethane to enhance π stacking. All the titrations that
were conducted in either dichloromethane or with 20% acetonitrile were used for structural and
speciation determinations only. The foldamer shows continual peak shifts (Figure 4b) upon
addition of chloride as the tetrabutylammonium (TBA+) salt (Fig. S24), which is consistent with
a 1:1 stoichiometry (Figure 3, yellow box). The internal protons (HA, HB, HC, HD) showed only
downfield movements consistent with anion binding.103-105 Protons on the azobenzenes showed
upfield movements consistent with π stacking106 arising from anion-induced folding into a
helix.107
K1:1
X–
+
Anions < 45 Å3
K2:1
+
β2
X–
2
+
Anions > 45 Å3
Figure 3. Binding equilibria involving small anions (yellow box) and large anions (red box).
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Larger anions were discovered to produce double helices (Figure 3, red box). Perchlorate
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is exemplary. Even though π-stacking is less favored in dichloromethane, H NMR signals for
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the double helices can still be seen (Fig. S42). Specifically, we see inflection points characteristic
of 2:1 stoichiometries in protons HC, HD, and HF, at 1, 3, and 1.5 equivalents (Fig. S43).
Consistently, the double helix is dominant in the ESI-MS (Figure 4g, CH2Cl2, 0.1 mM) with 0.5
equivalents of TBAClO4. This finding contrasts with Cl– (Fig. S53a) where the 1:1 dominates. In
this case, and based on our prior experience,108-109 the ESI-MS only provides a qualitative
assessment of the speciation preferences present in solution.
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While large anions were studied previously with aryl-triazole foldamers,30-31 it seems that
the solvents used inhibited formation of double helices. Jiang31 used a 10- and 20-residue
–
–
foldamer with bisulfate (HSO4 ) and phosphate (H2PO4 ), which should be large enough to
generate a double helix. However, only a single helix was observed. Their studies were
conducted in a 10:90 DMSO:THF mixture (ET(30) ~ 38). The solution polarity falls into the
regime (Figure 2a) where folding is disfavored. Craig30 used an aryl-triazole 9-mer with a range
–
of anions, including the large PF6 anion. In acetone (ET(30) = 42), they only observed a single
helix. Based on our observations here and the literature precedent,30-31 anion size appears to
cooperate with the solvophobic burial of π surfaces to drive double helix formation.
1
To approach conditions that favor folding (pure acetonitrile), but still retain sharp H
NMR peaks, a mixture of 20% acetonitrile in dichloromethane (ET(30) ~ 42) was used. Double
helix formation was enhanced (Figure 4e) over pure CD2Cl2. We expect and see that protons HA,
HE and HF are shifted upfield (Figure 4d) on account of being placed into a -stacked
environment (Figure 4e). By contrast, these same protons are not involved in π stacking in the
single helix (Figure 4f) and do not show inflection points with chloride (Fig. S27). With
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perchlorate, these protons show inflection points at 1.5, 1.75, and 6 equivalents (Figure 4c),
respectively. After this point they shift downfield indicative of less π stacking in the single helix
and eventual formation of the 1:1 species with excess anion. Of these, protons HA, HB, and HE
had not originally shown inflection points in the titration conducted in the less polar CD2Cl2.
From the number of peaks in the 1H NMR spectra (Figure 4c) and symmetry considerations, the
2:1 species is an intertwined double helix (Figure 4e) rather than a stacked pair of helices.
Anions that favor single helices can be identified from CD spectroscopy. Addition of one
equivalent of chloride led to the loss in the CD response of F1tt (150 μM, CH3CN, Figure 2c), as
observed32 with related aryl-triazole foldamers. Presumably, stronger CH hydrogen bonding to
the chloride ion disrupts the weak interactions that direct the chiral preferences of the
uncomplexed helix. This outcome contrasts with helicity inversions seen by Jeong when binding
anions to the single helix form of an indolocarbazole foldamer.80, 110 Overall, single-helix 1:1
complexes lose their CD response upon addition of 1 equivalent of anion.111
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In contrast to chloride, perchlorate shows almost no loss in CD signal intensity up to 5
equivalents (150 μM, CH3CN, Figure 2e). This observation is consistent with the production of
double helix (~60%) seen from the calculated speciation profile (Figure 2f). Presumably, chiral
preferences are retained in the double helix as a result of intertwining.
CD titrations (§S5) were undertaken to differentiate between anions that promote single
–
–
and double helices. The halides (Cl–, Br–, I–), nitrite (NO2 ) and nitrate (NO3 ) all showed a
drastic loss in the CD signal indicative of 1:1 single helices. Thiocyanate (SCN–) showed a
decrease in intensity (Fig. S18) but not to the extent of the halides, which is consistent with
–
–
–
–
–
intermediate behavior. The larger anions (BF4 , ClO4 , ReO4 , PF6 , SbF6 ) retained the CD
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signal indicative of double helix formation. These results reveal size dependent outcomes with
anions having a volume of 45 Å3 or larger forming double helices.
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Figure 4. (a) Structure of foldamer F1tt. (b) Normalized chemical shifts of HA and HJ with TBACl additions
1
(F1tt, 1 mM, CD2Cl2, 298 K, 600 MHz). (c) H NMR titration of TBAClO4 with F1tt (1 mM) and the (d)
normalized change in chemical shifts (HA, HB, HE, HF) with TBAClO4 (20:80 CD3CN:CD2Cl2, 298 K, 600
–
–
MHz). (e) DFT-optimized geometry of 2:1 double helix around ClO4 with HA, HB, HE, and HF labeled (ClO4
–
omitted for clarity) and (f) DFT-optimized geometry of the single helix around ClO4 ;location of protons HA,
–
HB, HE, and HF labeled (ClO4 omitted; ONIOM (B97D3/6-311+G(d):B97D3/6-31G(d)). (g) HR-ESI-MS of
F1tt (0.1 mM) with 0.5 equivalents of TBAClO4 (CH2Cl2).
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Quantification of Anion Binding Affinities and Helix Stabilities. The anion affinities
(Table 1) were measured in acetonitrile as a means to understand the recognition properties of
the foldamer in its most pre-organized starting state. On account of broad NMR peaks in the
titrations with acetonitrile (Figs. S28, S46), titrations were followed by UV-Vis spectroscopy
(§S8). The stability constants were quantified using an equilibrium-restricted factor analysis as
implemented in Sivvu.112 Ion pairing was ignored on account of being negligible under the
conditions examined (polar solvent and low concentration; see §S15). Binding equilibria
involved formation of either single (K1:1) or double helices (K1:1, K2:1, 2) as indicated by the
anion-induced CD responses and titrations. Consistently, inflection points in the UV-Vis
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titrations emerged (Fig. S76) for the anions predicted to form double helices. With SbF6 , an
–
–
–
inflection point was seen at 0.5 equivalents while BF4 , ClO4 , and PF6 all had inflection points
at 1.0 equivalents suggesting different levels of cooperativity. While UV absorbances of SCN–
–
1
and ReO4 anions precluded observation of inflections, they were seen in H NMR titrations
–
–
–
–
–
recorded in CD2Cl2. Therefore, SCN–, BF4 , ClO4 , ReO4 , PF6 and SbF6 were fit to double
–
–
helix binding models (Figure 3) while Cl–, Br–, I–, NO2 and NO3 were restricted to 1:1 binding
as a single helix.
Selectivity and Anion Affinity of F1tt . We observed trends in the equilibrium constants
associated with formation of single and double helices (Figure 5). Across the series of small
anions that form single helices, there is poor anion binding selectivity. While there is no size
–
selectivity across the halides, they are the preferred anions over the nitrogen oxides (NO2 ,
−
NO3 ), suggesting a modest shape-based selectivity.
Across the larger anions that form double helices, we see a number of trends. The
stabilities of the 1:1 single helices (Table 1, Figure 5) are seen to drop with increasing size of the
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–
–
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anions. This is true across the tetrahedral ions BF4 , ClO4 , and ReO4 (53 – 60 Å3) and for the
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–
–
two octahedral anions PF6 and SbF6 (73 and 83 Å3). Thus, across the entire series of anions
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9
–
(Cl– to SbF6 ), we see a general trend of decreasing affinity for the single helix. This behavior
matches Craig’s observations30 with an aryl-triazole 9-mer. In order to rationalize this loss in
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113-123
affinity, we first consider from our work on the cyanostar108-109,
and tricarb124-125
macrocycles that high-affinity binding of large anions is possible on the occasion when there is
complementary size matching between the anion and the cavity. We believe this size matching
can occur here given that the foldamer can expand its cavity to complement anion size; we verify
this expansion using density functional theory (DFT, vide infra). Consequently, we attribute the
general loss in affinity with anion size to a reduction in the size of the stabilizing  contact area
of the overlapping ends of the foldamer.
Table 1. Stability (K1:1, β2) and equilibrium (K2:1) constants for anions binding to F1tt (MeCN)a
ΔGK1:1
(kJ /mol)
–24 ± 1
–24 ± 1
–21 ± 2
–25 ± 1
–18 ± 2
–18 ± 3
–21 ± 1
–15 ± 1
–12 ± 1
–16 ± 1
–9 ± 2
ΔGK2:1
ΔGβ2
Cooperativity
Volumeb
(A3)
Anion
log K1:1
log K2:1
log β2
(kJ / mol)
(kJ / mol)
α = 4 K2:1 / K1:1
Cl–
Br–
20
29
4.2 ± 0.1
4.2 ± 0.2
3.6 ± 0.4
4.3 ± 0.2
3.2 ± 0.2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
–
NO2
33
-
-
I –
39
-
-
-
–
NO3
41 ± 1
49
-
-
-
SCN–
3.2 ± 0.6 4.5 ± 1.1 7.7 ± 1.7
3.7 ± 0.2 5.3 ± 0.6 9.0 ± 0.8
2.6 ± 0.1 4.7 ± 0.2 7.3 ± 0.1
2.1 ± 0.1 4.0 ± 1.0 6.1 ± 1.0
2.8 ± 0.2 3.9 ± 0.2 6.7 ± 0.2
1.6 ± 0.3 5.0 ± 0.2 6.6 ± 0.3
–26 ± 6
–30 ± 3
–27 ±1
–23 ± 6
–22 ± 1
–29 ± 1
–44 ± 10
–51 ± 5
–42 ± 1
–35 ± 6
–38 ± 1
–38 ± 2
80
–
BF4
53 ± 2
55 ± 1
60 ± 2
73 ± 2
83 ± 1
160
500
320
50
–
ClO4
–
ReO4
–
PF6
–
SbF6
10,000
aBinding constants determined from UV-Vis titrations. Errors represent the range of variation observed during
replicate titrations. bVolume method determination can be found in the Supporting Information §S15.
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The overall stability of the 2:1 double helical complexes (β2, Table 1) is highest with
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8
–
–
tetrafluoroborate (BF4 ; log β2 = 9.0 ± 0.8). Given that BF4 is one of the smallest anions to form
a double helix, it has one of the largest equilibrium constants for formation of the single helix
(log K1:1 = 3.7 ± 0.2). At the same time, the stepwise equilibrium describing the intertwining of a
free foldamer with the 1:1 complex (K2:1, Table 1) is seen to be almost invariant across the series
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–
of anions. As a consequence, the largest double helix stability seen with BF4 results from the
product of high 1:1 stability and the nearly constant equilibrium associated with foldamer
intertwining (K2:1). Across the rest of the anions we see a drop of almost three orders of
magnitude in stability (2), which largely follows K1:1.
log K
10
9
SCN–
–
BF4
8
–
β2
ClO4
–
–
SbF6
PF6
–
7
ReO4
6
5
Cl–
Br–
I–
K1:1
4
3
–
NO2
–
NO3
2
1
V
(Å3)
20
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40
50
60
70
80
Figure 5. Plot of equilibrium constants, log K1:1 (blue), and log β2 (pink), between various anions towards
foldamer F1tt in acetonitrile against increasing volume size. Anions titrated as TBA+ salts. Binding constants
determined from titrations conducted at foldamer concentrations ranging from 2–300 µM. Binding affinity
errors represent the range of variation observed during replicate titrations. Volume errors represent errors in
anion volume determination (Table S17).
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All the double helical complexes show high positive cooperativity ( >> 1.0), ranging 50
to 10,000. Thus, the binding of the first foldamer to the anion provides a significant enhancement
for the binding and intertwining of the second foldamer. This effect arises from the fact that, with
K2:1 remaining fairly constant, the decrease in K1:1 with increasing anion size leads to greater
cooperativity and, consequently, the dominance of double helices with larger anions. This
outcome was rationalized structurally with the aid of DFT-optimized geometries.
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(b)
(a)
(d)
(c)
31°
36°
Figure 6. DFT-optimized geometries using ONIOM (B97D3/6-311+G(d):B97D3/6-31G(d)): Top view of the
single helix formed around (a) chloride and (b) perchlorate. Side view of calculated double helices around (c)
chloride (experimentally unobserved) and (d) perchlorate.
Three Ways that Larger Anions Stabilize Double Helices. To help rationalize the size-
dependent preferences in double helix formation (Figure 5), we used DFT to calculate the
optimized geometries (Figure 6) of the single and double helices formed around chloride and
–
perchlorate. As the anion size increases from Cl– (20 Å3) to ClO4 (55 Å3), we see the cavity for
the single helix expands from 5.4 to 6.6 Å (HD•••HD distance between OTg-substituted
phenylenes). To accommodate the expansion, the overlapping ends of the foldamer have
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undergone a sliding motion of the π surfaces with the number of residues per turn increasing
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–
from seven (Cl–) to eight (ClO4 ). Thus, the size of the π contact area defined by the overlapping
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arms is reduced. This difference is expected to weaken the stability of the 1:1 helix with
perchlorate relative to chloride, correlating with the observed general trend with anion size. The
impact of π overlap area on affinity matches our design used here, and elsewhere,23, 32 to control
the number of π contacts and thus anion affinity with azobenzene photoisomerization.
We analyzed the underlying factors contributing to the differences between the single and
double helix formation. First, the number of short hydrogen bonds (Table S2) correlates with
helix preferences. With chloride, the double helix shows lengthening of all CH•••Cl– hydrogen
bonds; from having six that are less than 2.8 Å in the single helix to all of them being longer than
2.8 Å. With perchlorate, the opposite trend is observed. The number of short hydrogen bonds
increases from six to eight on going to the double helix. This structural analysis suggests that
enhanced hydrogen bonding favor the single helix with chloride and the double helix with
perchlorate.
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Second, torsion angles also contribute to helix selection. The double helix around
chloride (Figure 6c), which is not observed experimentally, is calculated to have a larger tilt
angle (36°) than perchlorate (31°, Figure 6d), which corresponds to increased aryl-triazole
torsion angles (Table S3). Larger torsion angles come at an energetic cost.126 The double helix
requires an average 2° change in each torsion angle for perchlorate, whereas chloride requires a
larger 5° change (Table S3). The smaller geometrical distortions help favor formation of double
helices64 with larger anions.
Third, the amount of π stacking surface area also enhances stabilization of the double
helix. Huc has shown64 that larger π stacking areas assist stabilization of double helices. Solvent
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quality also enhances this stability.4 In the single helix, the π stacking surface area (Table S5,
Figure 6) is larger around chloride (202 Å2) than perchlorate (169 Å2). For the double helices,
there is an increase in the π stacking surface area relative to two single helices of 1.8 and 2.2 for
chloride and perchlorate, respectively (Table S5). Consequently, perchlorate binding in the
double helix results in the burial of ~15% more π surface area than for chloride.
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In sum, the calculated structures support our observations. The double helix formed
around perchlorate shows an increase in the number of short H-bonds and a smaller energetic
cost to accommodate the torsion angles relative to the single helix. We also see a larger increase
in the π contact area for perchlorate relative to the areal change seen with chloride, consistent
with their different helical preferences. In contrast, the formation of the double helix around Cl–
results in a decrease in the number of short H-bonds and larger energetic penalties with larger
torsion angles relative to the single helix. These structural differences are consistent with
perchlorate favoring the double helix and chloride favoring the single helix.
We also calculated the relative stabilities of the single and double helices in the gas phase
(Table S6). We approximated the starting state of the foldamer as a prefolded helix despite the
likely existence of multiple accessible conformational states.127 We also omitted solvation
effects. Both the conformations and the impact of solvent on conformational distributions are
complex topics of ongoing investigation and outside the scope of our current analysis. Within
these limitations, we calculated (§S9) that formation of the double helix with chloride and
perchlorate have negative and positive cooperativity, respectively, consistent with experiment.
Selectivity and Anion Affinity of F1UV-PSS . After evaluating the impact of anion size on
the binding affinity and structure of the trans-trans state of the foldamer, we characterize the
allosteric control over function of the photofoldamer. The light-switched UV-PSS contains a
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mixture of the trans-trans, cis-trans, and cis-cis isomers in the ratio 16:29:55, respectively (Fig.
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S84). We did not observe double helix formation for any of the three isomers in a H NMR
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titration with TBACl (Fig. S85) consistent with the behavior of the pure trans-trans state.
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In order to observe formation of the double helix in the UV photostationary state, a H
NMR titration was conducted with TBAClO4 (1 mM, 20:80 CD3CN:CD2Cl2). As a means to
track all three isomers simultaneously, we conducted the titration on a solution composed of a
1:1:1 ratio of each isomer (Fig. S87). No inflection points were observed (Fig. S88) for the cis-
cis isomer, indicative of it only forming a single helix. As anticipated from earlier titrations
(Figures 4b and 4c), the trans-trans isomer showed double helix formation with an inflection
point seen at 20 equivalents with proton HH but not for any of the other protons. Hence, double
helix formation appears to be hindered within the isomeric mixture. The cis-trans isomer can
also participate in a double helix with protons HH and HI having inflection points at 10 and 4
equivalents, respectively. In the absence of inflection points for any other internal protons (HA,
HB, HC, HD) of the cis-trans isomer, the formation of a double helix is assumed to be of low
abundance. Additionally, it is unknown if the double helices are homo- or heterodimers (tt:tt,
tt:ct, ct:ct). As a result of weak double helix stabilities, along with the cis-cis isomer constituting
55% of the isomeric solution, the binding affinities in the UV-PSS determined by analysis of the
UV-Vis titrations (§S12) in acetonitrile were approximated by an averaged 1:1 binding model.
Our analysis of the binding data show that the unorganized state of the foldamer, F1UV-
PSS, shows no clear anion selectivity (Figure 7). This is surprising given the wide range in anion
sizes. However, it is consistent with the low anion selectivity previously observed by Jiang31
when binding anions spanning 20 to 63 Å3 in volume to a single helix. Presumably, the cavity of
the poorly organized foldamer, F1UV-PSS, expands to accommodate larger anions instead of
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recruiting a second foldamer to stabilize such anions. The steric bulk of the cis isomers likely
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also inhibit formation of the double helix.
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log KUV-PSS
5
I–
NO3
–
–
SbF6
BF4
Br–
4
3
2
1
–
ReO4
–
–
Cl–
PF6
–
ClO4
–
NO2
SCN–
V / Å3
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Figure 7. Binding constants (log K) of various anions towards foldamer F1UV-PSS in acetonitrile plotted against
increasing volume size. Anions titrated as the TBA+ salts. Binding constants determined from titrations
ranging from 5-100 µM. Binding affinity errors represent the range of variation observed during replicate
titrations. Volume errors represent errors in anion volume determination (Table S17).
Allosteric Control of the Function of Photofoldamers using Anions of Different Sizes.
With the discovery that small and large anions drive formation of different structures of the
photofoldamers, we then considered their different functions. Small anions lead to photodriven
binding and release of anions, while large anions enable photoswitching of the quaternary
structure by interconverting between chiral double helices and racemic single helices. To
highlight how the two functions are different from each other, we selected the working
conditions in our experiments. Our selection of different working conditions is no different from
the natural changes in the state of a cell, such as, between anabolic and catabolic states after
eating and during fasting, respectively. These changes lead to variations in the local
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concentrations of effectors to produce different allosteric effects, which we mimic with the
foldamer by altering local concentrations of different anions.
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The small anions form a single helix that can bind and release anions upon
photoirradiation,23 which is evaluated here across a broader range of anions for the first time (Cl–
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–
–
, Br–, I–, NO2 , and NO3 ). For these anions, the affinity swing is defined as the ratio of the two
binding constants (K1:1 / KUV-PSS). The largest affinity swing of 8-fold is seen with bromide (29
Å3) and the affinity swings decrease from there: NO2 6 (33 Å3), Cl– 5 (20 Å3), and I– 3 (39
–
Å3). Surprisingly, NO3 was found to have an inverse affinity swing of ½ such that the UV-PSS
–
binds the nitrate anion stronger than the trans-trans conformation. This outcome likely results
from poor shape matching between the trans-trans conformation and the trigonal planar nitrate
leading to weaker binding than to the cis-dominated UV-PSS.
As a read-out of the binding and release of the anions,23 we measured the conductivity of
a salt solution composed of the foldamer and one equivalent of TBACl (Table S12). The
conductivity depends on the diffusion coefficient and concentration of all ions in solution. The
difference in Cl– binding strengths between F1tt (log K = 4.2 ± 0.1) and F1UV-PSS (log K = 3.5 ±
0.1) changes the concentration of free Cl– ions in solution (Fig. S100) and, thus, solution
conductivity. A 1 mM solution of F1 displays the largest affinity swing in the presence of 1
equivalent of TBACl (Figure 8a) and was therefore chosen as the working condition best suited
to display the function. The chloride can be reversibly bound and released (Figure 8b) as
observed from the cycles in conductivity (Figure 8c) using 365 and 430 nm light to break and
remake the binding pocket, respectively, upon light-driven isomerization of the azobenzene.
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Cycle
Figure 8. (a) Change in the concentration of free chloride (Δ[Cl–]free) between the binding (F1tt) and release
(F1UVPSS) states of F1 (Δ[Cl–]free = tt[Cl–]free – UVPSS[Cl–]free), (b) mechanism of photoswitching, and (c)
change in conductivity of a solution of F1 (1 mM, MeCN, 293-295 K) with 1 equivalent of TBACl as
controlled using light. The dashed line corresponds to the conductivity of TBACl (1 mM) sans receptor.
The second function involves reversible chiroptical switching of the quaternary structure
between double and single helices, where we use light as a non-destructive and non-invasive
stimulus for the first time and read out the change in optical activity. Other stimuli, like acid-
base84 and the activator-inhibitor85 approach, have been used but they accumulate by-products,
thus reducing the life cycle of the systems. We have shown earlier that the double helix is
–
favored with BF4 (Figure 5) while the single helix is favored in the UV-PSS (Figure 7). There is
also the potential for the double to single helix conversion to produce changes in the
concentration of free anions in solution. However, under the working conditions we used, the
change in anion concentrations is shown from the binding constants (Fig. S102) to be minimal.
Instead, this condition produces the largest change in quaternary structure and corresponding
optical activity.
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Figure 9. Change in CD response controlled using photoisomerization (F1, 0.5 mM, 0.05 cm cuvette, 2
equivalents TBABF4, MeCN, 298 K).
The photoswitching between chiral double helices and racemic single helices was
observed using CD spectroscopy (Fig. S101). Upon the addition of 2 equivalents of TBABF4, a
solution dominated by the double helix is produced with a corresponding large CD response
(Fig. S101). Isomerization with 365 nm light, converts trans isomers to cis to produce a solution
dominated by CD-silent single helices resulting in a decrease in the CD response (Figure 9).
Residual CD response correlates with residual double helices. Replicate experiments with 0.5
and 1 equivalents reproduced the results (Figs. S103, S104).
The double helix can then be reformed from the single helix by isomerization with 430
nm light thus giving rise to a cycle. The double helix to single helix conversion was cycled
(Figure 9) several times to verify the reversibility promised by the use of light as a non-
destructive and non-invasive stimulus. While the photo-driven swing in cis and trans ratios and
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the binding affinities were not large enough to ensure high fidelity transformations, this is the
first example of reversible and repetitive photo switching between double and single helices.
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CONCLUSION
We have discovered anions as allosteric regulators whose different sizes control the
quaternary structure and corresponding function of anion-binding photofoldamers. With small
anions that form single helices, UV and visible light stimulation is used to control the anion’s
concentration in solution. Larger anions enable light-driven chiroptical switching between
hierarchical structures composed of chiral double helices and racemic single helices. This
allosteric behavior was built on our discovery that the volumetric size of the guest controls
formation of single and double helices. The emergence of guest size as a control parameter
appears to rely on the synergistic combination with a poor solvent for driving burial of π
surfaces, i.e., the solvophobic effect. With anions of larger size (> 45 Å3) a second foldamer is
recruited instead of further expansion of the size of the single helix’s cavity. Larger anions favor
the double helices over single helices on account of being able to form a greater number of
shorter hydrogen bonds, more favorable torsion angles, and burial of a larger  surface. Overall,
we describe a bioinspired system that displays allosteric regulation of both the structure and
function of an abiological photofoldamer. We believe these findings provide general design
principles facilitating the use of guest binding to allosterically regulate the function of a stimuli-
responsive foldamer.
ASSOCIATED CONTENT
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Experimental procedures, compound characterization and spectra, variable solvent CD, 1H NMR
titrations, UV-Vis titrations, CD titrations, mass spectrometry, isomerization experiments,
computational calculations, ion-pairing contribution, and the determination of anion volumes
supplied as Supporting Information (PDF).
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AUTHOR INFORMATION
Corresponding Author
*aflood@indiana.edu
Present Address
†Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-
Champaign, 405 North Mathews Avenue, Urbana, IL 61801, USA.
ACKNOWLEDGMENTS
This work was supported by the Chemical Sciences, Geosciences, and Biosciences Division of
the Basic Energy Sciences Program of the US Department of Energy Office of Science (DE-
FG02-09ER16068). The authors thank Xinfeng Gao from the Indiana University NMR facility,
Jonathan A. Karty from the Indiana University Mass Spectrometry Facility, and John Poehlman
from the Indiana University Electronics Instrument Services for support. We also thank
Katherine VanDenburgh for helpful discussions.
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