Alkaline earth metal ion induced coil-helix-coil transition of lysine-coumarin-azacrown hybrid foldamers with OFF-OFF-ON fluorescence switching

Article abstract of DOI:10.1002/chem.201202998

A new metal-ion-responsive and fluorescent foldamer, OPLM₈, composed of eight lysine-coumarin-azacrown units, has been designed and synthesized. The flexible OPLM₈ can be forced into a well-defined helix structure only upon the addition of alkaline earth metal ions. The structural change is based on the crown ether moieties being positioned in the requisite arrangement along the peptide chain, that is, at i, i+4 spacing, such that the alkaline earth metal ions can mediate the formation of four sandwich complexes between them. Moreover, varying the chelator-to-metal-ion ratio from 2:1 to 1:1 resulted in disassembly of the sandwich complexes leading to collapse of the helical structure to a random coil. These metal-ion-induced structural transitions could not only be monitored by the CD amplitude change but also easily probed by unique OFF-OFF-ON fluorescence intensity changes from 0.7-fold to 14-fold as the structure changed from the folded helix to a random coil. To further verify that the helix formation was indeed induced by metal-ion complexation, two kinds of control octamers with only four metal-ion chelators on the side chains were studied. One, which was capable of forming two sandwich complexes between the i and i+4 residues, displayed a negative Cotton couplet with the magnitude of its A value close to half that of OPLM ₈, and the second had four metal-ion chelators positioned in the same turn, and hence was incapable of forming intramolecular metal complexes and showed different induced CD signals. Collectively, the photospectroscopic data and the results of the control studies suggest that alkaline earth metal ions can efficiently promote the flexible octamer OPLM₈ into a well-organized helix by the formation of sandwich complexes between substituents at an i, i+4 spacing. Switching off the helix: An alkaline earth metal ion responsive and fluorescence-detectable foldamer (OPLM₈) has been designed and synthesized. Variation of the chelator-to-metal-ion ratio resulted in the assembly and disassembly of sandwich complexes between fluoroionophores with i, i+4 spacing on the peptide backbone, leading to the formation and collapse of the helical structure, and was probed by OFF-OFF-ON fluorescence switching (see graphic). Copyright

Full text of DOI:10.1002/chem.201202998

FULL PAPER
DOI: 10.1002/chem.201202998
Alkaline Earth Metal Ion Induced Coil–Helix–Coil Transition of Lysine–
Coumarin–Azacrown Hybrid Foldamers with OFF–OFF–ON Fluorescence
Switching
Yu-Chen Lin and Chao-Tsen Chen*[a]
Abstract: A new metal-ion-responsive
and fluorescent foldamer, OPLM8,
composed of eight lysine–coumarin–
azacrown units, has been designed and
synthesized. The flexible OPLM8 can
be forced into a well-defined helix
structure only upon the addition of al-
kaline earth metal ions. The structural
change is based on the crown ether
moieties being positioned in the requi-
site arrangement along the peptide
chain, that is, at i, i+4 spacing, such
that the alkaline earth metal ions can
mediate the formation of four sand-
wich complexes between them. More-
over, varying the chelator-to-metal-ion
ratio from 2:1 to 1:1 resulted in disas-
sembly of the sandwich complexes
leading to collapse of the helical struc-
ture to a random coil. These metal-ion-
induced structural transitions could not
only be monitored by the CD ampli-
tude change but also easily probed by
unique “OFF–OFF–ON” fluorescence
intensity changes from 0.7-fold to 14-
fold as the structure changed from the
folded helix to a random coil. To fur-
ther verify that the helix formation was
indeed induced by metal-ion complexa-
tion, two kinds of control octamers
with only four metal-ion chelators on
the side chains were studied. One,
which was capable of forming two
sandwich complexes between the i and
i+4 residues, displayed negative
a
Cotton couplet with the magnitude of
its A value close to half that of
OPLM8, and the second had four
metal-ion chelators positioned in the
same turn, and hence was incapable of
forming intramolecular metal com-
plexes and showed different induced
CD signals. Collectively, the photospec-
troscopic data and the results of the
control studies suggest that alkaline
earth metal ions can efficiently pro-
mote the flexible octamer OPLM8 into
a well-organized helix by the formation
of sandwich complexes between sub-
stituents at an i, i+4 spacing.
Keywords: fluorescence · foldam-
ers · helical induction · metal ions ·
sandwich complexes
Introduction
Functional groups capable of non-covalent or covalent in-
teractions, such as van der Waals, hydrophobic, hydrogen-
bonding, and electrostatic interactions, and metal-ion-coor-
dination interactions of varying strengths, are often judi-
ciously incorporated into either the backbones or side
chains of these molecules to facilitate the formation of heli-
cal structures. Thus, anion-responsive foldamers,[3] composed
The helix is one of the essential structural elements in
nature. Important biopolymers such as polypeptides and
proteins fold and self-assemble into these unique and hier-
archical structures, which play important roles in biocatalytic
functions, gene transcription, protein aggregation, and dis-
ease treatments.[1] The design of artificial helical structures
that closely mimic native helices can help to elucidate key
features of the structure and function of these helices, and
also allows the introduction of novel biological and chemical
reactivity into the relevant architectures. For example, syn-
thetic helical molecules have displayed great potential for
applications in chiral catalysts, chiral sensors, chiro-optical
switches, and chiral-based information storage.[2] Among
these molecules, polymers that fold into a helical form in re-
sponse to external stimuli are of particular interest.
of aryl strand building blocks with hydrogen-bond donors or
[4]
À
polar C H bonds, such as indolecarbazole or aryl-1,2,3-tri-
azole,[5] were found to fold into stable helical conformations
upon the binding of complementary anions. Metal-ion-in-
duced foldamers have been constructed from heterocycles[6]
such as pyrimidine–pyridine oligomers or a pyridine–oxazole
framework,[7] which provided coordination bonds to form
single- or double-stranded helicates in the presence of suita-
ble metal ions. Peptidomimetic foldamers derived from nat-
ural or unnatural amino acids have also been shown to
adopt stable helical structures through the introduction of
side-chain interactions in the regular spatial arrangement.[8]
In this context, Fujita et al. have demonstrated that aromat-
ic–aromatic interactions between aromatic amino acid resi-
dues and electron-deficient triazine units of a host cage pro-
moted the formation of short oligopeptides adopting a
stable helix.[9] Among them, foldamers that were mediated
by stronger interactions displayed higher stability.
[a] Dr. Y.-C. Lin, Prof. Dr. C.-T. Chen
Department of Chemistry, National Taiwan University
No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 Taiwan (R.O.C.)
Fax : (+886)2-23636359
E-mail: chenct@ntu.edu.tw
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202998.
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The helicity induced in the foldamers is typically probed
by circular dichroism, whereas fluorescence is more suitable
for monitoring the folding–unfolding transition as it pro-
vides highly sensitive detection, environmental sensitivity,
and real-time detection characteristics, thus allowing the de-
tection of minimal conformational changes.[10] However, syn-
thetic foldamers equipped with suitable fluorophores sensi-
tive to conformational changes have seldom been reported
in the literature. Only a few examples utilizing Fçrster reso-
nance energy transfer, fluorescence quenching by electron
transfer, or excimer formation as sensing mechanisms[11]
have hitherto been demonstrated. In our previous study, we
Figure 2. Chemical structure of the synthetic foldamer OPLM8 and a hel-
ical wheel representation of the OPLM8·4M2+ complex, which results
from metal-ion-assisted folding of OPLM8.
developed
a highly selective fluorescent chemosensor
(DEAC, Figure 1) that consisted of a [15]azacrown-5 ether
transitions (folding/unfolding) would be observable through
fluorescence changes. Moreover, the ketocoumarin fluoro-
phores, having favorable visible absorptions, would allow
monitoring of the extent of the helicity by circular dichroism
in the visible spectral region.
A metal-ion-responsive and fluorescence-detectable fold-
AHCTUNGTRENaGNUN mer, OPLM8, was thus designed and synthesized
(Figure 2). OPLM8 comprises of eight DEAC–lysine hybrid
residues. An oligo-l-lysine backbone was chosen because it
can be easily functionalized on the side-chain Ne-amino
group through amide bond linkages. OPLM8 could poten-
tially fold into a helical structure upon complexation with
metal ions capable of promoting the formation of intramo-
lecular sandwich complexes between the DEAC side chains
at the i and i+4 positions. A helical wheel representation of
the OPLM8·4M2+ complex is shown in Figure 2.
Figure 1. Schematic representation of highly ordered suprastructures in-
duced by sandwich complexes between DEAC-DO and metal ions.
(ionophore) and
a
ketoaminocoumarin (fluorophore).[12]
This molecular sensor showed a high affinity for the toxic
metal-ion Pb2+ through the formation of a 2:2 complex in-
volving coordination of the carbonyl groups present in the
fluorophore and ionophore moieties, which was accompa-
nied by the emission of fluorescence. Inspired by these com-
plexation features, we envisaged that an oligomer DEAC-
DO, constructed from a DEAC-derivatized monomer, could
possibly organize itself into the intramolecular helix-folded
DEAC-Helix and/or the intermolecular-ladder-like DEAC-
Ladder forms upon the complexation of appropriate metal
ions (Figure 1). At the same time, these conformational
Results and Discussion
Synthesis of OPLM8: OPLM8 was prepared by a solution-
phase synthesis[13] involving successive amide couplings of
OPLM1, OPLM2 (dimer of OPLM1), and OPLM4 (tetramer
of OPLM1), as shown in Scheme 1. The starting building
Scheme 1. Synthesis of OPLM8. Reagents and conditions: a) DCC, DMAP, HOBt, CH2Cl2, RT, 12–36 h; b) [PdACHTNUGTRNEGNU(PPh3)4] (10 mol%), N-methylaniline,
CH2Cl2, RT, 2–6 h; c) piperidine (20%)/DMF, RT, 10–30 min.
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Metal-Ion-Responsive Helix-Forming Lysine–Coumarin–Azacrown Foldamers
FULL PAPER
block, OPLM1, was first obtained from a DEAC derivative
with an N-methyl-N-glycine moiety at the C-7 position
(AcidAC) and an Fmoc/allyl-protected lysine residue (Ne-
amino-Boc1) by an amide coupling promoted by N,N’-dicy-
clohexyl carbodiimide (DCC) and hydroxybenzotriazole
(HOBt) according to known procedures.[13] Once the
OPLM1 monomer had been prepared, OPLM2, OPLM4,
and OPLM8 were subsequently obtained by iterative syn-
thetic steps involving selective deprotection of the allyl ester
and Fmoc groups (to provide the corresponding carboxyl-
and amino-terminal functional groups) followed by amide
coupling. Selective deprotections of the allyl and Fmoc
groups were achieved in high yields by using a palladium
were studied by UV/Vis and fluorescence spectroscopies
(Figure 3). In contrast to the distinct fluorescence emission
spectra, the dimer, tetramer, and octamers of OPLM1 exhib-
ited very similar UV/Vis absorption profiles, albeit with
slightly red-shifted wavelengths. The fluorescence emission
spectra showed, besides the emission peak corresponding to
the ketocoumarin moiety at 475 nm, a broad peak at around
550 nm that gradually intensified as the number of ketocou-
marin groups was increased. This emission peak is attributed
to the structural rigidity of the oligoACTHNUTRGNEUNG(l-lysine) backbone in
OPLMn, which restricts the segmental mobility as a result of
a certain degree of side chain–side chain interactions be-
tween coumarin moieties.
catalyst (e.g., [Pd
(PPh3)4]) and piperidine/dimethylforma-
Although DEAC, which constitutes the designated metal-
ion chelating site of OPLM, has been demonstrated to have
high specificity for Pb2+ ion,[12] it could display different
metal-ion specificity once incorporated into the oligomer,
because its disposition may no longer be optimal for form-
ing the same Pb2+ complexes as observed in solution. With
the aim of evaluating which metal ions are capable of form-
ing complexes with OPLM8, 17 metal ions (Li+, Na+, K+,
mide (DMF) solution (20%), respectively. The amide cou-
plings were then carried out as described for OPLM1. De-
tailed synthetic procedures and structural characterizations
can be found in the Supporting Information.[14]
Photospectroscopic properties of OPLMn (n=1, 2, 4, 8) in
the absence and presence of metal ions: The photospectro-
scopic features of OPLMn (n=1, 2, 4, 8) in CH2Cl2 solution
Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+
,
Ag+, Zn2+, Cd2+, Hg2+, and Pb2+, with perchlorate as the
counter ions) were investigated by high-throughput fluores-
cence screening in CH2Cl2 (Figure S3 in the Supporting In-
formation). The results indicated that OPLM8 does indeed
display different metal-ion specificities. Upon the addition
of seven of these metal ions, namely Na+, K+, Ag+, Ca2+
,
Sr2+, Ba2+, and Zn2+, OPLM8 exhibited noticeable fluores-
cence changes that were not observed with the other metal
ions.
Although the high-throughput results provided an initial
screening of potential candidates for inducing a conforma-
tional change in OPLM8, more detailed follow-up studies of
the influential metal ions were required to understand the
relationship between optical properties and metal-ion-bind-
ing modes. The absorption and emission spectra of OPLM8
in the presence of 4 and 10 equivalents of each of the influ-
ential metal ions (Na+, K+, Ag+, Ca2+, Sr2+, Ba2+, and
Zn2+) were recorded and the results can be found in the
Supporting Information (Figure S4 and Table S1). Based on
Figure 3. UV/Vis (open symbols) and normalized fluorescence emission
spectra (solid symbols) of OPLMn (n=1, 2, 4, 8; 10À6 m) in CH2Cl2
&
(OPLM1: lex =388 nm; OPLMn: lex =400 nm). OPLM1 ( ), OPLM2
~
*
(
),OPLM4 ( ),OPLM8 (N).
Figure 4. Proposed binding modes of DEAC derivatives to metal ions a) Na+, b) K+, c) Ag+, d) Ca2+, Sr2+, Ba2+, and e) Zn2+
.
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C.-T. Chen and Y.-C. Lin
the observed spectral changes, several complexation modes
could be proposed for the various metal ions (Figure 4). For
example, the addition of Na+ to OPLM8 gave rise to a
sharp emission band at 485 nm with 13-fold enhancement
(Figure S4a in the Supporting Information), as well as a
moderate red-shift in the absorption spectrum by about
20 nm, indicating that the carbonyl from chromen-2-one
moiety and the [15]azacrown-5 cavity of the DEAC deriva-
tive took part in the chelation (Figure 4a).[12] In the case of
K+, there was no appreciable change in the absorption, but
a 1-fold fluorescence enhancement with a shoulder at
550 nm, suggesting that two azacrown moieties of DEAC
sandwiched one K+ ion. The K+ cation is 40% larger than
the Na+ ion and cannot be accommodated well in the aza-
crown cavity; rather, it is known to form a sandwich com-
plex with two [15]azacrown-5 ether moieties (Figure 4b).[15]
This complexation mode drew two coumarin moieties close
together, resulting in fluorescence at 550 nm (Figure S4b in
the Supporting Information). The addition of Ag+ to
OPLM8 yielded relatively small red-shifts in the absorption
(ca. 11 nm) and fluorescence quenching, suggesting that
only the carbonyl groups of the DEAC derivative coordinat-
ed to Ag+ (Figure 4c and Figure S4c in the Supporting In-
formation). In contrast, upon the addition of divalent metal
ions such as Ca2+, Sr2+, and Ba2+ to OPLM8, pronounced
bathochromic shifts in the absorption in the range 30–40 nm
were generally observed. However, the addition of Zn2+
ions gave the largest red-shift (ca. 62 nm), suggesting the es-
tablishment of a strongly stabilized internal charge-transfer
(ICT) state upon complexation of Zn2+ ions with the two
carbonyl groups of the DEAC
ical suprastructure, changes in the circular dichroism signals
of OPLM8 before and after addition of the influential metal
ions were examined (Figure S5 and Table S1 in the Support-
ing Information). The results showed that only the addition
of Ca2+, Sr2+, and Ba2+ resulted in a net induced circular di-
chroism (ICD) response with negative signs of the exciton
Cotton effects, with no such effect being observed with Na+,
Ag+, K+, or Zn2+. The findings indicate that Ca2+, Sr2+
,
and Ba2+, having appropriate sizes, suitable surface charge
densities, and preferred coordination geometries,[17] effec-
tively cross-link two DEAC units via the azacrown and car-
bonyl groups, leading to the formation of a helical supra-
structure, whereas Na+, Ag+, K+, and Zn2+ are not suitable
mediators for the folding of OPLM8 into helical structures.
Titration of OPLM8 with Sr2+ ions to delineate the folding
process: The addition of Sr2+ resulted in the most pro-
nounced change in the circular dichroism spectrum (Fig-
ure S5e in the Supporting Information) among the alkaline
earth metal ions that were investigated, thus titrations of
foldACTHNUGRTNEUNGamer OPLM8, OPLM2, and OPLM4 with various
amounts of Sr2+ were performed (Figure 5 and Figures S6–
S8 in the Supporting Information) to delineate how the fold-
ing transition process of OPLM8 was influenced by an in-
creasing amount of alkaline earth metal ions. The correla-
tion of absorption intensity changes and number of metal-
ion equivalents (absorption isotherm, black squares in the
inset of Figure 5a) showed that saturation was reached upon
the addition of 4 equivalents of Sr2+ ions. The fluorescence
isotherm (gray circles in the inset) showed significant fluo-
derivative (Figure 4e).[12] While
the fluorescence intensity was
almost quenched in the pres-
ence of Zn2+ ions (Figure S4g
in the Supporting Information),
a unique fluorescence intensity
change pattern (OFF–OFF–
ON)[16] between 4 and 10 equiv-
alents of Ca2+, Sr2+, or Ba2+
was observed, indicating that
these cations must bind differ-
ently to Zn2+. The emission
spectra recorded in the pres-
ence of Ca2+, Sr2+, and Ba2+
ions showed
a
peak near
500 nm and a broad shoulder at
around 550–600 nm, strongly
suggesting that the DEAC de-
rivative moieties were aggregat-
ed to a significant extent upon
the addition of 4 equivalents of
these metal ions (Figure 4d and
Figure S4d–f in the Supporting
Information).
Figure 5. a) Overlaid absorption spectra of OPLM8 (2ꢁ10À6 m in CH2Cl2) and b) the corresponding overlaid
CD spectra upon the addition of increasing amounts of Sr2+. Insets show the correlation of the absorbance
To further clarify which
metal ions could induce the hel- 461 nm, with the amount of Sr
~
&
*
wavelength changes ( ) at 438 nm and fluorescence intensity changes ( ), and ICD signal changes ( ) at
(ClO4)2.
ACHTUNGTRENNUNG
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Metal-Ion-Responsive Helix-Forming Lysine–Coumarin–Azacrown Foldamers
FULL PAPER
rescence quenching upon the addition of less than 4 equiva-
lents of Sr2+, while further fluorescence enhancement was
observed at higher amounts of Sr2+. The CD titration spec-
tra[18] exhibited zero CD signal in the absence of Sr2+. As
the amount of Sr2+ was increased up to 4 equivalents (Fig-
ure 5b and inset therein), an intense ICD signal with nega-
tive first and positive second Cotton effects at 461 and
419 nm, respectively, was observed. However, at higher Sr2+
content (more than 4 equivalents), the amplitude of the
ICD signals decreased, indicating structural changes. It is
worth noting that the zero-crossing point of the ICD signal
at 438 nm corresponded well to the absorption spectral
region of the Sr2+·azacrown–coumarin complex.
Figure 6. Graphical illustration of conformational transition of OPLM8 in
the absence and presence of various equivalents of Sr2+ ions.
In the case of OPLM2, saturation was reached upon the
addition of 2 equivalents of Sr2+ ions, with a large fluores-
cence enhancement (Figure S7 in the Supporting Informa-
tion). The fluorescence titration profile appeared very differ-
ent from that of OPLM8 titrated with Sr2+, suggesting a dif-
ferent binding mode. Moreover, no ICD signal was observed
in the presence of various amounts of Sr2+ (Figure S9a in
the Supporting Information), reflecting the fact that the di-
peptide is too short to fold into any form of secondary struc-
ture even with the aid of strong metal-ion coordination.
When the tetramer OPLM4 was titrated with Sr2+, three
phases of fluorescence changes were observed. On the addi-
tion of up to 2 equivalents of Sr2+, only a small fluorescence
intensity increment was observed. However, the fluores-
cence at 500 nm increased rapidly when more Sr2+ was
added, and reached saturation at 4 equivalents of Sr2+. The
subsequent addition of more Sr2+ resulted in a decrease in
the intensity at 500 nm accompanied by the emergence of
an emission band at 550 nm (Figure S8 in the Supporting In-
formation). The corresponding CD titration spectra showed
that the ICD signals were split into negative peaks at 460
and 338 nm and a positive peak at 409 nm in the presence of
0.4–2.4 equivalents of Sr2+ and that further addition of Sr2+
resulted in sharply diminished signals at 338 and 409 nm as
well as a blue shift of the signal at 460 nm to 430 nm (Fig-
ure S9b in the Supporting Information). These observations
indicated that the tetramer was quite flexible and could
assume various secondary structures under the influence of
different amounts of Sr2+. In comparison with OPLM4,
OPLM8 displayed consistent changes in its absorp-
a maximum in the presence of 4 equivalents of metal ions.
No obvious fluorescence changes appeared, a result of the
close spatial proximity of the fluorophores (Helix, fluores-
cence self-quenching state, Figure 6). Upon the addition of
larger amounts of Sr2+ ions, the variation of the chelator-to-
metal ratio from 2:1 to 1:1 resulted in dissolution of the
sandwich complexes, leading to collapse of the helical struc-
ture (Random coil 2, as shown in Figure 6). Accordingly, the
CD intensity gradually decreased with a concomitant fluo-
rescence increase caused by gradual separation of the fluo-
rophores. The fluorescence OFF–OFF–ON changes and the
selectivity of the metal-ion-induced folding/unfolding transi-
tion of OPLM8 corroborated the preliminary results of high-
throughput fluorescence screening, thereby suggesting that
alkaline earth metal ion induced structural transitions could
be easily followed by changes in the fluorescence signals.
Verification of the helix stabilization by the formation of
sandwich complexes between substituents at an i, i+4
AHCTUNGTREGsNNUN pacing: To further verify that the helix formation was
indeed induced by metal-ion complexation between sub-
stituents at an i, i+4 spacing, two kinds of control octamers
were designed and synthesized. Their sequences are shown
in Figure 7. Each control octamer possessed only four
metal-ion chelators on the side chains, whereas the remain-
ing four were replaced with N-Boc protecting groups. Cate-
gory A octamers, comprising of 1,2,5,6- and 3,4,7,8-OPLM8,
could produce two sandwich complexes between the i and
i+4 residues and subsequently fold into a helix with some
tion, fluorescence, and CD spectra when titrated
with Sr2+, suggesting that the structural transitions
were more meaningful, and thus further investiga-
tions were focused on OPLM8.
Proposed metal-ion-induced structural transition of
OPLM8: Based on the titration results, a model for
the Sr2+-ion-induced conformational transition of
OPLM8 was proposed (Figure 6). In the presence of
4 equivalents of side-chain cross-linking agent (e.g.,
Ca2+, Sr2+, or Ba2+), four sandwich complexes
formed between the DEAC moieties and induced
Figure 7. Sequence representations of two categories of control octamers. Category A:
four metal-ion chelators are arranged at an i, i+4 spacing; Category B: four metal-ion
OPLM8 to fold into a left-handed helix along the
oligomer backbone, and the ICD intensity reached chelators are positioned in the same turn.
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2535

C.-T. Chen and Y.-C. Lin
stability. Category B octamers, comprising of 1,2,3,4- and
5,6,7,8-OPLM8, with four metal-ion chelators positioned in
the same turn, were incapable of forming intramolecular
metal complexes at the i, i+4 positions due to their unfavor-
able spatial disposition of the chelators. The two categories
of control octaACHTUNGTRENNUNGmers were obtained from the same starting
materials (Boc2 and OPLM2) by a selective deprotection
process and an amide coupling reaction, constructing differ-
ent sequences (refer to Schemes S3–S5 in the Supporting In-
formation).
The results of absorption, fluorescence emission, and cir-
cular dichroism experiments on these four control octamers
(see Figures 8 and 9) suggested that 1,2,5,6- and 3,4,7,8-
Figure 9. Absorption (top) and circular dichroism (bottom) spectra of
&
OPLM8 ( ), OPLM4 (N), and control octamers of Category B: 1,2,3,4-
(
) and 5,6,7,8-OPLM8 ( ) in CH2Cl2 (2ꢁ10À6 m) in the absence
~
*
OPLM8
(open symbols) and presence (solid symbols) of 2 equivalents Sr2+ (in the
case of OPLM8, 4 equiv Sr2+ were used).
and 334 nm and a positive peak at 418 nm upon the addition
of 2 equivalents of Sr2+ (Figure 9). The changes in the CD
spectra in response to Sr2+ were different from those of
OPLM8, suggesting that 1,2,3,4-OPLM8 and 5,6,7,8-OPLM8
may possibly have formed intrastrand sandwich metal com-
plexes between adjacent chelators because of the flexible
lysine side chains. Based on these control results, it can be
concluded that the helix formation was indeed induced by
the formation of four sandwich complexes between substitu-
ents at an i, i+4 spacing in OPLM8. Notably, OPLM4 and
1,2,3,4-OPLM8 displayed similar absorption changes and
Figure 8. Absorption (top) and circular dichroism (bottom) spectra of
&
*
OPLM8 ( ) and control octamers of Category A: 3,4,7,8-OPLM8 ( ) and
~
1,2,5,6-OPLM8
(
) in CH2Cl2 (2ꢁ10À6 m) in the absence (open symbols)
and presence (solid symbols) of 4 equivalents Sr2+
.
ICD signals in the presence of 0.4–2.4 equivalents of Sr2+
,
but different ICD profiles upon the addition of further Sr2+
(Figure 9 and Figure S9 in the Supporting Information).
These results were not surprising because the complexes
share some common structural features, such as four consec-
utive metal-ion chelators positioned in the same order, al-
though they differ in the peptide length. This observation
confirms that the length of a peptide influences the secon-
dary structure to a certain extent.
OPLM8 exhibited similar behavior to OPLM8 upon titration
with Sr2+ (albeit with smaller A values), whereas 1,2,3,4-
and 5,6,7,8-OPLM8 showed completely different folding
modes to that of OPLM8. The Sr2+-complexed structures of
both 1,2,5,6- and 3,4,7,8-OPLM8 (Figure 8) revealed a nega-
tive Cotton couplet centered around their absorption bands
at 438 nm. The A value of 3,4,7,8-OPLM8 was close to half
of that of OPLM8. This result was expected as the former
contained half the number of metal-ion chelators of the
latter. However, 3,4,7,8-OPLM8 apparently had a better
helix-promoting ability than 1,2,5,6-OPLM8 on the basis of
its higher A value. The difference in the folding ability may
arise from the bulky and long side-chain (metal-ion chela-
tor) on the N-terminal a-carbon of 1,2,5,6-OPLM8, which
did not adopt the adequate geometry to fold as in 3,4,7,8-
OPLM8 (Figure S10 in the Supporting Information).
Kinetics and stability: To assess the kinetics and stability of
the alkaline earth metal ion induced helical form of
OPLM8, a time-course experiment was carried out by fol-
lowing the ICD signals at 426 and 463 nm after the addition
of 4 equivalents of Sr2+ (Figure S11 in the Supporting Infor-
mation). It took only four minutes to reach the plateau of
CD enhancement that corresponds to a stable helix confor-
mation at room temperature.[19] This result clearly indicates
that the helical structure promoted by the strong coordina-
tion interaction between metal-ion chelators at i, i+4
In the case of the 1,2,3,4- and 5,6,7,8-OPLM8 controls of
category B, their ICD signals split into negative peaks at 468
AHCTUNGTREGsNNNU pacing and metal ions is formed rapidly and is quite stable.
2536
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ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 2531 – 2538

Metal-Ion-Responsive Helix-Forming Lysine–Coumarin–Azacrown Foldamers
FULL PAPER
to give OPLM1 as an orange solid (468 mg, 0.50 mmol). Yield 67%; m.p.
107–1098C; Rf =0.39 (CH2Cl2/MeOH, 95:5); H NMR (400 MHz, CDCl3/
Conclusion
1
drops CD3OD): d=7.76 (s, 1H), 7.74–7.70 (m, 4H), 7.57–7.56 (m, 2H),
7.35 (dd, J=8.0, 7.2 Hz, 2H), 7.30–7.25 (m, 3H), 6.72 (t, J=5.2 Hz; NH),
6.59 (d, J=8.8 Hz, 2H), 6.54 (d, J=10.8 Hz, 1H), 6.48 (s, 1H), 5.91–5.81
(m, 1H), 5.60 (d, J=8.0 Hz; NH), 5.28 (d, J=17.2 Hz, 1H), 5.21 (d, J=
10.0 Hz, 1H), 4.59 (d, J=6.0 Hz, 2H), 4.40–4.36 (dd, J=7.6, 7.2 Hz, 1H),
4.33–4.27 (m, 2H), 4.17 (t, J=7.2 Hz, 1H), 3.96 (s, 2H), 3.73 (t, J=
6.0 Hz, 4H), 3.64–3.55 (m, 16H), 3.27–3.22 (m, 2H), 3.09 (s, 3H), 1.70–
1.65 (m, 2H), 1.55–1.48 (m, 2H), 1.36–1.26 ppm (m, 2H); 13C NMR
(100 MHz, CDCl3): d=188.6, 171.3, 168.0, 158.9, 155.9, 155.4, 152.5,
151.1, 144.5, 143.2 (ꢁ2), 140.6, 131.8, 131.0, 129.6, 127.2, 126.6, 124.6 (ꢁ
2), 123.7, 120.5, 119.5, 118.4, 110.2, 109.8, 108.7, 98.0, 71.1, 70.1, 69.8,
68.0, 66.8, 65.8, 57.1, 53.9, 52.8, 47.2, 39.9, 39.0, 31.9, 29.1, 22.8 ppm; IR
(KBr): n˜ =1722, 1677 cmÀ1 (C=O); HRMS (ESI): m/z calcd for
C53H60N4O12: 944.4208; found: 945.4276 (M+H+).
In summary, our studies outline the structural design of a
metal-ion-responsive and fluorescence-detectable foldamer,
OPLM8, that is capable of undergoing folding/unfolding
transitions upon alkaline earth metal-ion complexation.
Even the short l-lysine-based octamer could fold into a
stable helical conformation through the coordination inter-
action between the metal-ion chelators and the alkaline
earth metal ions. The metal-ion-induced suprastructural
changes in the synthetic foldamer could be easily probed by
changes in the fluorescence intensities with concomitant
changes in the CD signals. In addition, we have demonstrat-
ed that the helix formation is indeed induced by the forma-
tion of sandwich complexes between groups at an i, i+4
spacing through systematic variation of the sequences in a
series of control foldamers. The magnitude of the circular
dichroism effect showed a linear correlation with the
number of sandwich complexes formed.
General procedure for selective deprotection of an allyl group using a
Pd0 catalyst:[21] The allyl-protected compound was dissolved in dry DMF
under oxygen-free N2 and then
a catalytic amount of [PdACHTUNGTRENNUNG(PPh3)4]
(10 mol%) and N-methylaniline (0.3 mol%) were added. The mixture
was stirred at ambient temperature for 8 h. After the reaction was com-
plete, the excess DMF was removed and the crude mixture was purified
by centrifugation. The residue was redissolved in a small volume of
CH2Cl2, precipitated with an eight-fold volume of hexane, and then puri-
fied by centrifugation at 5500 rpm for 15 min in a glass tube. The super-
natant was removed. The precipitate was redissolved in CH2Cl2 and the
washing procedure was repeated three times to obtain the pure com-
pound.
Experimental Section
General: All chemicals were purchased from commercial sources and
were used as received. Solvents used for syntheses were dried by stand-
ard literature methods before being distilled and stored under nitrogen
General procedure for selective deprotection of an Fmoc group by using
20% piperidine in DMF: The Fmoc-protected compound was dissolved
in CH2Cl2, and then 20% piperidine in DMF was added. The mixture
was stirred at ambient temperature for about 10–20 min (TLC monitor-
ing). The solvents were removed in vacuo and the crude mixture was pu-
rified by centrifugation. It should be noted that heating during drying of
the crude product causes an unwanted side reaction. The residue was re-
dissolved in a small volume of CH2Cl2, precipitated with an eight-fold
volume of hexane, and then purified by centrifugation at 5500 rpm for
15 min in a glass tube. The supernatant was removed. The precipitate
was redissolved in CH2Cl2 and the washing procedure was repeated three
times to obtain the pure compound.
1
over 4 ꢂ molecular sieves. H (400 MHz) and 13C (100 MHz) NMR spec-
tra were recorded on a Varian Mercury 400 spectrometer. 13C (200 MHz)
NMR spectra were recorded on a Bruker AVIII 800 MHz spectrometer.
Chemical shifts were referenced to selected residual proton peaks of the
deuterated solvents and are reported in dACTHNUTRGNEUNG
(ppm). 1H NMR data are re-
ported in the following order: chemical shift, multiplicity (s=singlet, d=
doublet, t=triplet, q=quartet, m=multiplet, br=broad), coupling con-
stant(s) in Hertz, number of protons. Melting points were determined on
a Fargo MP-1D melting point apparatus without correction. ESI mass
spectra were recorded on a Waters Micromassꢃ LCT Premier XE system.
MALDI-TOF mass spectra were recorded on a Bruker Autoflex III
TOF/TOF instrument using 4-hydroxy-a-cyanocinnamic acid (a-CHC)
and 2,5-dihydroxybenzoic acid (DHB) as matrices.
General procedure for amide coupling: Amides were prepared from the
corresponding carboxylic acids and amines through amide-bond forma-
tion. DCC (2.3 equiv), HOBt (2.3 equiv), and a catalytic amount of
DMAP (0.24 equiv) were added to a solution of the amine (1.0 equiv)
and carboxylic acid (1.02 equiv) in dry CH2Cl2. The products became
more viscous as the number of repeating units increased. The use of a
mixed solvent of CH2Cl2/DMF facilitated the coupling reaction and im-
proved the product yield. After confirming completion of the reaction by
TLC, the solvents were removed under vacuum and the residue was puri-
fied by flash column chromatography on silica gel eluting with different
ratios of CH2Cl2/MeOH to give the desired compounds.
Synthesis of compound AcidAC: A 0.5m aqueous lithium hydroxide solu-
tion (5.6 mL) was added to a solution of EsterAC (1.43 g, 2.52 mmol) in
MeOH (25 mL) and the reaction mixture was stirred for 2 h at reflux.
After confirming completion of the reaction by TLC, the reaction mix-
ture was neutralized with acidic resin, filtered, and concentrated to leave
a crude solid, which was purified by flash column chromatography on
silica gel eluting with CH2Cl2/MeOH/acetic acid (9:1:0.01) to give
AcidAC as an orange solid (1.19 g, 2.14 mmol). Yield 82%; Rf =0.14
(CH2Cl2/MeOH, 9:1); 1H NMR (400 MHz, CDCl3/drops CD3OD): d=
7.73 (s, 1H), 7.52 (d, J=8.8 Hz, 2H), 7.18 (d, J=8.8 Hz, 1H), 6.49–6.45
(m, 3H), 6.31 (s, 1H), 3.99 (s, 2H), 3.59–3.58 (m, 4H), 3.47–3.44 (m,
16H), 2.99 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=189.9, 170.9,
160.0, 156.2, 153.2, 151.3, 146.0, 131.9, 129.9, 123.6, 118.4, 110.2, 109.7,
108.0, 97.2, 70.5, 69.7, 69.3, 67.7, 53.5, 52.3, 39.3 ppm; IR (KBr): n˜ =1716,
1685 cmÀ1 (C=O); HRMS (ESI): m/z calcd for C29H34N2O9: 554.2264;
found 577.0837 (M+Na+).
Synthesis of OPLM2: Coupling of OPLM1-COOH (340 mg, 0.38 mmol)
with OPLM1-NH2 (266 mg, 0.37 mmol) in the presence of DCC (180 mg,
0.87 mmol), HOBt (117 mg, 0.87 mmol), and DMAP (11 mg, 0.09 mmol)
gave OPLM2 (518 mg, 0.32 mmol) as an orange solid. Yield 87%; m.p.
1
118–1208C; Rf =0.10 (CH2Cl2/MeOH, 95:5); H NMR (400 MHz, CDCl3/
drops CD3OD): d=7.75 (s, 1H), 7.71 (s, 1H), 7.66–7.62 (m, 6H), 7.59–
7.55 (NH), 7.50–7.48 (m, 2H), 7.41 (m, 2H), 7.34–7.16 (m, 4H), 6.55–6.50
(m, 6H), 6.41 (s, 2H), 6.10 (d, J=7.6 Hz; NH), 5.79–5.73 (m, 1H), 5.19
(d, J=16.8 Hz, 1H), 5.11 (d, J=10.4 Hz, 1H), 4.47–4.42 (br, 3H), 4.20 (d,
J=7.2 Hz, 2H), 4.14 (m, 1H), 4.07–4.03 (m, 1H), 4.00–3.93 (br, 3H),
3.68–3.50 (br, 40H), 3.26–3.18 (br, 4H), 3.10 (s, 3H), 3.03 (s, 3H), 1.78–
1.21 ppm (br, 12H); 13C NMR (100 MHz, CDCl3): d=189.4, 189.2, 172.0,
171.6, 169.0, 168.6, 159.7, 159.5, 156.3, 155.9, 153.2, 153.1, 151.4, 145.3,
145.0, 143.6, 143.4, 140.9, 132.1, 131.4, 130.0, 129.9, 127.4, 126.9, 126.8,
124.9, 124.0, 119.9, 119.7, 118.4, 110.4, 110.1, 108.6, 108.4, 97.9, 71.1, 70.1,
69.8, 67.9, 66.8, 65.6, 56.8, 56.6, 54.6, 52.8, 52.2, 47.0, 40.3, 40.0, 39.0, 38.4,
32.5, 30.7, 28.9, 28.5, 22.6, 22.3 ppm; IR (KBr): n˜ =1720, 1679 cmÀ1 (C=
Synthesis of OPLM1: DCC (353 mg, 1.74 mmol), HOBt (233 mg,
1.73 mmol), and a catalytic amount of DMAP (23 mg, 0.17 mmol) were
added to a solution of AcidAC (414 mg, 0.75 mmol) and Ne-amino-
[20]
Boc1 (315 mg, 0.77 mmol) in dry CH2Cl2 (18 mL) and the reaction mix-
ture was stirred at room temperature for 14 h. After confirming comple-
tion of the reaction by TLC, the mixture was filtered though a pad of
cotton to remove most of the by-product (N,N’-dicyclohexylurea). The
filtrate was concentrated and purified by flash column chromatography
on silica gel eluting with a gradient of CH2Cl2/MeOH (from 95:5 to 9:1)
Chem. Eur. J. 2013, 19, 2531 – 2538
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2537

C.-T. Chen and Y.-C. Lin
O); HRMS (BioTOF II): m/z calcd for C88H104N8O21: 1608.7316; found:
805.8643 ([M+2H+]/2).
[2] a) E. Yashima, K. Maeda, H. Iida, Y. Furusho, K. Nagai, Chem. Rev.
2009, 109, 6102–6211; b) B. M. Rosen, C. J. Wilson, D. A. Wilson,
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6540.
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3316–3325; b) H. Juwarker, K.-S. Jeong, Chem. Soc. Rev. 2010, 39,
3664–3674.
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Soc. 2011, 133, 13938–13941.
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Angew. Chem. Int. Ed. 2008, 47, 4926–4930; b) Y. Hua, A. H. Flood,
J. Am. Chem. Soc. 2010, 132, 12838–12840.
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11743; b) C. E. Schafmeister, J. Po, G. L. Verdine, J. Am. Chem. Soc.
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Chem. Soc. 2010, 132, 5564–5565.
Synthesis of OPLM4: Coupling of OPLM2-COOH (311 mg, 0.20 mmol)
with OPLM2-NH2 (262 mg, 0.19 mmol) in the presence of DCC (95 mg,
0.46 mmol), HOBt (13 mg, 0.10 mmol), and DMAP (3 mg, 0.02 mmol) af-
forded OPLM4 (341 mg, 0.12 mmol) as an orange solid. Yield 61%; m.p.
173–1768C; Rf =0.27 (CH2Cl2/MeOH, 9:1); 1H NMR (400 MHz, CDCl3/
drops CD3OD): d=7.70–7.08 (m, 31H), 6.52–6.34 (m, 17H), 5.72–5.65
(m, 1H), 5.13–5.03 (m, 2H), 4.39 (br, 2H), 4.30–4.16 (m, 3H), 4.08–3.83
(m, 12H), 3.69–3.50 (m, 80H), 3.24–3.00 (m, 20H), 1.71–1.23 ppm (m,
24H); 13C NMR (100 MHz, CDCl3): d=189.6 (ꢁ2), 171.9, 171.4, 169.1 (ꢁ
3), 159.8 (ꢁ3), 156.3, 153.2 (ꢁ2), 151.6, 145.3, 143.5, 143.3, 140.8, 132.1,
131.2, 130.0, 127.5, 126.8, 124.8, 124.0, 119.9, 119.6, 118.4, 110.4, 110.1,
108.4 (ꢁ2), 97.8, 71.0, 70.1, 69.8, 68.0, 66.8, 65.7, 56.3, 52.7, 46.9, 39.9,
38.8, 31.9, 31.1, 30.9, 29.6, 28.7, 28.5, 22.6 ppm; IR (KBr): n˜ =1719,
1667 cmÀ1 (C=O); HRMS (BioTOF II): m/z calcd for C158H192N16O39
2937.3533; found: 980.5125 ([M+3H+]/3).
:
Synthesis of OPLM8: Coupling of OPLM4-COOH (83 mg, 0.03 mmol)
with OPLM4-NH2 (72 mg, 0.03 mmol) in the presence of DCC (13 mg,
0.06 mmol), HOBt (1.8 mg, 0.01 mmol), and DMAP (0.4 mg,
0.003 mmol) afforded OPLM8 (102 mg, 0.02 mmol) as a yellowish-orange
solid. Yield 70%; m.p. 158–1628C; 1H NMR (400 MHz, CDCl3/drops
CD3OD): d=7.74–7.13 (m, 56H), 6.56–6.37 (m, 32H), 5.80–5.60 (m, 1H),
5.24–5.08 (m, 2H), 4.44 (br, 2H), 4.39–4.20 (m, 3H), 4.10–3.88 (m, 24H),
3.69–3.56 (m, 160H), 3.43–3.06 (m, 40H), 1.76–1.22 ppm (m, 48H);
13C NMR (200 MHz, CDCl3): d=190.0 (ꢁ3), 172.9 (ꢁ2), 172.4 (ꢁ3),
171.5 (ꢁ2), 169.4 (ꢁ5), 160.0 (ꢁ2), 156.5 (ꢁ2), 153.5 (ꢁ2), 151.8, 145.6 (ꢁ
3), 143.6 (ꢁ2), 141.0, 132.3 (ꢁ2), 130.2, 127.6, 127.0, 125.0 (ꢁ2), 124.2 (ꢁ
3), 120.1, 119.8, 110.7 (ꢁ2), 110.2, 108.6 (ꢁ3), 97.8 (ꢁ3), 71.0, 70.1, 69.8,
68.0, 66.8, 66.6, 56.2 (ꢁ2), 55.0, 53.4 (ꢁ2), 52.7, 46.9, 46.4, 39.9 (ꢁ3), 38.8
(ꢁ4), 31.8, 29.5 (ꢁ4), 28.8 (ꢁ3), 22.6 ppm (ꢁ4); IR (KBr): n˜ =1718,
1666 cmÀ1 (C=O); MALDI-TOF MS (matrix: a-CHC): m/z calcd for
C298H368N32O75: 5594.5966; found: 5595.702 (M+H+).
[10] a) C. A. Royer, Chem. Rev. 2006, 106, 1769–1784; b) X. Michalet, S.
Weiss, M. Jꢄger, Chem. Rev. 2006, 106, 1785–1813.
[11] a) Z. Zhong, Y. Zhao, Org. Lett. 2007, 9, 2891–2894; b) I. J. Lee,
B. H. Kim, Chem. Commun. 2012, 48, 2074–2076.
[12] C.-T. Chen, W.-P. Huang, J. Am. Chem. Soc. 2002, 124, 6246–6247.
[13] N. Solladiꢅ, A. Hamel, M. Gross, Tetrahedron Lett. 2000, 41, 6075–
6078.
[14] OPLM8 shows good solubility in CH2Cl2, CHCl3, and DMF, but its
NMR peaks in these solvents are quite broad and cannot be well re-
solved owing to
a
certain degree of aggregation. 13C NMR
(200 MHz) spectra of OPLMs were thus recorded on a Bruker
AVIII 800 MHz spectrometer to give better resolved carbonyl
peaks. The structural identity of OPLM8 was supported by detection
of the correct parent ion peak in the MALDI-TOF mass spectrum
with 4-hydroxy-a-cyanocinnamic acid (a-CHC; see the Supporting
Information, Figure S1).
Spectroscopic studies: High-throughput fluorescence screening of metal
ions was performed using a Wallac 1420 VICTOR2 plate reader. All solu-
tions were prepared in either spectroscopic grade CH3CN or CH2Cl2
without special efforts to exclude water or air.
UV/Vis, fluorescence, and circular dichroism titration studies: Absorp-
tion spectra were recorded on a Hewlett–Packard 8453A diode-array
spectrometer under the control of a Pentium PC running the manufactur-
er-supplied software package. Fluorescence spectra were obtained on a
Hitachi F-4500 spectrometer. CD spectra were recorded on a JASCO J-
810 spectropolarimeter using a quartz cell with an optical path length of
1 cm under nitrogen atmosphere. A typical titration experiment was car-
ried out as follows. A solution of the molecule of interest was prepared
in spectroscopic grade CH2Cl2 (2ꢁ10À6 m), and a 2 mL portion was trans-
ferred to a 1 cm quartz cuvette. A small aliquot of a stock solution of the
examined metal ion (1.6 mm) in spectroscopic grade CH3CN with per-
chlorate as the counterion was then added. The absorption and fluores-
cence spectra of the molecule of interest were both recorded as a func-
tion of the metal-ion concentration.
[15] P. D. Beer, A. D. Keefe, H. Sikanyika, C. Blackburn, J. F. McAleer,
J. Chem. Soc. Dalton Trans. 1990, 3289–3294.
[16] In the absence of Sr2+, the low fluorescence intensity was defined as
the “OFF” state. At 4 equivalents of Sr2+, the fluorescence intensity
increased slightly (about 0.7-fold), but was still defined as the
“OFF” state. At 10 equivalents, the fluorescence was enhanced 5-
fold, and was defined as the “ON” state.
[17] a) R. Murugavel, R. Korah, Inorg. Chem. 2007, 46, 11048–11062;
b) L. M. Teesch, J. Adams, J. Am. Chem. Soc. 1990, 112, 4110–4120.
[18] OPLM8 shows good solubility in CH2Cl2, CHCl3, and DMF. Howev-
er, metal-induced changes in circular dichroism were only observed
in CH2Cl2 and CHCl3; no such changes were observed in DMF. The
UV cut-off of these two solvents is near 240 nm. Thus, it was diffi-
cult to determine the backbone structures in these media since the
peptide bond absorption was in the far-UV region (180–240 nm).
[19] The helix that formed in solution was stable for at least 1 h.
[20] a) S. Wiejak, E. Masiukiewicz, B. Rzeszotarska, Chem. Pharm. Bull.
1999, 47, 1489–1490; b) P. Watts, C. Wiles, S. J. Haswell, E. Pombo-
Villar, Tetrahedron 2002, 58, 5427–5439; c) D. Andreu, S. Ruiz, C.
CarreÇo, J. Alsina, F. Albericio, M. ꢆ. Jimꢅnez, N. de La Figuera, R.
Herranz, M. T. Garcꢇa-Lꢈpez, R. Gonzꢉlez-MuÇiz, J. Am. Chem.
Soc. 1997, 119, 10579–10586.
Acknowledgements
The authors would like to thank the National Science Council of the Re-
public of China, Taiwan, and the National Taiwan University for financial
support, as well as Dr. S.-J. Huang and Ms. S.-L. Huang at the Instrumen-
tation Center, National Taiwan University, for their assistance with the
800 MHz NMR measurements.
[21] B. Holm, J. Broddefalk, S. Flodell, E. Wellner, J. Kihlberg, Tetrahe-
dron 2000, 56, 1579–1586.
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Thornton, Proc. Natl. Acad. Sci. USA 1996, 93, 13–20.
Received: August 23, 2012
Published online: November 28, 2012
2538
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ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 2531 – 2538