Synthesis and conformational characterization of tethered, self-complexing 1,5-dialkoxynaphthalene/1,4,5,8-naphthalenetetracarboxylic diimide systems

Article abstract of DOI:10.1021/ja0019225

Chemists are beginning to explore the abiotic folding of synthetic chains, and the term "foldamers" has been used to characterize oligomers with a strong inclination to adopt specific, compact conformations. The characterization of folded structure in solution is one of the difficult challenges facing the foldamer field. Aedamers were the first foldamers to make use of aromatic-aromatic interactions in water to direct folding and were designed to have several spectroscopic handles with which to probe folding conformations in solution. Herein is reported the synthesis and spectroscopic characterization of eleven aedamer dimers, with linkers chosen to provide a spectrum of lengths and flexibilities. The dimers, composed of one electron rich (1,5-dialkoxynaphthalene) and one electron deficient (1,4,5,8-naphthalenetetracarboxylic diimide) aromatic group tethered by a linker, are the smallest aedamer folding unit. The powerful spectroscopic handles associated with the stacked aedamer groups were exploited in a comprehensive spectroscopic analysis of conformation that included UV-vis absorption spectroscopy, fluorescence measurements (including time-resolved studies), as well as detailed NMR studies. The spectra were interpreted in the context of molecular modeling/spectral prediction and structural models were developed for the different dimers in aqueous solution. In most instances, the observed data was best described by an ensemble of predicted structures as opposed to one or few conformers. Thus, in the case of these aedamer dimers, "folding" does not appear to imply a two-state model with a rigid, unique conformation. Rather, the reported analysis indicates the data can best be described by a more dynamic model in which a given molecule spends its time in different folded conformations that are related by having a characteristic face-to-face stacking arrangement of the aromatic units.

Full text of DOI:10.1021/ja0019225

8898
J. Am. Chem. Soc. 2000, 122, 8898-8909
Synthesis and Conformational Characterization of Tethered,
Self-Complexing 1,5-Dialkoxynaphthalene/
1,4,5,8-Naphthalenetetracarboxylic Diimide Systems
Andrew J. Zych and Brent L. Iverson*
Contribution from the Department of Chemistry and Biochemistry The UniVersity of Texas at Austin,
Austin, Texas 78712
ReceiVed May 31, 2000
Abstract: Chemists are beginning to explore the abiotic folding of synthetic chains, and the term “foldamers”
has been used to characterize oligomers with a strong inclination to adopt specific, compact conformations.
The characterization of folded structure in solution is one of the difficult challenges facing the foldamer field.
Aedamers were the first foldamers to make use of aromatic-aromatic interactions in water to direct folding
and were designed to have several spectroscopic handles with which to probe folding conformations in solution.
Herein is reported the synthesis and spectroscopic characterization of eleven aedamer dimers, with linkers
chosen to provide a spectrum of lengths and flexibilities. The dimers, composed of one electron rich (1,5-
dialkoxynaphthalene) and one electron deficient (1,4,5,8-naphthalenetetracarboxylic diimide) aromatic group
tethered by a linker, are the smallest aedamer folding unit. The powerful spectroscopic handles associated
with the stacked aedamer groups were exploited in a comprehensive spectroscopic analysis of conformation
that included UV-vis absorption spectroscopy, fluorescence measurements (including time-resolved studies),
as well as detailed NMR studies. The spectra were interpreted in the context of molecular modeling/spectral
prediction and structural models were developed for the different dimers in aqueous solution. In most instances,
the observed data was best described by an ensemble of predicted structures as opposed to one or few conformers.
Thus, in the case of these aedamer dimers, “folding” does not appear to imply a two-state model with a rigid,
unique conformation. Rather, the reported analysis indicates the data can best be described by a more dynamic
model in which a given molecule spends its time in different folded conformations that are related by having
a characteristic face-to-face stacking arrangement of the aromatic units.
Introduction
peptides,4 sulfonopeptides,5 peptoids,6 oligocarbamates,7 oli-
gopyrrolidones,8 oligoureas,9 carbohydrate oligomers,10 oligo-
anthrilamides,11a,b crescent oligoamides,11c meta-linked pheny-
lacetylenes,12 and pyrimidine-pyridine oligomers.13 Hydrogen
bonding, conformational restriction, and solvophobic interactions
have all been used to advantage in the design of these folding
molecular systems.
Aedamers were the first foldamers to make use of aromatic-
aromatic interactions in water to direct folding.14 The aedamer
design is based on the face-to-face association of alternating
electron-rich “donor”, 1,5-dialkoxynaphthalene (DAN), and
electron-deficient “acceptor”, 1,4,5,8-naphthalenetetracarboxylic
diimide (NDI), aromatic moieties. The aromatic units are
connected by flexible linkages that are designed to allow a
pleated structure with a stacked core of aromatic units (Figure
1a). In aqueous solution, this folding is assumed to have a large
solvophobic driving force, which operates in concert with the
aromatic electron donor-acceptor complexation upon which
the aedamer name is based. Initial investigations were performed
on aedamers consisting of 2, 4, or 6 alternating aromatic units
linked through aspartate residues (Figure 1b).
Inspired by the remarkable variety of molecular architectures
found among biological macromolecules such as proteins and
nucleic acids, the synthesis of oligomers with predictable
secondary structure has attracted increasing interest in recent
years.1 Natural macromolecules are composed of linear chains
folded into compact structures, producing three-dimensional
ensembles of functional groups capable of complex tasks such
as binding and/or catalysis. Chemists are beginning to explore
abiotic folding of synthetic chains, and the term “foldamers”
has been used to characterize oligomers with a strong inclina-
tion to adopt a specific compact conformation.1a Examples
of foldamers include â-peptides,2 γ-peptides,3 vinylogous
* To whom correspondence should be addressed. E-mail: biverson@
utxvms.cc.utexas.edu.
(1) (a) Gellman, S. H. J. Acc. Chem. Res. 1998, 31, 173. (b) Seebach,
D.; Matthews, J. L. Chem. Commun. 1997, 2015. (c) Iverson, B. L. Nature
1997, 385, 113. (d) Borman, S. Chem. Eng. News June 16 1997, 32.
(2) (a) Barchi, J. J.; Huang, X.; Appella, D. H.; Christianson, L. A.;
Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 2000 122, 2711-2718.
(b) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman,
S. H. J. Am. Chem. Soc. 1999, 121, 6206. (c) Appella, D. H.; Barchi, J. J.;
Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309. (d)
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A key challenge facing the foldamer field is the characteriza-
tion of folded conformation in solution. Aedamers possess a
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10.1021/ja0019225 CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/31/2000

1,4,5,8-Naphthalenetetracarboxylic Diimide Systems
J. Am. Chem. Soc., Vol. 122, No. 37, 2000 8899
of one DAN and one NDI unit tethered by a linker. At the same
time, it was of interest to see how differences in linker flexibility
and length influence dimer folding.
Herein is reported the synthesis and spectroscopic charac-
terization of 10 folding aedamer dimers, with linkers chosen to
provide a spectrum of lengths and flexibilities (Figure 2). A
control dimer, compound 11, was also synthesized in which
folding is prevented through geometric constraints. Additionally,
water-soluble DAN and NDI monomers were synthesized and
their interactions characterized. A combination of UV-vis
absorption spectroscopy, fluorescence measurements (including
time-resolved studies), NMR, and molecular modeling/spectra
simulation are reported, enabling conclusions to be drawn
regarding the degree of folding, as well as the types of folded
conformations observed in the different molecules. Taken
together, the results and accompanying analysis provide a
comprehensive description of aedamer folding in solution,
paving the way for future investigations into the solution
structures of more complex aedamers and related foldamers.
Results
Monomer Studies. Water-soluble, uncharged analogues of
DAN and NDI (Figure 3) were synthesized (see the Supporting
Information). An NMR titration was carried out in D2O in which
the chemical shift of the NDI 12 aromatic protons was followed
as a function of DAN 13 concentration.15
The NMR titration data was examined using a Scatchard
analysis. Because of significant curvature apparent in the
Scatchard plot (Figure 4a), a combination of 1:1 and 2:1 binding
was postulated.17 Thus, the binding isotherm was fit with an
equation that includes the presence of both complexes (Figure
4b).18 Association constants of 1300 ( 200 M-1 and 200 (
100 M-1 were derived for the 1:1 and 2:1 complexes, respec-
tively. The corresponding upfield chemical shift changes (∆δ)
of -0.42 ( 0.10 ppm and -0.84 ( 0.02 ppm were determined
for the 1:1 and 2:1 complexes, respectively. Since the concen-
tration of 12 was kept constant while 13 was increased, it is
assumed that the 2:1 species corresponds to a 13-12-13
complex.19
Dimer Synthesis. Ten different folding aedamer dimers 1-10
were synthesized in an effort to provide a spectrum of linker
length and rigidity. For example, dimers 1-5 have 0-4 amino
acid residues between the DAN and NDI units. Dimers 6-8
are rigidified by virtue of having various bond rotations
prevented by cyclic amino acid units, while dimer 9 has an
overall cyclic structure. Dimer 10 is analogous to 2, except that
the N-terminus is not blocked with an acetamide group and the
aspartate was moved to the C-terminus in order to examine the
influence on folding of potential noncovalent cyclization due
Figure 1. (a) Schematic representation of folded aedamer structure.
(b) Chemical structure of aedamer oligomers.
number of powerful spectroscopic handles related to folding
by virtue of the stacked aromatic units that comprise the core
of the folded structure. In fact, inclusion of such spectroscopic
handles was an important consideration in the original aedamer
design. Nevertheless, a thorough understanding of aedamer
folding is complicated by the size and flexibility of the
molecules, especially the larger derivatives. Therefore, a series
of investigations were undertaken to characterize details of
folding in the smallest folding unit, namely the dimer, composed
(6) (a) Kirshenbaum, K.; Barron, A. E.; Goldsmith, R. A.; Armand, P.;
Bradley, E. K.; Truong, K. T. V.; Dill, K. A.; Cohen, F. E.; Zuckermann,
R. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303. (b) Armand, P.;
Kirshenbaum, K.; Goldsmith, R. A.; Farr-Jones, S.; Barron, A. E.; Truong,
K. T. V.; Dill, K. A.; Mierke, D. F.; Cohen, F. E.; Zuckermann, R. N.;
Bradley, E. K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4309. (c) Armand,
P.; Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L.; Dill, K. A.; Zuckermann,
R. N.; Cohen, F. E. Fold. Design 1997, 2, 369.
(7) (a) Paikoff, S. J.; Wilson, T. E.; Cho, C. Y.; Schultz, P. G.
Tetrahedron Lett. 1996, 37, 5653. (b) Cho, C. Y.; Moran, E. J.; Cherry, S.
R.; Stephans, J. C.; Fodor, S. P. A.; Adams, C. L.; Sundaram, A.; Jacobs,
J. W.; Schultz, P. G. Science 1993, 261, 1303.
(8) Smith, A. B., III; Guzman, M. C.; Sprengeler, P. A.; Keenan, T. P.;
Holcomb, R. C.; Wood, J. L.; Carroll, P. J.; Hirschmann, R. J. Am. Chem.
Soc. 1994, 116, 9947.
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(b) Nowick, J. S.; Mahrus, S.; Smith, E. M.; Ziller, J. W. J. Am. Chem.
Soc. 1996, 118, 1066.
(15) Significant self-association of the NDI derivative 12 was observed,
estimated to be ∼150 M-1 based on the concentration dependence of its
NMR spectrum.16 The DAN monomer 13 exhibited much less self-
association with an NMR-estimated dimerization constant of ∼10 M-1. Note
that both the NDI and DAN concentrations were kept low enough to avoid
any significant complications due to self-association.
(10) Szabo, L.; Smith, B. L.; McReynolds, K. D.; Parrill, A. L.; Morris,
E. R.; Gervay, J. J. Org. Chem. 1998, 63, 1074.
(11) (a) Hamuro, Y.; Geib, S. J.; Hamilton, A. D. J. Am. Chem. Soc.
1997, 119, 10587. (b) Hamuro, Y.; Geib, S. J.; Hamilton, A. D. Angew.
Chem. Int. Ed. Engl. 1994, 33, 446. (c) Zhu, J.; R. D.; Zeng, H.; Skrzypczak-
Jankun, E.; Zeng, X. C.; Gong, B. J. Am. Chem. Soc. 2000, 122, 4219.
(12) (a) Brunsveld, L.; Prince, R. B.; Meijer, E. W.; Moore, J. S. Org.
Lett. 2000, 2, 1525. (b) Prince, R. B.; Okada, T.; Moore, J. S. Angew. Chem.,
Int. Ed. 1999, 38, 233. (c) Prince, R. B.; Saven, J. G.; Wolynes, P. G.;
Moore, J. S J. Am. Chem. Soc. 1999, 121, 3114. (d) Nelson, J. C.; Saven,
J. G.; Moore, J. S.; Wolynes, P. G Science 1997, 277, 1793.
(13) Bassani, D. M.; Lehn, J.-M.; Baum, G.; Fenske, D. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 1845.
(16) Horman, I.; Dreux, B. HelV. Chim. Acta 1984, 67, 754.
(17) (a) Deranleau, D. A. J. Am. Chem. Soc. 1969, 91, 4050. (b) Tsukube,
H.; Furuta, H.; Odani, A.; Takeda, Y.; Kudo, Y.; Inoue, Y.; Liu, Y.;
Sakamoto, H.; Kimura, K. In ComprehensiVe Supramolecular Chemistry;
Atwood, J. L., Lehn, J. M., Eds.; Pergamon Press: New York, 1996; Vol.
8, Chapter 10.
(18) (a) Chudek, J. A.; Foster, R.; Twiselton, D. R.; J. Chem. Soc., Perkin
Trans. 2 1983, 1385. (b) Dodson, B.; Foster, R.; Bright, A. A. S.; Foreman,
M. I.; Gorton, J. J. Chem. Soc. B 1971, 1283.
(19) Preliminary experiments suggested that a 2:1 complex of the form
12-13-12 could also be observed when the NDI 12 was in excess, but
self-aggregation of 12 at higher concentrations precluded a meaningful
quantitative analysis in this concentration regime.
(14) (a) Lokey, R. S.; Iverson, B. L. Nature 1995, 375, 303. (b) Nguyen,
J. Q.; Iverson, B. L. J. Am. Chem. Soc. 1999, 121, 2639.

8900 J. Am. Chem. Soc., Vol. 122, No. 37, 2000
Zych and IVerson
Figure 2. Aedamer dimers synthesized for this study. 11 is a control compound in which folding is prevented through geometric constraints.
handle for cyclization.20 Dimers 2-5 were reacted with acetic
anhydride to cap the N-terminus, as in the original aedamer
design, while 6-8 were capped with succinic anhydride to
replace the negative charge not present in the linkage. The
synthesis of the control compound 11 is also included in the
Supporting Information.
NMR Chemical Shift Changes. Chemical shift changes
compared to the NDI 14 and DAN 15 reference compounds
for signals common to the 10 folding dimers 1-10 are listed in
Table 1. Spectra were recorded in phosphate buffered D2O, pD
7.0, at concentrations of 0.1 mM, where all signals were
essentially concentration independent. Resonances were assigned
through a combination of COSY and NOESY spectroscopy.
Figure 3. Neutral NDI (12) and DAN (13) compounds for NMR
titrations.
to a putative salt bridge between terminal groups. As a control,
compound 11, which is precluded from folding by virtue of the
geometric constraints imposed by the aromatic linker, was also
synthesized.
The aedamer dimers 1-10 were synthesized using standard
Fmoc-based solid-phase methodologies. The syntheses of DAN
and NDI-containing amino acids for solid-phase coupling are
included in the Supporting Information. Glycine-functionalized
Wang resin was used for all compounds, except 9 and 10, to
provide a C-terminal glycine residue. Aspartic acid-function-
alized Wang resin was used for 10. For 9, a Wang Tentagel
resin was functionalized with Fmoc-Asp-ODmb to provide a
Particularly dramatic were the changes in the aromatic region
of the spectra of 1-10 (Figure 5), in which all signals moved
upfield. The spectrum for the monomeric reference NDI 14
(Figure 5a) had a broad singlet at about 8.8 ppm corresponding
to the aromatic protons, while this area in the spectra of many
dimers contained two sets of signals, shifted upfield by as much
as -0.5 ppm compared to 14. For example, the aromatic NDI
signals of 1 (Figure 5c) and 8 (Figure 5j) displayed a particularly
large splitting of 70 and 100 Hz, respectively, while 3-5 (Figure
5e-g) exhibited little or no splitting. An intermediate splitting
(20) McMurray, J. S. Tetrahedron Lett. 1991, 32, 7679.

1,4,5,8-Naphthalenetetracarboxylic Diimide Systems
J. Am. Chem. Soc., Vol. 122, No. 37, 2000 8901
protons for all folding compounds except for 9.21 A portion of
a typical spectrum is shown is Figure 6. Couplings were
also observed between the linkage methylenes j and k with
the NDI aromatic protons for all folding compounds except
for 9.21 A number of other various long-range couplings
were also observed for some compounds. A more detailed
summary of the NOE data is available in the Supporting
Information.
UV-Vis Spectroscopy. UV-vis data are summarized in
Table 2. All 10 folding dimers showed significant absorbance
spectral shape changes in the UV-vis region compared to a
composite spectra derived from the superposition of isolated
NDI 14 and DAN 15 monomer spectra. A representative
example is shown in Figure 7. In particular, significant
hypochromism was observed for each dimer in the 260-400
nm region. This hypochromism varied from 50% for 7 to 31%
for 9 at 382 nm (see Table 2 for definition of % hypochromism).
Importantly, the hypochromism was concentration independent
in the range examined (<0.1 mM), indicative of monomeric
species. Upon heating to 80 °C, the molar extinction coefficients
at 382 nm (ꢀ382) increased by only about 4-6% for all folding
dimers. In contrast, the control compound 11 showed hypo-
chromism at room temperature analogous to compounds 1-10;
however, at 80 °C, the ꢀ382 of 11 increased dramaticallysover
40%.
A broad absorption band in the visible region, not observed
in 14 or 15, is observed for each of the folding dimers 1-10
(Table 2). The λmax for these so-called “charge-transfer” bands
was between 520 and 530 nm with the exception of 1, which
displayed a λmax near 510 nm. The molar extinction coefficients
for these bands at their respective λmax range from 200 to 400
M-1‚cm-1 (Table 2).
Fluorescence. The DAN unit of 15, which contains the same
linkage as 2 but no NDI moiety, exhibited a strong, structured
emission with a λmax of 330 nm (Figure 8) and a 10 ps lifetime
in aqueous phosphate buffer (pH ∼7). In contrast, shortened
lifetimes, as well as substantial or complete quenching of this
fluorescence, were observed for all dimers (Table 3). Where
observable, the dimer steady-state emissions had a shape very
similar to that of 15. Interestingly, the entire emission for 9
was shifted with its λmax observed at 308 nm compared to 330
nm for the other dimers.
Molecular Modeling. Unrestrained high-temperature mo-
lecular dynamics were used to produce a large set of random
unfolded conformations for each dimer.22 The unfolded struc-
tures were taken as a randomized set of starting structures that
were then “folded” by annealing with a weak distance restraint
between the NDI and the DAN units. The structures were then
subjected to geometry optimization without any restraints.23
Unfolded and distorted structures were discarded. This produced
nine ensembles of structures with an energy range of about 5-10
kcal/mol (see the Supporting Information). In some instances
(compounds 1, 2, and 6), the collection consisted largely of
structures with energies relatively close to that of the global
minimum found for that compound. However, some of the
collections, notably those for compounds 7 and 8, showed a
predominance of relatively higher energy structures.
Figure 4. (a) Scatchard plot of NMR titration experiment with NDI
12 and DAN 13 monomers in D2O. (b) NMR binding isotherm fit with
a 1:1 + 1:2 binding model.
was observed for the remaining dimers. The aromatic NDI
signals of 7 (Figure 5i) showed the greatest upfield shift of -0.5
ppm, while 9 (Figure 5k) showed the smallest change of about
-0.3 ppm.
The monomeric reference DAN 15 exhibited two doublets
(H-2, H-6 at 7.1 ppm and H-4, H-8 at 8.0 ppm) and one triplet
(H-3, H-7 at 7.5 ppm), each integrating to two protons (Figure
5b). In many dimer compounds the equivalent pairs were split
to give six total signals. This was especially prominent for 8
(Figure 5j) in which the ∆δ between H-4 and H-8 is nearly 1.1
ppm and in 1 (Figure 5c), where the ∆δ between H-2 and H-6
signals was greater than 0.7 ppm.
Significant changes were also observed for some linkage
signals (Table 1). For example, k in 1 was shifted by about
-0.7 ppm while j was shifted by less than -0.1 ppm compared
to the reference compound 15. Conversely, j was considerably
more shifted in 9 than k. Most of the other compounds exhibited
small upfield shifts for j and k with little change in other linkage
signals. An exception is 9 which displayed a ∆δ of about -0.7
for c. As expected for molecules containing a stereocenter and
exhibiting folding, many diastereotopic methylene groups were
observed, particularly in 9.
Control compound 11 displayed a very broad, unresolved
spectra at all concentrations examined, indicating extensive
aggregation at room temperature. This behavior is in distinct
contrast to the folding molecules 1-10, which displayed well-
resolved signals and chemical shifts essentially independent of
concentration (below ∼1 mM) indicative of monomeric species.
Higher temperature spectra of 11 were better resolved, but still
suggestive of substantial aggregation, and thus not appropriate
for comparison with 1-10.
(21) NOE spectroscopy was not performed on compounds 9 and 11 due
to the degree of aggregation at concentrations where long-range NOEs could
be observed.
(22) Compound 10 was excluded from the modeling studies since it has
the same linkage as 2 and 11 was also excluded because it cannot physically
fold in a manner analogous to 1-10.
(23) It was not appropriate to derive distance restraints from the NOE
data due to the poor quantitative value of these data as detailed in the “NOE
Spectroscopy” portion of the discussion section.
NOESY Spectroscopy. Weak intensity through-space cou-
plings were observed between the NDI and DAN aromatic

8902 J. Am. Chem. Soc., Vol. 122, No. 37, 2000
Zych and IVerson
Table 1. Chemical Shift Changes (ppm) for Dimers 1-10 Compared to NDI and DAN Reference Compounds 14 and 15a
a Shifts were measured at concentrations of 0.1 mm in phosphate-buffered D2O. At these concentrations the shifts were essentially concentration
independent. Entries with two values represent diastereotopic proton pairs.
For the purposes of this analysis, attention was focused
especially on the relative orientations of the aromatic units, since
the NMR analysis is based on these regions of the molecules.
Structures were defined as “folded” if the centers of the DAN
and NDI ring planes were within ∼5 Å of one another and the
angle between the planes was less than 30°. A coordinate system
was defined with its origin at the center of the NDI unit (Figure
9a). By setting a reference point at the center of the DAN unit,
the relative orientation of the DAN/NDI complex can be
described with three parameters: x offset, y offset, and DAN/
NDI axis angle (Figure 9). The DAN/NDI axis angle, R, is
defined as shown in Figure 9b. The distribution of each
parameter for each linkage is shown in Figure 10.
The types of orientations produced during the simulations
vary significantly between different linkages in some instances.
Notably, the dimer with the shortest linkage 1, displays the most
limited range of NDI/DAN orientations. Most of structures
generated with this linkage exhibit significant x offset of the
DAN unit away from the linkage side of the diimide. Also, the
range of R angles produced with this linkage is rather limited.
In contrast, a general trend toward greater orientation diversity
is seen with increasing linker length. For example, the dimer
with the longest linkage, 5, shows the widest array of offsets
and R values. The overall scatter of x offsets also becomes more
centered about the central reference point of the NDI with
increasing linker length. Some linkages show a weak partitioning
of lower and higher energy structures, defined as within or above
5 kcal/mol of the minimum respectively, along the x axis. This
partitioning is most pronounced in 7 and 8. For 8 and to a lesser
extent 7, all the low energy structures had a negative x offset
(left of the origin in Figure 9a, away from the linkage side of
the NDI) and the majority of low energy structures had a major
x offset.
empirical function was used to approximate the shielding/
deshielding effects of the aromatic rings24a while a separate
function was used for the carbonyls of the NDI unit.24b Details
of the calculations are discussed in the Experimental Section.
Shift changes were calculated for twelve protons, or sets of
equivalent protons, common to all dimers (values for diaste-
reotopic proton pairs were averaged). Thus, the simulated
chemical shift changes for any individual structure, or combina-
tion of structures, obtained from molecular dynamics could be
calculated and compared to the observed shift changes.
Single structures showed a wide range of agreement with
observed data (see the Supporting Information). Quality of fit
was quantified by the root-mean-square (rms) difference between
observed and calculated shift changes. In the case of 1, there
was a rough correlation between the rms fit and potential energy.
In no instance did the lowest energy structure found also have
the smallest rms difference. However, in many instances there
were relatively low energy structures with reasonably good rms
fits (>0.2 ppm).
Discussion
Monomer Studies. To obtain reliable parameters for complex
formation, a significant portion of the binding curve must be
obtained from a titration experiment.25 Previous binding titra-
tions using derivatives of NDI and DAN were hindered by
limited solubility in aqueous solvent and/or complications due
to the use of like-charged molecules.14a The increased solubility
of the neutral monomers, 12 and 13, allowed for near saturation
as seen in the binding isotherm shown in Figure 4b. A Scatchard
plot (Figure 4a) of the NMR titration data exhibited significant
curvature. Curved Scatchard plots are consistent with 2:1 or
higher order complex formation concurrent with 1:1 complex-
Chemical Shift Simulation. An algorithm was developed
to simulate the chemical shift changes caused by the anisotropy
of the NDI and DAN units. Briefly, these calculations derive
predicted chemical shift changes from the distance and angle
between a particular proton and the shielding entities. An
(24) (a) Abraham, R. J.; Fell, S. C. M.; Smith, K. M. Org. Magn. Reson.
1977, 9, 367. (b) Herranz, J.; Gonzalez, C.; Rico, M.; Nieto, J. L.; Santoro,
J.; Jimenez, M.; Bruix, M.; Neira, J. L.; Blanco, F. J. Magn. Reson. Chem.
1992 30, 1012.
(25) Deranleau, D. A.; J. Am. Chem. Soc. 1969, 91, 4044.

1,4,5,8-Naphthalenetetracarboxylic Diimide Systems
J. Am. Chem. Soc., Vol. 122, No. 37, 2000 8903
Figure 6. Portion of NOESY spectrum for dimer 5 showing long-
range couplings between NDI aromatic protons with DAN aromatic
protons and linkage methylenes j and k.
Table 2. Summary of UV-Vis Data for Dimers 1-10
% hypochromism charge transfer
molar extinction
compd
at 382 nma,b
(CT) λmax
coefficientc at CT λmax
1
2
3
4
5
6
7
8
9
38
45
39
43
49
43
50
49
31
40
510
526
526
520
520
528
526
526
526
526
410
330
260
360
370
260
390
180
210
340
10
a % hypochromism ) [1 - (molar extinction coefficient of dimer/
molar extinction coefficient of 14)] × 100%. ꢀ14 ) 26 600. b Measured
at concentrations of ∼30 µM or below. c M-1‚cm-1
.
Figure 7. UV spectra of dimer 1 compared to a composite spectra of
NDI 14 and DAN 15 reference compounds. Concentrations are 20 µM
in phosphate-buffered H2O. Molar extinction coefficients were con-
centration independent at these concentrations.
Figure 5. Aromatic region of 500 MHz 1H NMR of compounds 1-10,
(c)-(l) respectively, in phosphate buffered D2O at a concentration of
0.1 mM. Also shown, monomeric reference compounds NDI 14 and
DAN 15, (a)-(b) respectively, at 0.1 mM in buffered D2O.
ation.26 Note that the Scatchard plot is much more sensitive to
the possible presence of higher order complexes than the
Benesi-Hildebrand plot and should be used whenever there is
a possibility of multiple equilibrium.17a
Due to the curvature observed in the Scatchard plot (Figure
4a), a model presuming both 1:1 and 2:1 binding was used to
fit the NMR titration data27 (Figure 4b). Fitting experimental
data to multiple parameters has significant inherent error.
Nevertheless, the derived values provide a reasonable estimate
(26) Note that higher level complexation is not confirmed or ruled out
by the observed Scatchard plot curvature, but is merely consistent with
such observations.

8904 J. Am. Chem. Soc., Vol. 122, No. 37, 2000
Table 3. Summary of Fluorescence Data for Dimers 1-10
Zych and IVerson
proposed for the dimer molecules.28 The magnitude of the NDI
aromatic proton ∆δ derived for a 1:1 DAN/NDI complex (-0.42
( 0.10 ppm) is comparable to those observed for the dimer
compounds (-0.3 to -0.5 ppm) 1-10.30 Thus, it can be
proposed that NDI and DAN have a substantial face-to-face
interaction in aqueous solution, leading to the prediction that
the dimer molecules will adopt relatively stably folded, compact
structures in water.
compd
rel quantum yielda
lifetime (ps)
15
1
2
3
4
5
6
7
8
1.00
0.02
0.02
0.01
0.10
0.09
0.00
0.00
0.00
∼0.1b
0.00
10.0
7.2
7.5
6.7
9.7
9.4
4.6
3.4
5.4
6.5
4.7
1H NMR Chemical Shift Changes. The chemical shift
changes observed in all 10 folding dimers are entirely consistent
with face-to-face association of the NDI and DAN units. All
9
10
28
∆δ for the DAN and NDI aromatic signals are upfield
compared to the reference compounds 14 and 15 (Table 1). The
observed chemical shifts, therefore, argue against significant
contributions from conformations with edge-to-face association
of the NDI and DAN units.
a Excitation λ ) 296 nm. b Estimated due to difference in emission
max; 15 -330 nm, 10 -310 nm.
λ
Chemical shift changes can contain significant conformational
information and have been previously employed in this
manner.11a,31 The aedamer system is particularly well suited to
such an approach due to the strong anisotropy of the aromatic
units and their close proximity to one another in folded
structures. Shift calculations give considerable insight into how
different NDI/DAN geometries can affect the chemical shifts
and splitting patterns of the aromatic region of aedamer NMR
spectra. Unlike NOESY spectroscopy, chemical shifts can easily
be obtained even at very low sample concentrations. This is
advantageous when self-association at higher concentrations is
a significant problem.
The change of the NDI aromatic signal from a broad singlet,
as in the reference compound 14, to an apparent AA′BB′ pattern
(two distorted “doublets”) in many of the dimers (Figure 5c-l)
is attributed to differential shielding along the NDI x axis caused
by DAN complexation (Figure 11). Each “doublet” is assigned
as a pair of protons, A and A′ for example, made equivalent
due to fast rotation of the NDI unit about the x axis.32 Four
different signals would be expected if fast rotation on the NMR
time scale was not occurring, unless the average complex was
very nearly symmetrical about either the x or y axes. Since four
different signals were not observed for any of the 10 folding
dimers, fast rotation, as opposed to all symmetrical complexes,
is the more plausible explanation for the AA′BB′ pattern.
The chemical shift difference between AA′ and BB′ can be
interpreted as an indication of the average offset along the x
axis of the DAN/NDI coordinate system (Figure 9). A significant
shift difference between AA′ and BB′ signals of the NDI
residues is consistent with the DAN being substantially offset
from the origin along the x axis (Figure 11a). Conversely, a
small or absent shift difference is consistent with the average
DAN position being more centered around the origin (Figure
11b) of the NDI. In line with expectation, dimers with the
Figure 8. Emission spectra of DAN reference compound 15 at a
concentration of 2.0 µM in phosphate buffered H2O.
(28) Upfield chemical shifts are experienced in the regions of space
directly above aromatic and carbonyl pi systems while downfield shifts are
experienced in the plane of the π system.29
(29) Gunther, H. NMR Spectroscopy, 2nd ed.; John Wiley & Sons: New
York, 1995; 78-93.
(30) It is not appropriate to quantitatively compare the ∆δ values derived
for the complex with those for dimers 1-10 since the geometry of the
intramolecular dimer complex may vary significantly from that of the
intermolecular monomer complex.
(31) (a) Hunter, C. A.; Packer, M. Chem. Eur. J. 1999, 5 1892. (b)
Schneider, H.-J.; Rudiger, V.; Cuber, U. J. Org. Chem. 1995, 60, 996. (c)
Schneider, H.-J. Recl. TraV. Chim. Pays-Bas 1993, 112, 412. (d) Abraham,
R. J.; Rowan, A. E.; Mansfield, K. E.; Smith, K. M. J. Chem. Soc., Perkin
Trans. 2 1991, 515. (e) Tachiyashiki, S.; Yamater, H. J. Chem Soc. Dalton
Trans. 1990, 13. (f) Fukazawa, Y.; Ogata, K.; Usui, S. J. Chem. Soc. 1988,
110, 8692.
Figure 9. Coordinate system and orientation parameters for NDI/DAN
interaction. (a) x and y offset. (b) NDI/DAN axis angle R.
of the association constants and chemical shift changes for NDI/
DAN complexes in water. Significantly, the upfield shifts of
the NDI aromatic protons observed in titrations of 12 and 13
are consistent with face-to-face, or parallel, complexation, as
(27) Higher order complexation, beyond 2:1, cannot be rigorously ruled
out. However, the assumption of a 2:1 + 1:1 binding model seems
reasonable where the concentration of one component is in considerable
excess through most of the titration.
(32) Note that it is not possible to assign which of the two “doublets” is
actually AA′ or BB′.

1,4,5,8-Naphthalenetetracarboxylic Diimide Systems
J. Am. Chem. Soc., Vol. 122, No. 37, 2000 8905
Figure 10. Distribution of NDI/DAN orientation parameters for molecular modeling ensembles of dimers 1-9. Left panels: x and y offset, each
circle represents the center of the DAN unit. Right panel: NDI/DAN axis angle R. Filled circles and bars represent structures within 5 kcal/mol of
the lowest energy conformer found for that compound. Open circles and bars represent structures with energies 5 kcal/mol or more above the
lowest energy conformer. The linkage is attached to the right side of the NDI outline. See Figure 9 for definition of orientation parameters.
longest linkers such as 3-5 (Figure 5e-g) have NDI signals
consistent with nearly centered DAN complexation while the
dimers with shorter and/or more rigid linkers, such as 1 and 8,
display spectra consistent with a large offset (Figure 5c,j).
The splitting of DAN signals is correspondingly influenced
by the stacking orientation in the dimers. For the monomeric
species, three pairs of equivalent protons are observed, even
when the alkoxy groups are different as in 15 (Figure 5b).
However, unsymmetrical complexation in the dimers produces
significant splittings. For example, H-6 (Figure 5c) of 1 is shifted
by about -1.0 ppm, while H-2 is only shifted by -0.3 ppm
compared to the same signals in 15. The large upfield shift
experienced by H-6 indicates that, in a significant number of
important conformers, this proton must be near the strong
shielding zone of the NDI naphthalene core while H-2 must be
Figure 11. Schematic representation of DAN offset relative to NDI.
(a) “Large” x offset. (b) “Small” x offset.

8906 J. Am. Chem. Soc., Vol. 122, No. 37, 2000
Zych and IVerson
considerably more remote. A similar, but more dramatic
example is the difference between H-4 and H-8 in compound 8
(Figure 5j). Here, H-4 is shifted by -0.4 ppm while H-8 is
shifted by nearly -1.5 ppm, suggesting that in a significant
number of conformers, H-8 must be directly above the shielding
zone of the NDI naphthalene core. Dimers 2 and 6-10 also
exhibit major splitting in one or more of their DAN proton pairs
consistent with significant stacking asymmetry. As with the NDI
aromatic signals, dimers with longer linkages, 3-5 display little
splitting indicating a higher degree of symmetry in the intramo-
lecular NDI/DAN complex.
a two state model, a large temperature increase would be
expected to help favor the unfolded state if the two states haVe
similar free energies. The lack of temperature dependence
therefore suggests that the folded/unfolded equilibrium strongly
favors the folded state.
Charge-transfer (CT) bands are also observed for all folding
dimers consistent with NDI/DAN complexation14a (Table 2).
The CT λmax varies slightly among almost all of the different
dimers, implicating some variation in the relative NDI/DAN
geometry, since orbital interactions are dependent on orientation.
Only 1 exhibits a significantly different CT λmax of about 510
nm suggestive of a unique NDI/DAN geometry. This could be
correlated with the relatively limited, and unique, NDI/DAN R
angles available with this linkage as predicted by molecular
mechanics (Figure 10).
The control compound 11 shows hypochromism at room
temperature, but at 80° C, the hypochromism decreases dramati-
cally. This degree of temperature dependence was not observed
with any of the folding dimers, 1-10. In addition, compound
11 was the only molecule exhibiting a very broadened NMR
spectra indicating extreme aggregation. The temperature de-
pendent change in hypochromism can be explained with the
reasonable assumption that intermolecular aromatic-aromatic
interactions are more easily disrupted at higher temperature for
entropic reasons compared to the intramolecular interactions
of the folding systems 1-10. In this way, compound 11 serves
as an important control that illustrates behavior expected if any
of the folding dimers did not exist primarily in the proposed
folded, compact conformations in solution. Thus, such a
dramatic change in behavior for 1-10 vs 11 can be viewed as
further evidence that 1-10 exist in largely and/or completely
folded states in solution.
The chemical shifts of the linker methylene groups also
provide an indication of NDI/DAN orientation. For example,
in compound 1, k is shifted by -0.7 ppm while j is shifted by
only about -0.1 ppm relative to the same signals in the reference
compound 15. This large difference is consistent with k being
much closer to the shielding zone of the NDI compared with j
in a significant number of important conformers. Note that this
is consistent with the ∆δ difference between DAN H-2 and H-6
since H-2 is adjacent to j and H-6 is adjacent to k. Interestingly,
the reverse order of ∆δ magnitude is observed for dimer 9,
suggesting that j and DAN H-2 must be closer in this case.
NOESY Spectroscopy. The observed long-range couplings
are consistent with all 10 of the folding dimers existing in a
compact, folded state. In particular, numerous cross-peaks were
observed for all of the folding dimers between the aromatic NDI
protons and the DAN aliphatic as well as aromatic protons. In
addition, the patterns of cross-peaks was somewhat different
for the different compounds. As with the other spectroscopic
data, the most straightforward interpretation of these findings
leads to a face-to-face folding model.
Fluorescence. If energy transfer mechanisms are ignored, the
extensive quenching of DAN fluorescence observed for all
dimers is also consistent with a predominance of folded
conformations in aqueous solution. The very weak (essentially
not observable in steady-state experiments) luminescence in
compounds 6-8, 10 is attributed to nearly complete complex-
ation of the NDI and DAN units. Complexed DAN is not
expected to exhibit luminescence since CT complexes have low
energy states available for very fast nonradiative decay.33,34
Quenching can occur by excitation of a preformed complex
(static quenching), by NDI complexation of an excited DAN
(dynamic quenching) or a combination of the two. If only
dynamic quenching is occurring, then the relationship I/Io )
τ/τo should hold true where I is the emission intensity of the
DAN in the presence of NDI, Io is the emission intensity of the
DAN in the absence of NDI, τ is the emission lifetime of the
DAN in the presence of NDI, and τo is the emission lifetime of
the DAN in the absence of NDI.35 Since I/Io >>> τ/τo is
observed, most of the quenching is attributed to complexed
DAN. Thus, even dimers exhibiting lifetimes similar to 15 and
measurable steady state emission intensities are likely to exist
in a predominantly complexed state.
Unfortunately, there are several factors that prevent an
unambiguous structure assignment for the different dimers based
solely on a quantitatiVe analysis of the observed NOE data.
First, the molecular modeling, as discussed in more detail below,
suggests that numerous nearly isoenergetic conformational states
may be available to some compounds. Thus, any observed
coupling may actually be the average of several couplings in
significantly varying conformations. In addition, the equivalence
of the NDI aromatic AA′ and BB′ protons, caused by rotation
of the NDI around the x axis complicates much of the detailed
distance information contained in any thru space couplings with
these protons (Figure 11). Finally, concentrations must be kept
relatively low to avoid significant self-association, thus increas-
ing signal/noise ratio problems. Note that having to use low
concentrations also introduced the complication of longer mixing
times, resulting in spin-diffusion concerns.
The NOEs listed are qualitatiVely useful, however, in evaluat-
ing any potential structures or ensemble of structures derived
from the predicted/observed 1H NMR chemical shifts. In other
words, if a proposed structure or ensemble of structures predicts
a particular strong NOE, it should be observable. Conversely,
there should not be an observed NOE that is not predicted by
a proposed structure or ensemble of structures.
Unfortunately, any quantitative analysis of the quenching data
is precluded by the complicating possibility of a third quenching
mechanism, resonance energy transfer. There is significant
overlap of the DAN emission with the NDI absorption.
UV-Vis Spectroscopy. Significant UV hypochromism,
compared to the isolated NDI and DAN moieties in 14 and 15,
respectively, is observed for all of the dimers (Table 2), again,
entirely consistent with face-to-face NDI/DAN complexation14a
in the folded dimers. In theory, subtle variations in the observed
degree of hypochromism could reflect differences in either the
amount of complex present or the geometry of the complexes.
Very little change is seen in this hypochromism over the range
of 25-80 °C with any of the folding dimers 1-10. Assuming
(33) Ashton, P. R.; Ballardini, R.; Balzani, V.; Boyd, S. E.; Credi, A.;
Gandolfi, M. T.; Gomez-Lopez, M.; Iqbal, S.; Philip, D.; Preece, J. A.;
Prodi, L.; Ricketts, H. G.; Stoddart, J. F.; Tolley, M. S.; Venturi, M.; White,
A. J. P.; Williams, D. J. Chem. Eur. J. 1997, 3, 152.
(34) Such quenching would be unresolvably fast in this experiment where
resolution was on the order of 50 ps.
(35) MacKenzie, R. E.; Fory, W.; McCormick, D. B. Biochemistry 1969,
8, 1839.

1,4,5,8-Naphthalenetetracarboxylic Diimide Systems
J. Am. Chem. Soc., Vol. 122, No. 37, 2000 8907
Figure 12. Low energy conformations of dimer 1 that, in combination,
Figure 13. Low energy conformations of dimer 8 that, in combination,
provide a good fit with the observed spectroscopic features.
provide a good fit with the observed spectroscopic features.
Additionally, dimers with the shortest and most rigid linkers
display the greatest degree of quenching, perhaps consistent with
energy transfer playing a dominant role in these systems.
However, note that dimer 6, which has the same linkage length
as 2 but is presumably more rigid, exhibits almost no detectable
emission in contrast to 2. The identical linkage lengths should
nearly equalize resonance energy transfer effects between the
two compounds suggesting that the observed complete quench-
ing in the more rigid dimer indicates a slightly greater degree
of complexation. Dimer 10 also has the same linkage length as
2 and presumably the same approximate magnitude of resonance
energy transfer effects. Nevertheless, 10 displays no detectable
emission, unlike 2. This could be interpreted as a greater degree
of complexation compared to 10 due to the formation of an
intramolecular salt-bridge. The red-shift in the emission of 9
compared to all other dimers is likely attributed to fewer
nonradiative decay mechanisms available to the rigidified cyclic
structure.
Detailed Analysis of Individual Dimers. By combining the
predicted information from the molecular modeling and chemi-
cal shift calculations with the experimentally observed spec-
troscopic data, a number of conclusions could be reached
regarding the detailed conformations of the different dimers.
In each case, attempts were made to fit (1) one or more low
energy structures, (2) selected groups of structures, or (3) the
entire ensemble of predicted structures to the observed spec-
troscopic data. NOE data were useful as a qualitative tool to
help make decisions about the feasibility of structural predic-
tions.
Compound 1. A combination of the two structures in Figure
12 or the entire conformational collection predict the observed
spectra equally well. It is interesting to note that the two
structures in Figure 12 correspond, in terms of the x offset, with
the two major “clusters” seen in the entire conformation
collection (Figure 10). Thus, considering only the two structures
may be a simplified way to view the entire ensemble. Overall,
the observed spectra is consistent with significant asymmetry
in the NDI/DAN complex and x offset (note the relatively large
difference in delta shift for AA′ versus BB′) of the DAN unit
away from the linkage side of the NDI. A large x offset and
limited number of NDI/DAN axis angles might explain the
observed altered CT λmax (510 nm).
Compound 2. The entire collection of conformations gives
a better match with the observed data than any single low energy
structure or combination of such conformers. The NDI/DAN
complex is rather asymmetric as seen by many differential shift
changes. However, the difference between AA′ and BB′ is
significantly less than that observed for 1, suggesting that the x
offset is somewhat diminished. This trend is also seen in the
conformational mapping (Figure 10).
Compounds 3-5. Averaging over the entire conformational
collection gave a good fit to the observed data in each case.
Here, a clear trend toward greater symmetry in the NDI/DAN
complex with increasing linkage length (Figure 5e-g) was
observed. This structural interpretation of the data is consistent
with the intuitive notion that dimers with the more flexible
linkers should be thought of as extremely dynamic structures
in which aromatic stacking is maximized in the time-averaged
structure.
Compound 6. Again, the averaged collection best satisfies
the observed data. 6 displays a shift pattern generally similar
to 2, and the calculated structures, as evidenced by the NDI/
DAN orientation parameters, are similar as well between these
two compounds. The most significant spectral difference
between 6 and 2 is that both the predicted and observed spectra
suggest a slightly more symmetrical complex of 6 compared to
2, manifested in a smaller AA′ vs BB′ difference and near
overlap of DAN H-3 and H-8. Compound 6 has the same linkage
as 2 with the exception of the ring system on the amino acid
residue R carbon of 6. Thus, it is interesting that the incorpora-
tion of the presumably rigidifying cyclopentane unit seems to
have only minor effects on the conformation of the system.
Compound 7. The averaged ensemble of structures again
gave the best match with the observed data. Overall, the degree
of asymmetry and x offset are on the order of that observed for
2 or 6.
Compound 8. In this instance a 1:1 combination of two low
energy conformers provides the best prediction of the observed
data (Figure 13). Note that both types of conformers have the
cyclic linkage element in a presumably low energy chair
conformation and that the average x offset of the two is
substantial, in agreement with the very large shift difference
seen between AA′ and BB′.

8908 J. Am. Chem. Soc., Vol. 122, No. 37, 2000
Zych and IVerson
Compound 9. The lack of observable long-range through-
space couplings, at concentrations where aggregation is minimal,
hinders the analysis of this structure. However, chemical shift
changes can still provide considerable information about major
structural features. Interestingly, the aromatic region of 9 closely
resembles that of 1, except for the reversal of order for former
equivalent pairs (in descending ppm: dimer 1; H-8, H-3, H-4,
H-7, H-2, H-6, dimer 9; H-4, H-7, H-8, H-3, H-6, H-2). Also,
both compounds show major shift changes for the O-CH2-
CH2- methylenes of the DAN unit. However, for 1 signals, k
and l are shifted downfield, while for 9 it is j and i that are
most affected. All of this suggests a significantly offset structure,
but in the opposite direction as that proposed for 1. This may
seem surprising since both linkages of 9 are of equal length
and constitution compared with 1. However, they do have the
opposite N to C directionality, which apparently must account
for the observed asymmetry. Single structures, combinations,
and the entire ensemble all gave a poor reproduction of the
observed data. The reasons for this failure are unclear. It is
possible that the “folded” conformational space of this com-
pound was not adequately sampled due to its rigid, restrained
nature. Alternatively, the particular conformations of this
compound may fortuitously exaggerate inaccuracies in the
functions used to simulate the shift changes.
systems. In general, longer linkages allowed for more sym-
metrical face-to-face aromatic stacking, while shorter or more
rigid linkages caused greater asymmetry/offset of the stacked
complex. The equilibrium between unfolded/folded states may
be driven toward folding by more rigid linkages, but even the
longest and most flexible linkages apparently produced a clear
predominance of folded conformations based on all of the
spectroscopic data. This latter point was emphasized by the
observed high level of aggregation and unique temperature-
dependent spectra of the control compound 11, a molecule that
is geometrically prevented from folding. Any of the dimers (1-
10) in the study that were not predominantly folded in solution
would have been expected to mimic more the observed behavior
of 11.
The characterization of solution conformation represents a
key element of foldamer research. The preceding analysis has
demonstrated that the powerful spectroscopic handles associated
with the stacking of aromatic units in folded aedamers can be
exploited to obtain a dynamic, working model of folded structure
in aqueous solution. The analysis described in this study can
therefore be considered a blueprint for the design and structural
characterization of more elaborate, and ultimately functional,
aedamers and related foldamers.
Experimental Section
Compound 10. A conformational analysis was not carried
out for this compound since it has the same linkage as dimer 2.
Nonetheless, it is interesting to examine its spectra compared
to compound 2 (Figure 5d,l). Compound 10 was synthesized
with a free N-terminus to allow for the possibility of noncovalent
cyclization thru salt-bridge formation. No direct evidence of
such cyclization was observed. The compounds exhibited very
similar NMR and UV spectra. There was some difference in
the observed fluorescence; 10 displayed essentially complete
quenching in steady-state experiments, while 2 showed a small
residual emission. Also, 10 had a significantly shortened
fluorescence lifetime compared to 2. All this suggests that 10
has a distribution of conformers very similar to 2 in solution,
but may have an equilibrium that favors folded states to a
somewhat greater extent.
General Methods. Commercially available reagents and solvents
were used without further purification unless noted otherwise. DMA
and DMF for peptide couplings were stored under dry argon with 4 Å
molecular sieves. Organic solvent solutions were dried over Na2SO4
and evaporated on a rotary evaporator under reduced pressure. Flash
chromatography was performed using 230-400 mesh silica gel. All
aqueous solutions were prepared in 50 mM sodium phosphate buffer
(pH ∼7).
Spectroscopy. (NMR) Routine 1-D spectra were recorded on a
Varian UNITY+ 300s. All other spectra were recorded on a Varian
INOVA 500. Chemical shifts are reported in ppm relative to tetram-
ethylsilane (TMS) for organic solvents and relative to (CH3)3SiCD2-
CD2CO2Na (TSP) in aqueous solution. Mixing times for NOE
experiments were 500 milliseconds. Concentrations for NOE experi-
ments were kept between 1.5 and 2.0 mM. UV-vis spectra were
recorded on a Hewlett-Packard 8452A diode array spectrophotometer.
Steady-state fluorescence measurements were performed on a Perkin-
Elmer LS50 spectrofluorimeter. Emission intensities were corrected for
differences in absorbance at the excitation wavelength (296 nm) to give
relative quantum yields. Fluorescence lifetimes were measured by time-
correlated single-photon counting using a mode-locked, synchronously
pumped, cavity-dumped dye laser. The excitation wavelength used was
296 nm while the emission was monitored at a variety of wavelengths
between 320 and 420 nm. Decay profiles were fit to a single
exponential.
Conclusions
All 10 of the folding dimers, 1-10, exhibited spectroscopic
properties fully consistent with a predominance of folded
structures in solution, although the exact orientation of the NDI/
DAN complex was linkage dependent. A simple interpretation
of observed spectra did not lead to realistic structural prediction.
Rather, molecular modeling, including chemical shift simulation,
combined with the spectroscopy provided an approach to
characterizing solution structure. In most instances, the observed
data were best explained by a large ensemble of structures as
opposed to one or few conformers. Thus, in the case of these
aedamer dimers, “folding” does not appear to imply a two-state
model with a rigid, uniquely folded structure, but rather a
dynamic situation in which the molecule spends the majority
of its time in different folded conformations. These folded
conformations are far from random for a given molecule,
however, in that they share the key structural feature of having
the aromatic units stacked in a face-to-face orientation.
NMR Titrations. 16 D2O solutions were prepared with 12 kept at
a constant concentration of 0.20 mM and 13 varying from 0.60 mM to
62.27 mM. The ∆δ of the aromatic protons of 12 was calculated by
subtracting from the δ of 12 only at 0.20 mM (8.75 ppm). For the
assumption of concurrent 1:1 and 2:1 complexation the binding isotherm
was fit to eq 118
where K1 is the equilibrium constant for the formation of 12-13, K2 is
the equilibrium constant for the formation of 13-12-13, ∆δ1 is the
shift of the 12-13 complex, ∆δ2 is the shift of the 13-12-13 complex,
and [13]o is the total concentration of 13. The data were fit using
Kaleidagraph 3.0.536 with the restraint ∆δ2 ) 2∆δ1.
The robustness of the aromatic stacking motif in water has
been demonstrated by extensive modification of the aedamer
linkage without disruption of the overall folded structure. Such
behavior in aqueous solution is particularly significant since it
will allow for consideration of interactions with biological
(36) Synergy Software, 1994, 2457 Perkiomen Ave, Reading, PA 19606.

1,4,5,8-Naphthalenetetracarboxylic Diimide Systems
J. Am. Chem. Soc., Vol. 122, No. 37, 2000 8909
Molecular Modeling. All modeling was performed in HyperChem37
using the MM+ force field. Programming was developed in Excel38
4.0 Macro Language to automate annealing/optimatization opera-
tions in HyperChem and to return data (atomic coordinates, energies,
etc.) to Excel spreadsheets for calculations. Structures were truncated
after methylenes k and c (j and d for compound 1) to make the
calculations more expedient. Conformer collections: For each dimer
examined, 10 random starting structures were each subjected to 10
ps of unrestrained molecular dynamics at 1000 K with snapshots taken
every ps to give 100 structures for annealing. A weak distance restraint
was then applied between the centers of the NDI and DAN units while
the simulation temperature was lowered to 300 K over 5 ps. The
structures were then refined by unrestrained geometry optimization
(Fletcher-Reeves conjugate gradient, 0.01 kcal/mol). Chemical shift
simulation: The chemical shift change produced by each “benzene”
ring of the DAN and NDI units was approximated by eq 2.24a The
chemical shift change induced by each carbonyl of the NDI unit was
approximated by eq 3.24b The total ∆δ for each NDI proton under
consideration (aromatic, c, and d) was calculated as the sum of the
shifts induced by each of the two DAN “benzene” rings (eq 4). The
total ∆δ for each DAN proton under consideration (aromatic, j, k,and
l) was calculated as the sum of the shifts induced by each of the two
NDI “benzene” rings and each of the four NDI carbonyls (eq 5). For
methylenes, the averaged position of the two protons was used for all
equations.
∆δcarbonyl ) 9.13(Ga) - 3.08(Gb)
(3)
3
Ga ) (1-3 cos2 θ)/Ra
3
Gb ) (1-3 cos2 ω)/Rb
∆δNDI ) ∆DAN Ar ring1 + ∆DAN Ar ring2
(4)
∆δDAN ) ∆NDI Ar ring1 + ∆NDI Ar ring2 + ∆NDI CO1 + ∆NDI CO2
+
∆
NDI CO3 + ∆NDI CO4 (5)
Acknowledgment. We thank the Robert A. Welch Founda-
tion (F-1188), the National Institutes of Health (R01 GM55646),
and the Alfred P. Sloan Foundation (BR-3583) for financial
support of this work. We would also like to thank Mark S.
Cubberley for his review of the manuscript, Steve Sorey for
2-D NMR experiments, Don O’Connor for time-resolved
fluorescence measurements, and Vladimir Guelev for many
lively discussions.
∆δAr ring ) 27.6(1-3 cos2 θ*)/r3
(2)
Supporting Information Available: Synthetic schemes and
full experimental for 1-15, tables of NOE data, and additional
molecular modeling data. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA0019225
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