Báo cáo khoa học: Well-defined secondary structures Information-storing molecular duplexes and helical foldamers based on unnatural peptide backbones pot

REVIEW ARTICLE
Well-defined secondary structures
Information-storing molecular duplexes and helical foldamers based on unnatural
peptide backbones
Adam R. Sanford, Kazuhiro Yamato, Xiaowu Yang, Lihua Yuan, Yaohua Han and Bing Gong
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY, USA
Molecules and assemblies of molecules with well-defined
secondary structures have been designed and characterized
by controlling noncovalent interactions. By specifying
intermolecular interactions, a class of information-storing
molecular duplexes have been successfully developed. These
H-bonded molecular duplexes demonstrate programmable,
sequence-specificity and predictable, tunable stabilities.
Based on these highly specific molecular zippers (or glues),
a systematic approach to designing self-assembled structures
is now feasible. Duplex-directed formation of b-sheets,
block copolymers and templated organic reactions have
been realized. By specifying intramolecular noncovalent
interactions, a backbone-rigidification strategy has been
established, leading to unnatural molecular strands that
adopt well-defined, crescent or helical conformations.
The generality of this backbone-rigidification strategy has
been demonstrated in three different classes of unnatural
oligomers: oligoaramides, oligoureas and oligo(phenylene
ethynylenes). Large nanosized cavities have been created
based on the folding of these helical foldamers. Tuning the
size of the nanocavities has been achieved without changing
the underlying helical topology. These helical foldamers can
serve as novel platforms for the systematic design of nano-
structures.
Keywords: backbone rigidification; duplex; foldamer;

folding; helix; hydrogen bond; nanocavity; noncovalent;
self-assembly; template.
Introduction
The assembly and folding of biomolecules are arguably two
of the most important features in nature. There is no doubt
that without the ability of nature to form very stable
aggregates of small molecules, or to form well-defined
secondary, tertiary and even quaternary structures of
macromolecules, life could not exist as we know it. For
example, the formation of duplex DNA represents one of
the most elegant and best known examples of both self-
assembly and folding of biomacromolecules [1]. The folding
of polypeptide chains into secondary and eventually a
bewildering array of tertiary structures results in protein
molecules that are responsible for most of the biological
interactions and functions found in nature.
A logical next step is to mimic nature and create
nonbiologically derived molecules that either fold into
well-defined secondary structures, or assemble into larger
architectures. Since the early 1990s there has been a great
deal of literature devoted to these types of biomimetic
structures that involve intermolecular self-assembly and/or
intramolecular folding of unnatural molecules, and their
potential applications [2–15]. The focus of a growing
number of research groups including our own is to achieve
structures whose properties may lead to multitudes of
applications both in and out of the biological realm.
The development of most of the unnatural assembling and
folding structures has been inspired by peptide and protein
structures. For example, the pioneering studies of Gellman

and Seebach on b-peptides, a class of unnatural peptidomi-
mimetic folding oligomers, have demonstrated the feasibility
of folding unnatural oligomers into well-defined conforma-
tions. Extending the concept of b-peptideshasledtoother
peptidomimetic foldamers such c-peptides [16,17], d-pep-
tides [18,19] and oxa-peptides [20]. The early investigation on
molecular association of nucleobases by Jorgensen and
Zimmerman has led to the development of highly specific
H-bonded pairs that have been used in specifying inter-
molecular interactions [21–23]. Although there has been a
great deal of work in both molecular self-assembly and
folding, the focus of this review primarily covers the work
completed and ongoing in our laboratory. For a more com-
prehensive view of these two fields there have been a number
of excellent reviews published in recent years concerning
molecular self-assembly and folding [2–15,25–29,39].
Hydrogen-bonded duplexes
Nature has, of course, a monopoly on the most complex
self-assemblies of molecules. In nature, the cooperative
action of many noncovalent attractions often leads to highly
specific recognition events, resulting in thermodynamically
stable assemblies. The unfavorable loss in entropy is usually
Correspondence to B. Gong, Department of Chemistry, 811 Natural
Sciences Complex, University at Buffalo, State University of New
York, Buffalo, NY 14260, USA.
Fax: + 1 716 6456963, Tel.: + 1 716 6456800 ext 2243,
E-mail: ffalo.edu
Abbreviations: A, acceptor; D, donor; m-PE, oligo(meta-phenylene
ethynylene).
(Received 17 December 2003, revised 6 February 2004,

accepted 2 March 2004)
Eur. J. Biochem. 271, 1416–1425 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04062.x
offset by the large numbers of cooperative enthalpic
interactions within the assembled structures, such as in
duplex DNA.
Much effort has been expended recently to produce self-
assembling molecular assemblies between two or more sep-
arate molecules. Inspired by nucleic acids, a powerful
approach to self-assembly involves the design of recognition
units (modules) that store and retrieve information in a
digital fashion[2]. Modules withhigh specificity and strength,
when tethered to various structural components, can serve as
information carriers for instructing the formation of a variety
of self-assembling structures. Compared to systems whose
assembly depends directly on the structural features of the
corresponding molecular components, such an approach
is more versatile because structural units that are either
incompatible or only randomly associate with one another
can now be forced to assemble. The corresponding modules
thus act as templates fororganizingand assembling structural
components in specific sequential and spatial arrangements.
A great majority of themodules have usedthe hydrogen bond
as the primary stabilizing force due to the predictable
directionality and strength of this noncovalent interaction.
During the last decade, hydrogen-bonded complexes
based on rigid heterocycles with multiple H-bonding donor
(D) and acceptor (A) sites have received the most attention
as recognition modules [2]. Systems based on various
H-bonded modules have been reported. For example,
Whitesides et al. described multicomponent structures

based on the cyanuric acid–melamine motif [24–26]. The
groups of Zimmerman [30–32] and Meijer [33–35] reported
heterocyclic complexes with arrays of H-bond donors
and acceptors. Krische [36] and Hunter [37] developed
H-bonded duplexes. Ghadiri et al. [38,39] reported the
assembly of modified cyclic peptides utilizing eight hydro-
gen-bonds while Rebek et al. reported on the assembly of
curved monomers that assembled into a form reminiscent of
a tennis ball [40,41].
In spite of their increasingly wide applications, the use of
heterocycle-based modules may suffer from several compli-
cations. Most heterocyclic systems are accompanied by
tautomerism [30], resulting in loss of information and
reduction of the observed association constants. In addition,
one must consider that in heterocycle-based modules,
secondary electrostatic interactions, due to the proximity
of adjacent H-bond donors and acceptors, often complicate
the predictability of the strength of the designed complexes.
We have developed our own approach for designing
molecular recognition modules using molecular duplexes
that are not only sequence-specific, but also with program-
mable sequences and tunable stabilities [3].
Molecular duplexes: programmable, information-storing
molecules
We recently developed a class of highly stable molecular
duplexes that are characterized by their by sequence-
specificity and tunable stability: one single strand carrying
an arrangement (sequence) of H-bond donors and acceptors
specifically recognize another strand with a corresponding
complementary H-bonding sequence (Fig. 1) [3]. These

duplexes adopt an extended, tape-like conformation. The
H-bonding sequences of these duplexes are readily
programmable. Single strands with almost any donor–
acceptor arrangement can be designed and prepared based
on straightforward amide/peptide chemistry. These
duplexes also showed regio-specificity and can thus act as
molecular manipulators.
Specifically, the molecular strands involve oligoamides
consisting of meta-substituted benzene rings linked by
glycine residues. The amide O and H atoms act as H-bond
donor and acceptor sites. Various arrangements of the
amide O and H atoms lead to Ôhydrogen bonding sequencesÕ
that allow the specific association of two single strands
carrying complementary seqences [3].
The structure of a six-H-bonded duplex is shown in Fig. 2
to illustrate the specific design: the duplex consists of two
identical oligoamide strands carrying self-complementary
H-bonding arrays of AADADD. Connecting the aromatic
building blocks with glycine linkers in different order leads
to oligoamide strands carrying different arrays (sequences)
of H-bond donors and acceptors. As a result, duplexes with
both self-complementary and complementary H-bonding
sequences have been designed and characterized. Our study
has demonstrated that these molecular duplexes are char-
acterized by predictable, tunable affinity, programmable
sequence-specificity and convenient synthetic availability,
making them ideal as recognition modules for the instructed
assembly of various structural units.
Four H-bonded duplexes [3,42]. The first generation
duplexes were designed around a four-H-bond platform

with self-complementary H-bonding sequences of DDAA
and DADA (Fig. 3). Of note are the six-membered inter-
molecular hydrogen bonds that not only serve to block any
undesirable intermolecular H-bond interactions, but also
force a molecular conformation conducive to dimerization.
Dimerization for both complexes was studied and
confirmed through 1D, 2D and variable temperature
Fig. 1. A schematic illustration of a H-bonded molecular duplex
(molecular zipper).
Fig. 2. A six-H-bonded, self-complementary duplex.
Ó FEBS 2004 Well-defined secondary structures of unnatural oligomers (Eur. J. Biochem. 271) 1417
NMR experiments, and vapor pressure osmometry studies.
Association constants between these homodimers were
determined based on concentration-dependent chemical
shifts of the amide protons. The dimerization constant for
the DADA homodimer was found to be > 4.4 · 10
4
M
)1
while the dimerization constant for the DDAA dimer was
found to be  6.5 · 10
4
M
)1
.
The amino acid linker showed very little effect on the
strength of the dimerization as a whole. For example, when
the glycine used in initial tests was replaced with a bulkier
phenylalanine, the dimerization constant was found to be
 4 · 10

4
M
)1
, within the ±10% error associated with the
NMR method used in determining the constant. All three
types of compounds, regardless of D–A sequence and
amino acid spacer, exhibit very similar association constants
and can be considered to have roughly the same stabilities.
This is in contrast to H-bonded complexes based on rigid
heterocycles, whose stabilities are influenced by secondary
electrostatic interactions and are thus not only determined
by the number of H-bonds, but also depend on the specific
arrangement of the H-bond donors and acceptors. The
sequence-independent stabilities of our duplexes are easily
explained by the larger distance (>5 A
˚
) between their
adjacent intermolecular H-bonds than those (<2.3 A
˚
)of
the H-bonded pairs of rigid heterocycles. Thus, by adjusting
the number of intermolecular H-bonds, the stability of a
duplex can be controlled accordingly.
For example, compared to the four-H-bonded duplexes,
the dimerization constant of a two-H-bonding molecule was
found to be dramatically lower ( 25
M
)1
) [42]. Thus, if the
number of H-bond D/A sites is increased, the overall

stability of the corresponding duplex should also increase.
This led to the investigation of even longer, more complex
duplexes. The investigations also focussed on the creation of
duplexes of consisting of two different but complementary
strands.
Six H-bonded Duplexes [3,43]. Thenextstepinthe
course of investigation involved six-H-bonded molecular
duplexes. Initially, compounds 1 and 2 were designed and
synthesized as complementary pairs with only one allowable
mode of dimerization (Fig. 4). Initial indications of dime-
rization were apparent as the solubilities of separate
solutions of 1 (< 1 m
M
)and2 ( 10 m
M
) were low but
that of the 1 : 1 mixture of the two in the same solvent was
much higher ( >>100 m
M
). This phenomenon presuma-
bly was due to the shielding of the highly polar amide
groups on the Ôsticky edgeÕ of each single strand.
The formation of the six-H-bonded duplex was conclu-
sively confirmed by 2D NMR data showing critical
interstrand NOEs in chloroform. In addition, the duplex
could even be detected by straight phase (SiO
2
) TLC.
Using 10% dimethylformamide in chloroform as the
eluant, differences in R

f
values between 1 (R
f
¼ 0.00),
2 (R
f
¼ 0.10) and the 1 : 1 mixture (R
f
¼ 0.96) clearly
indicated the formation and high stability of the duplex.
Further NMR studies showed significant downfield shifts
of the amide protons of the 1 : 1 mixture of 1 and 2
in comparison to the separate solutions of the single strands.
Attempts to determine the dimerization constant by NMR
failed as no upfield shift of the amide protons was detected
upon dilution in CHCl
3
to a concentration as low as 1 l
M
.
Isothermal titration calorimetry was employed to deter-
mine the association constant via titration of 1 with 2
in chloroform. The result can only be estimated to
be ¼ 1 · 10
9
M
)1
in chloroform, in agreement with NMR
results. A more accurate value of K
a

was determined when
the titration was carried out in chloroform containing 5%
dimethyl sulfoxide, resulting in an association constant of
3.5 · 10
6
M
)1
.
In addition to the above complementary pairs of
duplexes, a self-complementary, six-H-bonded duplex were
also studied (see Fig. 2). Again, as with other six-H-bonded
duplex, NMR experiments failed to determine an associ-
ation constant due to the high strength of the association.
By assuming a 10% dissociation, a lower limit of
4.5 · 10
7
M
)1
wasestimatedfortheK
a
. Interstrand NOEs
were observed under a wide range of concentration
conditions, indicating little dissociation even in highly
dilute solution. Simple observation on a TLC plate indica-
ted even greater stability than that of a similar non-self-
complementary six-H-bond duplex. Virtually no tailing,
indicative of dissociation, was observed on the TLC
medium for the self-complementary duplex while the non-
self-complementary strand showed significant tailing. To
determine a more accurate association constant, a pyrene-

labeled derivative of this self-complementary system was
studied by a fluorescence method. The dimerization con-
stant was determined to be (6.8 ± 4.1) · 10
9
M
)1
. Such a
strong stability and the feature of self-complementarity
gives this compound an interesting potential for the
synthesis of supramolecular, high molecular mass homo-
polymers [44].
The sequence-specificity of the six-H-bond duplex was
probed with the inclusion of mismatched binding sites along
the backbone of a duplex (Fig. 5) [45]. This study provided a
unique look at the importance of sequence-specificity on
these unnatural self-assembly systems. The mismatched
pairs were studied with 1D and 2D NMR and isothermal
titration calorimetry. The results were compared to the
Fig. 3. Self-complmentary, four-H-bonded duplexes.
Fig. 4. Example of six-H-bond, complementary duplex 1•2.
1418 A. R. Sanford et al. (Eur. J. Biochem. 271) Ó FEBS 2004
parent complementary duplex. The ÔmismatchedÕ strands
still assemble as do the ÔmatchedÕ strands, but with much less
stability. Isothermal titration calorimetry experiments
revealed that the mismatched duplexes exhibited stabilities
40 times less than that of the corresponding matched pairs.
Duplex foldamers [46]
Progress with our four- and six-H-bonding duplexes led to
the development of a new class of compounds with eight
H-bonding sites. Single strands 4 and 5, one with two

DDAD modules and the other with two AADA modules
linked in a head-on fashion, were originally designed to
form supramolecular polymers. Similarly, a self-comple-
mentary strand consisting of two ADAD modules linked
head-to-head was also designed. If these strands adopted an
extended conformation, the directionality of the unsym-
metrical four-H-bond module would enforce a partial
overlap of the molecular strands, leading to the formation
of supramolecular polymers. Instead, it was discovered that
each eight-H-bonding strand associated with one separate
complementary strand; however, like many biomacromole-
cules, the molecular strands adopted a well-defined, folded
conformation (Fig. 6).
Noncovalent, templated formation of b-sheets [3,47]
The duplexes described above can be viewed as mimics of
two-stranded b-sheets consisting of extended b-strand
mimetics. The interstrand distance in these duplexes should
be nearly the same ( 5A
˚
) as that found in b-sheets as both
involve backbone amide groups in forming interstrand
H-bonds. The duplexes, when tethered to natural peptide
strands, may serve as templates to bring the peptide strands
into close proximity. In addition, the programmable
sequence-specificity of the duplexes allows the design of
unsymmetrical H-bonding sequences, which ensures the
precise registration of the amino acid residues of the
attached natural peptide strands, leading to the formation
of two-stranded b-sheets. Therefore, the organizational
stability of the H-bonded duplexes provided an opportunity

for nucleating and stabilizing b-sheets, a feature that could
help provide critical insight into such structures. As opposed
to studies on b-sheets based on b-hairpins, the nature of
such an assembly is intermolecular, making it possible to
pair peptides of various lengths and sequences by simply
mixing the corresponding templated peptides (Fig. 7). The
resultant mixing of complementary hybrid duplex strands
would force the otherwise flexible peptide chains to form
b-sheets.
To evaluate the strategy of nucleation and stabilization of
b-sheets, we designed four hybrid chains, each consisting
of a tripeptide segment coupled with a region capable of
forming a four-H-bonded duplex (Fig. 8). The H-bonding
sequence of the duplex was designed to be unsymmetrical to
direct the peptide chains to the same (or different) end of
the template. Hybrids 6a and 6b were paired with hybrids 7a
and 7b.
As a control, hybrid 6c was designed. Pairing 6c to the
corresponding hybrid strands led to the attachment of the
tripeptide chains to the opposite end of the duplex template.
H
1
NMR results of duplexes of 6a with 7a and 7b,aswell
as 6b with 7a and 7b exhibited very sharp, well-defined
resonances indicative of well-defined overall structures.
Mixtures of 6c with its corresponding partner showed no
such resolution, exhibiting broad, nearly indistinguishable
resonances. Also observed was the noticeable chemical shift
of the H
1

NMR resonances in only the complementary
hybrid strands in comparison to single hybrid strands and
the tripeptide strands alone.
Two-dimensional NMR (NOESY) studies provided
conclusive evidence of duplex and b-sheet formation.
NOE contacts were observed between opposite amino acid
residues. In the case of ‘plain’ tripeptide mixtures or
duplexes with ‘wrongly’ attached tripeptides, no NOE
contacts were observed.
Fig. 5. An example of a duplex containing a mismatched ‘binding’ site.
Fig. 6. Single strands 4 and 5. Each strand consisting of two identical
four-H-bonding halves linked in a head-on fashion, were originally
designed to (A) form supramolecular polymer, but were found to (B)
adopt folded (stacked) conformations upon associating with each
other.
Fig. 7. Schematic representation of b-sheet nucleation and stabilization
by a four H-bond duplex template.
Ó FEBS 2004 Well-defined secondary structures of unnatural oligomers (Eur. J. Biochem. 271) 1419
Further investigations based on H-bonded duplexes
Supramolecular block copolymers. The extraordinary spe-
cificity and stability of our H-bonded duplex is again
demonstrated recently in the design of supramolecular
block copolymers. By attaching three polystyrene and three
poly(ethylene glycol) chains to the two strands of a duplex
derived from 1 and 2, a total of nine block coploymers were
created by simply mixing the polymer-tethered templates
(Fig. 9). NOESY study confirmed that the duplex template
was precisely matched as expected from the H-bonding
sequences. The successful noncovalent linking of the
polystyrene and poly(ethylene glycol) chains was confirmed

by size exclusion chromatography. More exciting were the
results from the atomic force microscopy that revealed
microphase separation typical of covalent block copolymers
by these supramolecular block copolymers. Figure 9B
shows one such atomic force microscopy image of a pair
carrying polystyrene 21 000 and poly(ethylene glycol) 6000
chains.
A duplex-templated organic reaction
Olefin metathesis reactions have found applications in a
wide variety of fields [48,49]. Intermolecular olefin meta-
thesis involving two different olefins can be complicated by
the fact that a mixture of three products can result when the
two reacting olefins have similar stabilities. The use of our
duplex strands can solve this problem. By tethering two
separate olefins on two complementary strands, the olefins
Fig. 8. Hybrid duplex strands.
Fig. 9. Block copolymers. (A) Design of supramolecular block copolymers based on the six-H-bonded hetero-duplex 1•2. Mixing three templated
polystyrene chains with three complementarily templated poly(ethylene glycol) chains leads to nine block copolymers. (B) The AFM image showing
microphase separation of one of the supramolecular block copolymers.
1420 A. R. Sanford et al. (Eur. J. Biochem. 271) Ó FEBS 2004
were brought into close proximity by the pairing of the
duplex strands. Metathesis reactions have been carried out
between two olefins at concentrations (1 or 2 m
M
)thatare
otherwise too low for intermolecular metathesis reactions
to occur. As a result, only the desired, hetero-crosslinked
products were formed in high (>90%) yields [40].
Intramolecular self-assembly: helical
foldamers

In recent years there has been intense interest in creating
oligomers and/or polymers with unnatural backbones that
display stable, well-defined conformations. Pioneering
reports from Gellman [7,50] and Seebach [51,52] on
helically folding b-peptides opened the floodgates for a
rush of reports on other unnatural helical structures.
From the c-peptides reported by Hanessian [53] and
Seebach to the oligo(pyridine dicarboxamides) reported
by Huc and Lehn [54] to the folding oligo(m-phenylene
ethynylenes) reported by Moore [55,56] and the helical
aromatic oligoureas by Tanatani [57], folding structures
are obviously not monopolized by nature itself. We have
reported novel helical foldamers based on the enforced
folding of oligoarylamides and oligo(phenylene ethyny-
lenes) that exhibit well defined helical secondary structures
that have great potential in both materials science and
biological application.
Oligoamide foldamers
Our approach to the design of helical foldamers involves
the development of oligoarylamides with rigid, crescent
backbones [58,59]. These oligoamides can also be viewed
as aromatic c-peptides. The initial focus was placed on
oligomers consisting of building blocks derived from
2,4-dihydroxy-5-nitrobenzoic acid (or meta-disubstituted
building block). When derivatives of this molecule were
connected via an amide bond, it was reasoned that the
bifurcated (three-centered) H-bond, consisting of the two
intramolecularly H-bonded five and six membered rings,
would limit the rotational freedom of the aryl-amide bonds.
The three-centered H-bonds rigidify the overall backbone

and thus force a crescent shape, for example, on the
tetramer shown in Fig. 10. The resulting three-centered
hydrogen bond was found to be highly stable from both
theoretical and experimental evidence [59].
The crescent and/or helical conformations should be
reinforced by the interplay of multiple factors such as the
rigidity of the benzene ring and amide groups, the propen-
sity of the amide bonds to adopt the trans conformation,
and eventually (as chain length increased to over one turn)
favorable p–p stacking interactions between overlapping
phenyl rings. This indeed was the case as indicated by 2D
NMR and X-ray crystallographic data from oligomers
consisting of two, three, four, five and six residues. The
structure proved itself to be very stable in both the solid
state and in solution. From this data it is clear that the
orientation of the amide oxygens yielded an interior cavity
that is electronegative and hydrophilic. Once the conforma-
tion of the amide backbone was confirmed, the extension of
the backbone beyond the length of a single turn, and thus a
helix was attempted [60].
Indeed, combining two tetramers with a symmetrical
residue derived from 4,6-dihydroxyisophthalic acid, resulted
in nonamers of more than one full helical turn [60]. Side
chains, however, were found to play a critical role in the
solubility of the corresponding oligomers. It was found that
oligomers carrying short side chains such as methyl or
isopropyl groups had rather limited solubility. Long alkyl
chains, such as the linear octyl or dodecyl groups, were
adopted to impart solubility. However, it was discovered
that when side chains longer than a methyl group were

placed on adjacent residues of an oligomer, this led to a
distortion/twisting of the helix, primarily from steric inter-
actions between side chains. This problem was solved by
designing oligomers containing methyl side chains on every
other residue. Recently, a building block carrying one long
alkyl and one methyl side chain became available, making
it possible to construct oligomers using a single building
block.
The folded structures were rigorously characterized by
both 2D and X-ray crystallography. The helices show
excellent stability in organic solvents and our recent results
show that folding also occurred in very polar (and hydrogen
bond disrupting) solvents such as water and dimethyl
sulfoxide. The persistence of the highly favorable three-
centered H-bonds, which act to rigidify the backbone and
lead to the overall folded conformations of the oligoamides,
was further demonstrated by extremely slow amide proton–
deuterium exchange rates.
While in nature, large cavities of nanometer scale are
usually found at the tertiary or quarternary structural level
of proteins, we have been able to create and tune the
nanocavities while maintaining the same helical topology of
our foldamers [60]. One of the highly attractive features of
oursystemistherelativeeaseinwhichtheinteriorcavity
Fig. 10. A tetrameric crescent oligoamide (A) and its crystal structure (B). For clarity, the octyl groups are replaced with green dummy atoms in the
crystal structure.
Ó FEBS 2004 Well-defined secondary structures of unnatural oligomers (Eur. J. Biochem. 271) 1421
can be altered. It was initially envisioned that by merely
incorporating a certain proportion of residues derived from
2,3-hydroxy-4-nitrobenzoic acid (or simply para-disubsti-

tuted building block) into the oligomer, the curvature of the
corresponding backbone would be decreased, leading to an
increase in the size of the interior cavity (Fig. 11). For
example, a 21-mer (Fig. 12) consisting of alternating meta-
and para-building blocks was found to fold into a helical
conformation of slightly more than one spiral turn. The
existence of a helical conformation was supported by end-
to-end NOEs and by NOEs between each amide proton
and the protons on two of its neighboring side chains.
A computer model of the 21-mer constructed based on
parameters from the X-ray structures of short oligomers of
the same system revealed a helix with an interior cavity of
over 30 A
˚
across, the largest thus far created by unnatural
foldamers [60].
Extension of this porous foldamer system beyond our
original explorations is at the forefront of our current focus.
As indicated by NMR and X-ray crystallographic data, the
interior channel lined by amide oxygens has potential for
application. Short oligomers may act as membrane-bound
carriers for ions and small molecules; longer oligomers may
fold into nanotubes with hydrophilic channels. Oligomers
with lengths matching the thickness of the lipid bilayer could
act as channels for transporting ions and small molecules
(Fig. 13). The side chains, which point radially outward on
the exterior of a helix, should help the integration of our
porous foldamers into lipid bilayers.
Probing the stability of our backbone-rigidified helical
foldamers in polar solvents will be one of our future

studies. Oligomers that fold stably in aqueous media are
of great interest due to their biological significance and
their potential for the development a variety of bio-related
materials. The incorporation of triethylene glycol side
chains and aliphatic chains terminated with carboxyl
groups are the strategies we intend to use to render the
corresponding oligomers reasonably soluble in aqueous
media.
If an oligoamide backbone could fold into well-defined
and robust conformation based on backbone rigidification,
could the same strategy of backbone rigidification be
applied to different unnatural backbones? Initial work in
our laboratory involving oligoureas have shown promise.
Fig. 11. Adjusting cavity size by tuning the curvature of a backbone.
Fig. 12. A 21mer (approximately half is shown) with an interior cavity
of > 30 A
˚
.
Fig. 13. Schematic representation of ion channel based on our porous
helix.
1422 A. R. Sanford et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Folding oligoureas
The backbones of this class of oligomers involve benzene
rings linked by N,N¢-disubstituted urea groups (Fig. 14).
The presence of ester groups ortho to the urea N atoms leads
to the formation of a intramolecularly H-bonded, six-
memberd ring, which, combined with the preferred (cis, cis)
conformation of the urea group, leads to the rigidification of
the oligourea backbone. The meta-disubstituted benzene
rings, in combination with the nonlinear urea groups,

enforce a curved conformation. Molecular modeling studies
showed that, for oligomers with more than four residues, a
helical conformation results. (Fig. 14A) The interior of the
helix is characterized by oxygen atoms from the urea
functionality, which lead to hydrophilic cavities of  4A
˚
across. Urea linkages have the advantage of being chem-
ically robust and resistant to most natural enzymes.
In contrast to the large cavity of the crescent oligoamides
mentioned above, the much smaller sizes of the neutral,
electrostatically negative cavities of the oligoureas are ideal
for binding and/or transporting ions. Our ultimate goal is to
develop these foldamers into novel ion carriers, and
eventually, into ion channels. Oligoureas from the dimer
to the hexamer have been prepared. Extensive studies based
on computational, 1D and 2D NMR studies have con-
firmed the design principle. Our latest preliminary studies on
metal ion binding have identified a tetramer that selectively
binds sodium ions. During the revision of this manuscript,
a paper published by Meijer, et al. came to our attention
that reports helical polyureas based on design principles
very similar to those described here [61].
As shown in Fig. 14B, replacing one of the ester group on
a residue with an ether group results in five-membered
hydrogen-bonded rings. The backbone of the corresponding
oligomer should be more flexible due to the weaker H-bond
in the five-membered ring.
The driving forces that facilitate the folding of these
oligoureas are virtually identical to those for the folding of
oligoamides mentioned above: (a) two localized intramole-

cular hydrogen bonds that lead to the rigidification of each
of the cis, cis-urea linkages and (b) the aromatic stacking
interaction that further stabilized the helical conformation.
Folding oligo(phenylene ethynylenes) [62]
The strategy of backbone rigidification has also been also
applied to oligo(meta-phenylene ethynylenes) (m-PEs).
Solvent-driven folding of oligophenylacetylenes carrying
Fig. 14. Folding oligourea (general structures).
Fig. 15. A backbone-rigidified hexa (phenylene ethynylenes).
Fig. 16. Tuning the cavity of backbone-rigidified m-PE foldamers. A larger cavity can be produced by incorporating one (left) or two (right) para
substituted residues.
Ó FEBS 2004 Well-defined secondary structures of unnatural oligomers (Eur. J. Biochem. 271) 1423
polar side chains has been well established by Moore and
coworkers [56,57]. The Moore system relied on polar side
chains to effect a hydrophobic collapse of the PE backbone,
leading to helical structures that were denatured in nonpolar
solvents. Based on the m-PE system, a completely different
folding strategy was achieved by introducing an intra-
molecular H-bond that restrict the rotational freedom of
the backbone (Fig. 15).
Folded m-PE oligomers, from dimers to heptamers, were
observed in nonpolar solvents such as chloroform. The
folded conformations were confirmed by X-ray and
2D-NMR studies. As anticipated, the 2D
1
HNMR
(NOESY) spectrum of a hexamer showed end-to-end NOEs
that could only be explained by a helical confirmation of this
oligomer. Similar to the crescent oligoamides, the cavity size
of the backbone-rigidified m-PEs can be tuned by changing

the connectivity of some of the backbone units (Fig. 16).
Placing the H-bonds and the corresponding side chains
inward should lead to functional cavities.
Conclusions
While progress has been made in mimicking the basic
features of biological systems, there is still a great deal of
work to be done. For all intents and purposes, molecular
self-assembly and molecular folding are still in their
infancy. Nearly all unnatural systems, while elegant and
fascinating in their own regard, still fall well short of
the shear complexity and grandeur of constructs seen in
nature. The recognition, interaction and folding of bio-
molecules, particularly biomacromolecules, have inspired
most of the currently known unnatural systems. By
mimicking natural systems, the scientific community has
witnessed the breathtaking progress made the relevant
fields over the past decade. The advantage of unnatural
systems, however, is not limited to mimicking the
structures and functions of biological molecules. The
potential of unnatural self-assembling or folding molecules
may ultimately lie with the scientists, who are only limited
by their imagination.
Along these lines of thinking, we have succeeded in
synthesizing and characterizing novel, and potentially useful
self-assembly and folding systems that draw inspirations
from nature, which may be applied to both natural and
unnatural settings. Both our information-storing duplexes
and helical foldamers have already shown exciting poten-
tials in a variety of different applications, and are motivating
us toward the development of new systems.

Acknowledgements
We thank the NASA (NAG5-8785), NSF (CHE-0314577), NIH
(R01G
M
63223), ONR (N000140210519) and ACS-PRF (37200-AC4)
for financial support.
References
1. Saenger, W. (1984) Principles of Nucleic Acid Structure,Springer,
New York.
2. Zimmerman, S.C. & Corbin, P.S. (2000) Heteroaromatic modules
for self-assembly using multiple hydrogen bonds. Struct. Bond. 96,
63–94.
3. Gong, B. (2001) Specifying non-covalent interactions: sequence-
specific assembly of hydrogen-bonded molecular duplexes. Synlett
582–589.
4. Sijbesma, R.P. & Meijer, E.W. (2003) Quadruple hydrogen bon-
ded systems. Chem. Commun., 5–16.
5. Prins, L.J., Reinhoudt, D.N. & Timmerman, P. (2001) Non-
covalent synthesis using hydrogen bonding. Angew. Chem. Int. Ed.
Engl. 40, 2383–2426.
6. Seebach, D. & Matthews, J.L. (1997) b-Peptides: a surprise at
every turn. Chem. Commun., 2015–2022.
7. Gellman, S.H. (1998) Foldamers: a manifesto. Acc. Chem. Res. 31,
173–180.
8. Stigers, K.D., Soth, M.J. & Nowick, J.S. (1999) Designed mole-
cules that fold to mimic protein secondary structures. Curr. Opin.
Chem. Biol. 3, 714–723.
9. Gong, B. (2001) Crescent oligoamides: From acyclic ÔmacrocyclesÕ
to folding nanotubes. Chem. Eur. J. 7, 4336–4342.
10. Hill, D.J., Mio, M.J., Prince, R.B., Hughes, T.S. & Moore, J.S.

(2001) A field guide to foldamers. Chem. Rev. 101, 3893–4011.
11. Cheng, R.P., Gellman, S.H. & DeGrado, W.F. (2001) b-Peptides:
From structure to function. Chem. Rev. 101, 3219–3232.
12. Cubberley, M.S. & Iverson, B.L. (2001) Models of higher-order
structure: Foldamers and beyond. Curr. Opin. Chem. Biol. 5,
650–653.
13. Patch, J.A. & Barron, A.E. (2002) Mimicry of bioactive peptides
via non-natural, sequence-specific peptidomimetic oligomers.
Curr. Opin. Chem. Biol. 6, 872–877.
14. Schmuck, C. (2003) Molecules with helical structure: how to build
a molecular spiral staircase. Angew. Chem. Int. Ed. Engl. 42,
2448–2452.
15. Sanford, A.R. & Gong, B. (2003) Evolution of helical foldamers.
Curr. Org. Chem. 7, 1649–1659.
16.Hanessian,S.,Luo,X.H.,Schaum,R.&Michnick,S.(1998)
Design of secondary structures in unnatural peptides: Stable
helical c-tetra-, hexa-, and octapeptides and consequences of
a-substitution. J. Am. Chem. Soc. 120, 8569–8570.
17. Seebach, D., Brenner, M., Rueping, M. & Jaun, B. (2002) c
2
-, c
3
-,
and c
2,3,4
-Amino acids, coupling to c-hexapeptides: CD spectra,
NMR solution and X-ray crystal structures of c-peptides. Chem.
Eur. J. 8, 573–584.
18. Szabo, L., Smith, B.L., McReynolds, K.D., Parrill, A.L., Morris,
E.R. & Gervay, J. (1998) Solid phase synthesis and secondary

structural studies of (1 fi 5) amide-linked sialooligomers. J. Org.
Chem. 63, 1074–1078.
19. Hungerford, N.L., Claridge, T.D.W., Watterson, M.P., Aplin,
R.T., Moreno, A. & Fleet, G.W.J. (2000) Tetrahydrofuran amino
acids: Secondary structure in tetrameric and octameric carbo-
peptoids derived from a
D
-allo-5-(aminomethyl) tetrahydrofuran-
2-carboxylic acid. J. Chem. Soc., Perkin Trans. 1, 3666–3679.
20. Yang,D.,Qu,J.,Li,B.,Ng,F F.,Wang,X C.,Cheung,K K.,
Wang, D P. & Wu, Y D. (1999) Novel turns and helices in
peptides of chiral a-aminoxy acids. J. Am. Chem. Soc. 121,
589–590.
21. Jorgensen, W.L. & Pranata, J. (1990) Importance of secondary
interactions in triply hydrogen-bonded complexes – Guanine-
cytosine vs uracil-2,6. J. Am. Chem. Soc. 112, 2008–2010.
22. Pranata, J., Wierschkem, S.G. & Jorgensen, W.L. (1991) OPLS
potential functions for nucleotide bases – relative association
constants of hydrogen-bonded base-pairs in chloroform. J. Am.
Chem. Soc. 113, 2810–2819.
23. Murray, T.J. & Zimmerman, S.C. (1992) New triply hydrogen-
bonded complexes with highly variable stabilities. J. Am. Chem.
Soc. 114, 4010–4011.
24. Seto, C.T. & Whitesides, G.M. (1990) Self-assembly based on
the cyanuric acid melamine lattice. J. Am. Chem. Soc. 112,
6409–6411.
1424 A. R. Sanford et al. (Eur. J. Biochem. 271) Ó FEBS 2004
25. Whitesides, G.M., Mathias, J.P. & Seto, C.T. (1991) Molecular
self-assembly and nanochemistry – a chemical strategy for the
synthesis of nanostructures. Science 254, 1312–1319.

26. Whitesides, G.M., Simanek, E.E., Mathias, J.P., Seto, C.T., Chin,
D.N., Mammen, M. & Gordon, D.M. (1995) Noncovalent
synthesis – using physical-organic chemistry to make aggregates.
Acc. Chem. Res. 28, 37–44.
27. Conn, M.M. & Rebek, J. (1997) Self-assembling capsules. Chem.
Rev. 97, 1647–1668.
28. Philp, D. & Stoddart, J.F. (1996) Self-assembly in natural and
unnatural systems. Angew. Chem. Int. Ed. Engl. 35, 1155–1196.
29. Archer, E.A., Sochia, A.E. & Krische, M.J. (2001) The covalent
casting of one-dimensional hydrogen bonding motifs: toward
oligomers and polymers of predefined topography. Chem. Eur.
J. 7, 2059–2065.
30. Corbin, P.S. & Zimmerman, S.C. (1998) Self association without
regard to prototropy. A heterocycle that forms extremely stable
quadruply hydrogen-bonded dimers. J. Am. Chem. Soc. 120,
9710–9711.
31. Murray, T.J. & Zimmerman, S.C. (1992) New triply hydrogen-
bonded complexes with highly variable stabilities. J. Am. Chem.
Soc. 114, 4010–4011.
32. Fenlon, E.E., Murray, T.J., Baloga, M.H. & Zimmerman, S.C.
(1993) Convenient synthesis of 2-amino-1,8-naphthyridines,
building-blocks for host-guest and self-assembling systems. J. Org.
Chem. 58, 6625–6628.
33. Beijer, F.H., Sijbesma, R.P., Kooijman, H., Spek, A.L. & Meijer,
E.W. (1998) Strong dimerization of ureidopyrimidones via
quadruple hydrogen bonding Self-assembly mediated by the
donor-donor-acceptoracceptor-acceptor-donor (DDA-AAD)
hydrogen-bonding motif: Formation of a robust hexameric
aggregate. J. Am. Chem. Soc. 120, 6761–6769.
34. Beijer, F.H., Kooijman, H., Spek, A.L., Sijbesma, R.P. & Meijer,

E.W. (1998) Self-complementarity achieved through quadruple
hydrogen bonding. Angew. Chem. Int. Ed. Engl. 37, 75–78.
35. Folmer, B.J.B., Sijbesma, R.P., Kooijman, H., Spek, A.L. &
Meijer, E.W. (1999) Cooperative dynamics in duplexes of stacked
hydrogen-bonded moieties. J. Am. Chem. Soc. 121, 9001–9007.
36. Archer, E.A. & Krische, M.J. (2002) Duplex oligomers defined via
covalent casting of a one-dimensional hydrogen-bonding motif.
J. Am. Chem. Soc. 124, 5074–5083.
37. Bisson, A.P., Carver, F.J., Eggleston, D.S., Haltiwanger, R.C.,
Hunter,C.A.,Livingstone,D.L.,McCabe,J.F.,Rotger,C.&
Rowan, A.E. (2000) J. Am. Chem. Soc. 122, 8856–8868.
38. Ghadiri, M.R., Kobayashi, K., Granja, J.R., Chadha, R.K. &
McRee, D.E. (1995) The structural and thermodynamic basis for
the formation of self-assembled peptide nanotubes. Angew. Chem.
Int. Ed. Engl. 34, 93–95.
39. Hartgerink, J.D., Clark, T.D. & Ghadiri, M.R. (1998) Peptide
nanotubes and beyond. Chem. Eur. J. 4, 1367–1372.
40. Szabo, T., Hilmersson, G. & Rebek, J. (1998) Dynamics of
assembly and guest exchange in the tennis ball. J. Am. Chem. Soc.
120, 6193–6194.
41. Rebek, J. (1999) Reversible encapsulation and its consequences in
solution. Acc. Chem. Res. 32 (4), 278–286.
42. Gong, B., Yan, Y., Zeng, H., Skrzypczak-Jankunn, E., Kim,
Y.W., Zhu, J. & Ickes, H. (1999) A new approach for the design of
supramolecular recognition units: Hydrogen-bonded molecular
duplexes. J. Am. Chem. Soc. 121, 5607–5608.
43. Zeng, H., Miller, R.S., Flowers, R.A. & Gong, B. (2000) A highly
stable, six-hydrogen-bonded molecular duplex. J. Am. Chem. Soc.
122, 2635–2644.
44. Zeng, H.Q., Yang, X.W., Brown, A.L., Martinovic, S., Smith,

R.D. & Gong, B. (2003) An extremely stable, self-complementary
hydrogen-bonded duplex. Chem. Comm. 1556–1557.
45. Zeng,H.,Ickes,H.,Flowers,R.A.&Gong,B.(2001)Sequence
specificity of hydrogen-bonded molecular duplexes. J. Org. Chem.
66, 3574–3583.
46. Yang, X.W., Martinovic, S., Smith, R.D. & Gong, B. (2003)
Duplex foldamers from assembly-induced folding. J. Am. Chem.
Soc. 125, 9932–9933.
47. Zeng, H., Yang, X., Flowers, R.A. & Gong, B. (2002) A non-
covalent approach to antiparallel b-sheet formation. J. Am. Chem.
Soc. 124, 2903–2910.
48. Connon, S.J. & Blechert, S., (2003) Recent developments in olefin
cross-metathesis. Angew. Chem. Int. Ed. Engl. 42, 1900–1923.
49. Trnka, T.M. & Grubbs, R.H. (2001) The development of
L2X2Ru¼CHR olefin metathesis catalysts: an organometallic
success story. Acc. Chem. Res. 34, 18–29.
50. Dado, G.P. & Gellman, S.H. (1994) Intramolecular hydrogen-
bonding in derivatives of b-alanine and c-amino butyric-acid –
model studies for the folding of unnatural polypeptide backbones.
J. Am. Chem. Soc. 116, 1054–1062.
51. Seebach, D., Overhand, M., Kuhnle, F.N.M., Martinoni, B.,
Oberer, L., Hommel, U. & Widmer, H. (1996) b-Peptides: Syn-
thesis by Arndt-Eistert homologation with concomitant peptide
coupling. Structure determination by NMR and CD spectroscopy
and by X-ray crystallography. Helical secondary structure of a
b-hexapeptide in solution and its stability towards pepsin Helv.
Chim. Acta 79, 913–941.
52. Seebach, D., Ciceri, P.E., Overhand, M., Jaun, B., Rigo, D.,
Oberer, L., Hommel, U., Amstutz, R. & Widmer, H. (1996)
Probing the helical secondary structure of short-chain b-peptides.

Helv. Chim. Acta 79, 2043–2066.
53.Hanessian,S.,Luo,X.H.,Schaum,R.&Michnick,S.(1998)
Design of secondary structures in unnatural peptides: stable helical
c-tetra-, hexa-, and octapeptides and consequences of a-substitu-
tion. J. Am. Chem. Soc. 120, 8569–8570.
54.Berl,V.,Huc,I.,Khoury,R.G.,Krische,M.J.&Lehn,J.M.
(2000) Interconversion of single and double helices formed from
synthetic molecular strands. Nature 407, 720–723.
55. Hill, D.J. & Moore, J.S. (2002) Helicogenicity of solvents in the
conformational equilibrium of oligo(m-phenylene ethynylene)s:
implications for foldamer research. Proc. Natl Acad. Sci. USA 99,
5053–5057.
56. Nelson, J.C., Saven, J.G., Moore, J.S. & Wolynes, P.G. (1997)
Solvophobically driven folding of nonbiological oligomers.
Science 277, 1793–1796.
57. Tanatani, A., Kagechika, H., Azumaya, I., Fukutomi, R., Ito, Y.,
Yamaguchi, K. & Shudo, K. (1997) Helical aromatic urea and
guanidine. Tetrahedron Lett. 38, 4425–4428.
58. Zhu, J., Parra, R.D., Zeng, H., Skrzypczak-Jankun, E., Zeng,
X.C. & Gong, B. (2000) A new class of folding oligomers: Crescent
oligoamides. J. Am. Chem. Soc. 122, 4219–4220.
59. Parra,R.D.,Zeng,H.,Zhu,J.,Zheng,C.,Zeng,X.C.&Gong,B.
(2001) Stable three-center hydrogen bonding in a partially rigidi-
fied structure. Chem.Eur.J.7, 4352–4357.
60.Gong,B.,Zeng,H.,Zhu,J.,Yua,L.,Han,Y.,Cheng,S.,
Furukawa, M., Parra, R.D., Kovalevsky, A.Y., Mills, J.L.,
Skrzypczak-Jankun, E., Martinovic, S., Smith, R.D., Zheng,
C., Szyperski, T. & Zeng, X.C. (2002) Creating nanocavities of
tunable sizes: Hollow helices. Proc. Natl Acad. Sci. USA 99,
11583–11588.

61. van Gorp, J.J., Vekemans, J.A.J.M., Meijer, E.W. (2004) Facile
synthesis of a chiral polymeric helix; folding by intramolecular
hydrogen bonding. Chem. Commun. 60–61.
62. Yang, X., Brown, A.L., Furukawa, M., Li, S., Gardinier, W.E.,
Bukowski,E.J.,Bright,F.V.,Zheng,C.,Zeng,X.C.&Gong,
B. (2003) A new strategy for folding oligo(m-phenylene ethyny-
lenes). Chem. Comm. 56–57.
Ó FEBS 2004 Well-defined secondary structures of unnatural oligomers (Eur. J. Biochem. 271) 1425