Folding and Assembly of Short α, β, ?-Hybrid Peptides: Minor Variations in Sequence and Drastic Differences in Higher-Level Structures

Article abstract of DOI:10.1021/jacs.9b06094

Multilevel protein structures typically involve polypeptides of sufficient lengths. Here we report the folding and assembly of seven short tetrapeptides sharing the same types of α-, β-, and aromatic ?-amino acid residues. These are two sets of hybrid peptides, with three members in one set and four in the other, having complementary hydrogen-bonding sequences that were hypothesized to pair into linear H-bonded duplexes. However, instead of undergoing the anticipated pairing, the initially examined three oligomers, 1 and 2a or 2b, differing only in their central αβ hybrid dipeptide sequence, do not associate with each other and exhibit distinctly different folding behavior. Experiments based on NMR and mass spectrometry, along with computational studies and systematic inference, reveal that oligomer 1 folds into an expanded β-turn containing an unusual hybrid α/β-amino acid sequence composed of glycine and β-alanine, two α- A nd β-amino acid residues that are conformationally most flexible, and peptides 2a and 2b adopt a noncanonical, extended helical conformation and dimerize into double helices undergoing rapid conformational exchange or helix inversion. The different central dipeptide sequences, αβ vs βα, result in drastically different intramolecular H-bonding patterns that are responsible for the observed folding behavior of 1 and 2. The revealed turn and double helix have few natural or synthetic counterparts, and provide novel and unique folding prototypes based on which chiral α- A nd β-amino acids are incorporated. The resultant derivatives 1a, 1b, 2c, and 2d follow the same folding and assembling behavior and demonstrate the generality of this system with the formation of expanded β-turns and double helices with enhanced folding stabilities, hampered helix inversion, as well as defined and dominant helical sense. This work has demonstrated the unique capability of synthetic foldamers in generating structures with fascinating folding and assembling behavior. The revealed systems offer ample opportunity for further structural optimization and applications.

Full text of DOI:10.1021/jacs.9b06094

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Article
Folding and Assembly of Short #, #, #-Hybrid Peptides: Minor Variations
in Sequence and Drastic Differences in Higher-Level Structures
Yukun Zhang, yulong zhong, Alan L. Connor, Daniel P Miller, Ruikai Cao, Jie Shen, Bo Song, Erin
Shammel Baker, Quan Tang, Surya V.S.R.K. Pulavarti, Rui Liu, Qiwei Wang, Zhong-lin Lu, Thomas
Szyperski, Huaqiang Zeng, Xiaopeng Li, Richard D Smith, Eva Zurek, Jin Zhu, and Bing Gong
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 05 Aug 2019
Downloaded from pubs.acs.org on August 5, 2019
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Folding and Assembly of Short α, β, γ-Hybrid Peptides: Minor Vari-
ations in Sequence and Drastic Differences in Higher-Level Struc-
tures
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⊥
,#
Yukun Zhang,†, Yulong Zhong, ¶,# Alan L. Connor, ¶ Daniel P. Miller,¶ Ruikai Cao,¶ Jie Shen,║ Bo
Song,§ Erin S. Baker,‡ Quan Tang,Δ Surya V. S. R. K. Pulavarti,¶ Rui Liu,Δ Qiwei Wang,† Zhong-lin Lu,Δ
Thomas Szyperski,¶ Huaqiang Zeng,║ Xiaopeng Li,§ Richard D. Smith,‡ Eva Zurek,¶ Jin Zhu,†,* Bing
Gong¶,*
† Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China
¶ Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260, USA
⊥ University of Chinese Academy of Sciences, Beijing 100049, China
║ Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos 138669, Singapore
§ Department of Chemistry, University of South Florida, Tampa, Florida 33620, USA
‡ Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99354, USA
Δ College of Chemistry, Beijing Normal University, Beijing 100875, China
ABSTRACT: Multilevel protein structures typically involve polypeptides of sufficient lengths. Here we report the folding and
assembly of seven short tetrapeptides sharing the same types of α-, β-, and aromatic γ-amino acid residues. These are two sets of
hybrid peptides, with three members in one set and four in the other, having complementary hydrogen-bonding sequences that were
hypothesized to pair into linear H-bonded duplexes. However, instead of undergoing the anticipated pairing, the initially examined
three oligomers, 1 and 2a or 2b, differing only in their central αβ hybrid dipeptide sequence, do not associate with each other and
exhibit distinctly different folding behavior. Experiments based on NMR and mass spectrometry, along with computational studies
and systematic inference, reveal that oligomer 1 folds into an expanded β-turn containing an unusual hybrid α/β-amino acid se-
quence composed of glycine and β-alanine, two α- and β-amino acid residues that are conformationally most flexible; and peptides
2a and 2b adopt a non-canonical, extended helical conformation and dimerize into double helices undergoing rapid conformational
exchange or helix inversion. The different central dipeptide sequences, αβ vs βα, result in drastically different intramolecular H-
bonding patterns that are responsible for the observed folding behavior of 1 and 2. The revealed turn and double helix have few
natural or synthetic counterparts, and provide novel and unique folding prototypes based on which chiral α- and β-amino acids are
incorporated. The resultant derivatives 1a, 1b, 2c, and 2d follow the same folding and assembling behavior and demonstrate the
generality of this system with the formation of expanded β-turns and double helices with enhanced folding stabilities, hampered
helix inversion, as well as defined and dominant helical sense. This work has demonstrated the unique capability of synthetic
foldamers in generating structures with fascinating folding and assembling behavior. The revealed systems offer ample opportunity
for further structural optimization and applications.
INTRODUCTION
synthetic foldamers can accommodate a much wider range of
building blocks and covalent linkages,17 and offer designs that
are limited only by one’s imagination. Consequently, oligo-
meric molecules with a great variety of primary structures can
be made available based on the basic principles of physical
organic and synthetic chemistry, which allows distinct folding
behavior and folded structures that mimic or differ from those
of biomacromolecules to be uncovered.
The folding of biomacromolecules has inspired the devel-
opment of foldamers,1 i.e., oligomers having abiotic back-
bones with defined conformations, for mimicking biological
folding or uncovering folding and other behavior not seen in
nature. The majority of known foldamers adopt helical con-
formations reminiscent of the α-helix and other single-
stranded helices, with representative systems including pep-
tidomimetic foldamers such as β-,2,3 γ-,4 and δ-peptides,5 ali-
phatic oligoureas,6 and peptoids;7 and abiotic foldamers such
as oligo(phenylene ethynylenes),8 aromatic oligoamides,9 hy-
drazides,10 and oligoureas,11 as well as other foldamers con-
sisting of aromatic residues.12 In contrast, non-helical folda-
mers, like those adopting turns,13 sheets,14,15 or other confor-
mations,16 are still rare. Compared to biological foldamers,
Herein we report the folding and assembly of oligomers 1
and 2, hybrid tetrapeptides having α-, β-, and aromatic γ-
amino acid residues and differing only in their central αβ di-
peptide segments (Figure 1). In spite of their short length, the-
se hybrid peptides exhibit distinctly different folding and as-
sembling behavior, as shown by their lack of mutual associa-
1
tion, their H-bonded amide protons, and their dissimilar H
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mers and polymers,22 was initially chosen and incorporated
NMR spectra, which, though initially perplexing, could even-
1
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5
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8
tually be understood based on an interactive feedback loop
between experimental analysis, computational studies, and
systematic inference. Two novel secondary structures, one
being an unusual hairpin or an expanded β-turn with a loop
consisting of an α- and a β-amino acid residue, and the other
an extended helix that associates into interconverting double
helices, have been identified. The folding and assembly of
these hybrid peptides, which involve multi-structural levels
typically associated with proteins, are surprisingly sophisticat-
ed and found with few other synthetic foldamers.
into 1 and 2a. Four other oligomers, 1a/b and 2c/d, derived
from 1 and 2a/b are also designed and studied (see Figure 8).
Apparently, other side (R) chains and end (R’ and R’’) groups
can also be incorporated. If adopting extended conformations,
oligomers 1 and 2 should pair into duplex 1•2 by forming five
intermolecular H-bonds (Figure 1b) involving amide protons a
and d of 1, and a’, c’, and d’ of 2. Protons b and e of 1, and
protons b’ and e’ of 2, being parts of highly favorable six-
membered H-bonded rings, should remain intramolecularly H-
bonded under most conditions as demonstrated by numerous
previous, including our own, studies.18b,23
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We previously developed H-bonded duplexes composed of
linear oligoamide strands carrying arrays of H-bond donors
and acceptors.18 With their programmable sequence-specificity,
our H-bonded duplexes served as molecular association units
for forming templated β-sheets,19 supramolecular block copol-
ymers,20 and for the specification of chemical reactions.21
While hetero- and homo-duplexes with even numbers of H-
bonds have been well characterized,18 duplexes having odd
numbers of inter-strand H-bonds, which will only allow het-
ero-duplexes to form, have not been made.
RESULTS AND DISCUSSION
Observations on the surprising lack of interaction be-
1
tween 1 and 2, dissimilar H NMR spectra, and conforma-
tional exchange. The anticipated association of 1 and 2 was
first examined by comparing the chemical shifts of protons a
and d of 1, and a’, c’, and d’ of 2, to those of the same protons
in the 1:1 mixture of the two oligomers. Forming the H-
bonded duplex 1•2 should result in large (1 to 2 ppm) down-
field shifts of these amide protons.28 Surprisingly, compared to
the amide 1H resonances of single strands 1 and 2a, these pro-
tons exhibited small, mostly negligible changes in their chem-
ical shifts upon mixing the two oligomers (1:1) at 1 mM, 10
mM, and 50 mM (Table S1). The insignificant shifts in the
resonances of these five protons indicate very weak, if any, H-
bonding interaction between the two oligomers.
R
R
(a)
h
A
f
h1
B
O
O
b
c
e
g1
g
a
H
N
H
N
O
O
H
N
O
O
j
1
N
m
N
H
l
i
k
f1
d
O
N
H
O
O
R
O
R
O
h'
h'1
Compared to proton a of 3 (1 mM in CDCl3) that appears
at 7.22 ppm and undergoes negligible H-bonding at this con-
centration,18b the signals of protons a and d of 1, and a’ and d’
of 2a (or 2b), also at 1 mM in CDCl3, show very large (1.8 to
2.8 ppm) downfield shifts, indicating that, although 1 and 2 do
not associate with each other, their amide protons do engage in
H-bonding with H-bond donors of their own molecules.
b'
e'
g'
a'
g'1
H
N
O
H
N
i'
A'
B'
R'' 2
R'
N
H
N
m'
k'
l'
j'
f'1
f'
c'
d'
O
H
H
O
R
R
(b)
h
h1
O
O
c
b
g1
e
g
H
H
O
H
O
j
In addition to the apparent lack of H-bonding interactions
A
B
N
N
N
1
l
m
between 1 and 2a, the H NMR spectra of the two peptides
N
N
i
f
k
f1
d
a
bear no similarity (Figure 2). The spectrum of 1 contains sharp,
well-dispersed signals indicating the presence of a well-
defined species (Figure 2a). The fact that the amide and aro-
O
H
O
H
O
c'
d'
a'
O
H
N
H
N
O
H
N
j'
f'
f'1
B'
l'
k'
m'
1
matic H NMR signals of 1 can be completely assigned to the
R''
N
H
N
H
R'
i'
backbone protons suggest that the two racemic 2-ethylhexyl
side chains do not result in observable diastereotopic signals
for the backbone protons of 1.
A'
e'
b'
g'1
O
O
g'
O
O
R
O
R
h'
h'1
duplex 1•2
By contrast, the spectrum of 2a contains both sharp and-
broadened peaks (Figure 2b), including the severely broadened
signals of protons i’ and k’, the broadened resonances of pro-
tons c’ and d’, and the moderately broadened peaks of protons
f’, j’ and l’. The resonances of the other amide and aromatic
protons of 2a remain sharp. The presence of both sharp and
broad peaks in the spectrum of 2a rules out the formation of
poorly defined higher aggregates. Instead, the line-broadening
observed with only some of the proton resonances of 2a is
typical of chemical exchange caused by conformational inter-
conversion in the microsecond to millisecond time scale.24
R
O
2a: R'= CN, R'' = -CH(CH3)2
2b: R = CH3, R'= -n-C4H9,
R'' = -n-C5H11
b
H
O
N
N
H
n or n1
a
1,2a, 3: R = -CH2CH(C2H5)(C4H9)
O
3
Figure 1. (a) Structures of hybrid peptides 1 and 2. (b) The ex-
pected H-bonded duplex 1•2. Labels of most hydrogens and the
two aromatic residues are shown. Compound 3 corresponds to the
aromatic residues of 1 and 2.
Oligomer 2b, which shares the same amino acid sequence
with 2a but has methyl side chains and linear end groups,
Tetrapeptides 1 and 2a/b (Figure 1a) differ in the sequenc-
es of their central α/β-dipeptide segments, i.e., βAla-Gly of 1
and Gly-βAla of 2, that are flanked by the same aromatic γ-
amino acid residues at their C- and N-termini. The racemic 2-
ethylhexyl group, widely used in solubilizing various oligo-
1
gives a H NMR spectrum that only differs from that of 2a in
the chemical shifts of protons a’ and m’ (Figures S1) due to
the electron-withdrawing cyano group of 2a. Like those of 2a,
the peaks of protons i’ and k’ of 2b are severely broadened;
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and the signals of c’, d’, and j’ are broadened. The same con-
intramolecularly H-bonded. Given its sharp 1H resonances,
1
2
3
4
5
6
7
8
formational interconversion observed with both 2a and 2b,
which differ in their side chains and end groups, can only be
attributed to the same tetrapeptide backbone shared by the two
oligomers. The very similar 1H NMR spectra of 2a and 2b also
suggest that their different side chains have no influence on
the resonances of their backbone protons.
peptide 1 seems to exist as a single species. From 3 to 25 mM
in CDCl3, amide protons c’ and d’ of 2a shift downfield by
0.16 and 0.13 ppm, respectively and proton a’ shows an insig-
nificant shift (Table S2b). The concentration-dependent shifts
observed with protons c’ and d’ imply that 2a self-associates
via intermolecular H-bonding interactions.
o
The role of temperature. From 0 to 45 C in CDCl3, the
CHCl3
f1
f
(a)
signals of protons a and d of 1 (5 mM) shift upfield by 0.33
and 0.22 ppm, respectively (Table S3a), suggesting that the
intramolecular H-bonds involving protons a and d are sensi-
tive to rising temperature. The small upfield shift (~0.06 ppm)
of proton c indicates that H-bonding involving proton c is neg-
ligible at 5 mM. From 0 to 45 oC, the resonances of protons c’
and d’ of 2a (5 mM in CDCl3) exhibit noticeable upfield shifts
of 0.42 and 0.41 ppm, respectively, which points to weakened
H-bonding and indicates that protons c’ and d’ are intermolec-
ularly H-bonded (Table S3b). Proton a’ shows a small (<0.1
ppm) shift, implying that it may either be intramolecularly H-
bonded or not H-bonded.
h1
e
h
9
g1
d
a
g
b
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11
12
13
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16
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c
ppm
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
m
l
n1
k
n
j
i
At -45 oC, all of the 1H resonances of 2a and 2b turn sharp
5.0
4.5
4.0
3.5
3.0
2.5 ppm
1
(Figures S2), which confirms that the broadened H resonanc-
CHCl3
(b)
es of 2a and 2b at room temperature are due to conformational
f'1
b'
interconversion that slows down with decreasing temperature.
o
h'
e'
At -45 C, the protons of each of the Gly and βAla methylene
a'
g'
h'1
groups, i.e., protons i’, j’, or k’, appear as two well separated
peaks, indicating that bond rotation involving these methylene
carbons is restricted due to slowed conformational intercon-
version, resulting in non-equivalent protons.
g'1
f'
c'
d'
ppm
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
The effect of solvent polarity. The H-bonding interactions
of 1 and 2 were further studied in CDCl3 containing DMSO-d6.
Among the amide protons of 1, proton c shows a downfield
shift of ~1 ppm as the ratios of DMSO-d6 increased from 0 to
15% (Figure 3a and Table S4a), confirming that proton c is
exposed to solvent molecules. In comparison, the resonances
of the other amide protons exhibit much smaller shifts, indicat-
ing that 1 adopts a conformation in which these protons are
shielded from solvent molecules and are intramolecularly H-
bonded. The amide protons of 2a responded very differently to
changing solvent polarity (Figure 3b and Table S4b). At 5 mM
with 0 to 15% DMSO-d6, the signals of protons c’ and d’ un-
dergo 1.04 ppm and 0.58 ppm, respectively, upfield shifts,
indicating that the intermolecular H-bonds between the mole-
cules of 2a are weakened or interrupted by DMSO molecules.
In contrast, the signal of proton a’ shows a noticeable (0.30
ppm) downfield shift, indicating that proton a’ of 2a is ex-
posed to solvent molecules.
m'
n'
n'1
i'
l'
j'
k'
5.0
4.5
4.0
3.5
3.0
2.5 ppm
Figure 2. 500-MHz partial 1H spectra of (a) 1 (5 mM) and, (b) 2a
(5 mM) at 25 oC in CDCl3.
The H-bonded amide protons that do not lead to the asso-
ciation of 1 and 2, along with the different 1H NMR spectra of
the two otherwise similar hybrid peptides, suggest that H-
bonds involving the amide protons of 1 or 2 mainly act by
either stabilizing folded conformation(s) or facilitating the
self-association of each peptide.
(a)
(b)
a
a'
Distinct H-bonding behavior and the dimerization of 2.
To understand the unexpected and puzzling lack of association
between 1 and 2, inter- and intramolecular H-bonding interac-
tions involving the amide protons of these oligomers were
systematically examined against concentration, temperature,
and solvent polarity.
d
d'
b
e
c
b'
e'
c'
The influence of concentration. Among the amide hydro-
gens of 1, only proton c shows a significant (0.66 ppm) down-
field shift from 0.1 to 25 mM in CDCl3 (Table S2a), indicating
that this proton becomes increasingly involved in intermolecu-
lar H-bonding with rising concentration. Similar to the signals
of protons b and e that are intramolecularly H-bonded, the
resonances of protons a and d also exhibit very small shifts in
this concentration range, suggesting that these protons are
Figure 3. Chemical shift δNH of amide protons of (a) 1 (5 mM)
and (b) 2a (5 mM) versus percent of DMSO-d6 in CDCl3.
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1
Evidence from H NMR studies thus indicates that, except
for proton c, the other amide protons of 1 are intramolecularly
H-bonded. Proton a’, which shows no shift with changing
concentration or temperature but moves downfield with in-
creasing proportion of DMSO, seems to be exposed to solvent
molecules and does not participate in the self-association of 2a.
Protons c’ and d’, with their resonances shifting noticeably
with changing concentration, temperature, and ratio of polar
solvent, must be involved in intermolecular H-bonding that
facilitates the self-association of 2a.
same or different segments of the two molecules are H-bonded
together. Each dimer of 2A has its four intermolecular H-
bonds arranged roughly in a plane that separate the two con-
stituent molecules, while the dimers based on 2B have their
two constituent molecules wrapping around each other, form-
ing double helices.
1
2
3
4
5
6
7
8
R
(a)
O
e
O
H
B
N
N
c
d
O
H
HN
1A
a
O
H
N
O
9
Dimerization of 2. Protons c’ and d’ of 2b show concentra-
tion-dependent shifts (Figure S3) that fit satisfactorily into a
dimerization model,18b giving a dimerization constant of 7 x
103 M-1, a value close to those of linear 4-H-bonded duplexes
we reported before,18b implying that tetrapeptides 2 may be
undergoing dimerization via four intermolecular H-bonds.
N
H
A
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13
14
15
16
17
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19
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22
23
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b
O
O
R
R
O
b'
O
H
O
A'
N
N
(b)
R'
N
a'
c'
H
O
H
R
2A
e'
O
H
O
B'
N
R''
N
d'
O
H
That oligomer 2 indeed undergoes dimerization was con-
firmed by examining 2b with electrospray ionization-mass
spectrometry (ESI-MS) and travelling wave ion mobility-mass
spectrometry (TWIM-MS). In CHCl3 with different ratios of
CH3OH (Figure S4a), the dimers of 2b exist as the major spe-
cies in the least polar solvent (CHCl3/CH3OH = 9/1); in
CHCl3/CH3OH (1/1), the dimers and monomers of 2b exist in
similar abundance; in CHCl3/CH3OH (1/9), oligomer 2b exists
mainly as its monomers. These results are fully consistent with
the H-bonded nature of 2b dimer. In addition, tandem mass
spectrometry indicated that the dimers of 2b fully associate
into the monomers at 22 V (Figure S4b).
R
R
O
b'
b'
O
O
H
O
H
O
O
A'
A'
N
N
N
N
R'
R'
N
N
H
H
c'
c'
O
a'
O
O
d'
H
H
a'
H
a'
c'
O
H
N
H
N
N
R'
O
R''
N
R
N
A'
H
H
B'
O
b'
O
e'
O
O
R
R
R
e'
H
or
e'
H
N
O
O
O
O
B'
B'
N
R''
R''
N
N
H
d'
d'
O
H
O
c'
a'
H
d'
H
N
O
O
N
H
H
N
N
R'
R''
N
A'
2A2
2A1
B'
O
H
b'
O
e'
O
O
R
O
R
a'
c'
H
N
O
H
N
N
O
R
R'
A'
b'
H
O
O
The effects of concentration, temperature, and solvent po-
larity on the resonances of amide protons suggest that
tetrapeptide 1 folds into a monomeric conformation that is
stabilized by H-bonds involving protons a and d, while proton
c does not engage in intramolecular H-bonding. Peptide 2,
with its protons c’ and d’ engaging in intermolecular H-
bonding, exists as dimers as confirmed by mass spectrometry.
Besides, peptide 2 undergoes conformational interconversion
that slows down at low temperature.
2B
R
O
e'
O
H
N
B'
R''
N
d'
H
O
R
R
O
e'
O
b'
H
H
O
O
A'
B'
O
N
H
d'
N
R''
R'
N
N
a'
N
O
O
c'
H
H
c'
a'
a'
H
N
c'
H
N
H
N
O
H
O
N
N
H
R'
N
H
R'
A'
A'
O
O
b'
O
O
O
b'
O
R
R
R
R
or
e'
e'
O
O
H
N
H
N
O
O
B'
B'
N
H
d'
R''
N
R''
H
d'
O
O
a'
Likely conformations and assembly of 1 and 2. Based on
the results and conclusions from NMR and mass spectral stud-
ies, the likely conformations of 1 and 2 are deduced. As shown
in Figure 4a, to exist as monomers with protons a and d, but
not c, being intramolecularly H-bonded, the only reasonable
conformation of peptide 1 is hairpin 1A in which the two aro-
matic residues are H-bonded. Hairpin 1A is an unusual, ex-
panded β-hairpin with a loop consisting of an α-amino acid
(glycine) residue and a β-amino acid (β-alanine) residue.
H
c'
d'
O
H
N
O
H
N
R'
N
b'
N
R''
N
A'
O
H
B'
H
O
O
e'
O
O
2B2
2B1
R
R
Figure 4. Illustration of the possible conformations of (a) 1 and
(b) 2 deduced from data based on 1D H NMR and mass spectral
studies. H-bonds are shown as dashed lines. Some of the bonds
are not drawn in proportion for clarity.
1
Revelation of the conformations and assembly of 1 and
2 based on computational studies. Monomer 1A and the four
possible dimers of 2 were optimized with dispersion-corrected
density functional theory (DFT) computations25 carried out
with the ADF software package.26 Further computational de-
tails can be found in the Supporting Information. Confor-
mation 1A was optimized to a hairpin structure, i.e., a novel
expanded β-turn28 involving an 11-atom, intramolecularly
hydrogen-bonded ring with a hybrid α/β amino acid sequence
consisting of Gly and βAla residues (Figure 5a). The O···H
distances in the two intramolecular H-bonds involving protons
a and d are 1.93 Å and 1.99 Å, respectively, while proton c
does not engage in intramolecular H-bonding.
The likely conformations of 2 are presented in Figure 4b.
Protons c’ and d’, along with the two benzamide oxygen at-
oms of 2, constitute an array of four H-bond donors and accep-
tors. To dimerize, peptide 2 has to adopt a bent conformation,
which is only possible if the ethylene linker of the βAla resi-
due dissects the array of the four H-bond donors and acceptors
into two segments, one involving the Gly residue flanked by
proton c’ and a benzamide oxygen; and the other being the
aromatic residue B’ flanked by proton d’ and the other ben-
zamide oxygen. Two different bent conformations, 2A and 2B,
are possible. In 2A, N-Hc’ and N-Hd’ bonds, and the two ben-
zamide C=O bonds point to the same side; in 2B, N-Hc’ and
N-Hd’ bonds, along with their adjacent C=O bonds, point to
different directions.
The computationally optimized structures and relative en-
ergies of dimers 2A1, 2A2, 2B1, and 2B2 (Figure S5) indicate
that 2B1 and 2B2 are much more stable (ΔδE > 16-21
kcal/mol, see Table S5) than either 2A1 or 2A2. Comparing
Two different dimers, 2A1 and 2A2, or 2B1 and 2B2,
could be formed based on 2A or 2B, depending on whether the
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the optimized structures of the four dimers indicate that in 2A1 placed into close proximity in the hairpin conformation of 1. A
1
2
3
4
5
6
7
8
or 2A2, oligomer 2 adopts a strained, bent conformation, and
the aromatic rings and amide linkages are not positioned to
allow the existence of any stacking interactions; while in 2B1
or 2B2, oligomer 2 adopts a relaxed, extended helical confor-
mation that engages in stacking interactions involving the ar-
omatic rings and amide groups. The energetic and structural
differences revealed by computational studies thus suggest
that 2 most likely exists as conformation 2B1 and 2B2, two
extended helices.
very strong NOE between protons c and i was also observed,
which, along with a strong NOE between c and d, demon-
strates that the Gly and βAla residues constitute the loop of the
expanded β-turn. In addition, NOEs between remote protons a
and f1, a and l, d and f, and d and i further support the hairpin
conformation adopted by 1.
Three inter-proton distances determined based on NOEs
(with the mixing time selected from a NOE buildup) between
protons a and f1, d and f, and m and l of 1 and the same three
distances based on energy-minimized conformation 1A show
good agreement, with errors of 1.2, 3.1 and 4.8% (Table S6).
The computations were performed in gas phase at 0 K, and the
NMR experiments were in CDCl3. So solvent and finite tem-
perature effects may be responsible for the differences. These
results demonstrate that the computationally optimized hairpin
does reflect the folded structure.
9
Between the two possible dimers of 2B, dimer 2B1 is 4.5
kcal/mol more stable than 2B2. Comparing the optimized
structures of 2B1 and 2B2 indicates that in dimer 2B1 (Figure
5b), the two pairs of benzene rings are placed within stacking
distance of each other (C-C distances of ~3.3-4.1 Å, cf. 3.9-4.1
Å calculated for the benzene dimer); while in 2B2 (Figure S5),
only one pair of benzene rings is involved in π-π stacking in-
teractions, and the two molecules are twisted considerably to
allow intermolecular H-bonds to form. Thus, peptide 2a or 2b
most likely exists as double helix 2B1 that gains its stability
from both H-bonding and aromatic stacking interactions.
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The expanded β-turn revealed with 1 represents a simple
but extraordinary hairpin motif since the α/β dipeptide se-
quence, consisting of Gly and βAla, the conformationally most
flexible α- and β-amino acid residues, results in a highly flexi-
ble loop. In fact, the expanded β-turn adopted by 1 is very
unusual given that the formation of known natural or expand-
ed β-turns requires the presence of at least one turn-promoting
or conformationally constrained residue, such as aminoisobu-
tyric acid (Aib) or Pro, in their dipeptide loops.28
(a)
(b)
O
H
N
O
O
H
N
N
H
R'
N
H
A'
A
O
O
O
O
R
O
R
Figure 5. (a) Energy-minimized structures of 1A and, (b) dimer
2B1. The side chains of 1 and 2 and the end groups R’ and R’’ of
2 are replaced with methyl groups. Hydrogen bonds are indicated
as dashed dark green lines. All H atoms, except for those of amide
groups, are removed for clarity. The intramolecular H-bonded
rings involving the βAla or Gly residue and aromatic residue A or
A’ are shown under each optimized structure.
The behavior of 1 and 2 can be explained by the different
intramolecular H-bonds involving the βAla or Gly residue and
aromatic residue A or A’ (Figure 5, bottom). In 1, the NH
group of the βAla residue is part of an intramolecularly H-
bonded six-membered ring that involves the phenolic oxygen
of residue A; In 2, the NH bond attached to residue A’ forms a
intramolecularly H-bonded six-membered ring and another
five-membered ring involving the carbonyl group of the Gly
residue, which constitutes a three-center H-bond that is espe-
cially robust as demonstrated by our previous studies.27 Such a
three-center H-bond enforces coplanarity on residues A’ and
Gly in 2, as revealed by the structure 2B1 (Figure 5b), which
allows 2 to only adopt an extended helical conformation. In
contrast, no such three-center H-bond involving residues A
and βAla of 1 exists, which offers sufficient flexibility for the
βAla-Gly segment of 1 to serve as the loop of hairpin 1A.
Validation of hairpin and double helical conformations.
Figure 6. Partial NOESY spectra containing major NOEs re-
vealed with 1 (5 mM, 500 MHz, 25 C, mixing time: 500 ms) in
CDCl3. Major NOEs are indicated with arrows with the darker
arrows indicating strong NOEs and the lighter ones indicating
NOEs of medium strength.
Conclusive evidence confirming the folded conformations of
o
1
hybrid peptides 1 and 2 is provided by 2D H NMR spectra.
The NOESY spectrum of 1 recorded in CDCl3 contains multi-
ple NOEs that are fully consistent with hairpin 1A. The most
diagnostic NOEs include strong ones between protons l and m,
and f and f1 (Figure 6), indicating that these protons are indeed
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The NOESY spectra of both 2a and 2b, recorded at -45 oC,
at which all of the H resonances are well dispersed, are very
generality of the folding and assembling behavior observed
with 1 and 2, L-alanine and L-β-homoalanine was introduced
to give oligomers 1a and 1b, or 2c and 2d (Figure 8).
1
1
2
3
4
5
6
7
8
similar. As shown in Figure 7, the NOESY spectrum of 2b
contains strong NOEs between protons a' and g'1, c’ and f', f'1
and k', and j' and l'. Other NOEs include two with medium
strength between c' and d', d' and l' (Figure S6). All strong and
medium NOEs can be accounted for by the optimized confor-
mation of dimer 2B1. For example, the strong NOEs between
protons c’ and f’, and j’ and l’ are consistent with the predicted
pairing of the two H-bonding segments of 2B1; those between
protons a’ and g’1 are diagnostic of the stacked aromatic rings.
Expanded β-turns with tunable stability. The NOESY
spectra of 1a and 1b reveal multiple strong NOEs between
remote protons (Figures S7 and S8). These NOEs, similar to
those that reveal the hairpin conformation of 1, demonstrate
that 1a and 1b fold into the same expanded β-turns.
The relative folding stability of 1, 1a, and 1b were probed
by comparing temperature-dependent shifts of the resonances
of protons a and d that are intramolecularly H-bonded in the
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o
Comparing computed inter-proton distances of 2B1 with
those derived from the NOEs (with the mixing time selected
from a NOE buildup) of 2b revealed that the computed dis-
tances are consistent with the NOE-derived distances, with
errors between computed and measured values ranging from
2.8% to 9.6% (Table S7a). In contrast, the inter-proton dis-
tances based on structure 2B2 and the NOE-based distances
show very large discrepancies, with errors ranging from 7.4%
to 190.8% between computed and measured values (Table
S7b). These results demonstrate that the optimized double
helix 2B1 reflects the double-helical conformation of 2 in so-
lution, i.e., tetrapeptides 2 exist as double helix 2B1, not 2B2.
hairpin conformations. From 0 to 45 C, the smallest upfield
shifts, 0.24 and 0.18 ppm, respectively, were given by 1b,
followed by 0.33 and 0.22 ppm for 1, and 0.33 and 0.27 ppm
for 1a (Table S8). These results show that replacing βAla of 1
with L-β-homoAla enhances the stability of the hairpin con-
formation of 1b, while replacing the Gly residue with L-Ala
gives 1a with a hairpin conformation that is slightly less stable.
That the folded structure of 1b is indeed more stable than
that of 1 was shown by monitoring ROE contacts between
terminal protons l and m that are brought into close proximity
in the hairpin conformations. ROESY spectra recorded in
CDCl3 containing different proportions of CD3OD at 0 oC
indicate that for 1, the end-to-end ROE contact between pro-
tons l and m could be detected with up to 55% CD3OD (Figure
S9); while for 1b, the same ROE remained in the solvent with
up to 95% CD3OD (Figure S10). These results indicate that
introducing a L-β-homoAla residue greatly enhances the sta-
bility of the resultant β-turn. It is expected that screening addi-
tional β-amino acid residues could uncover expanded β-turns
with stability in highly competitive media, even water.
R
O
R
O
(a)
c
e
b
O
H
N
H
N
O
H
N
l
m
H3C
N
H
N
H
CH3
a
d
O
O
X
O
Y
1a: X = CH3, Y = H; 1b: X = H, Y = CH3
R = -CH2CH2CH(CH3)2
R
R
(b)
O
O
e
b
O
H
N
O
Y
i
O
H
N
R'
N
H
N
H
N
H
R''
k
f1
j
f
a
c
d
O
X
O
2c: X = CH3, Y = H; 2d: X = H, Y = CH3
R= CH3; R' = n-C5H11; R'' = n-C6H13
Figure 8. Structures of hybrid peptides (a) 1a and 1b derived
from 1, and (b) 2c and 2d derived from 2.
Figure 7. Partial NOESY spectra containing major NOEs re-
vealed with 2b (5 mM, 400 MHz, -45 oC, mixing time: 500 ms) in
CDCl3. Major NOEs are indicated with arrows.
Double helices with biased handedness and enhanced sta-
bility. The presence of chiral amino acid residue in 2c or 2d
should result in two possible diastereomeric double helices
with different stabilities, and thus bias the helical sense for the
double helices of 2c or 2d. Consequently, the rapid helix in-
version shown by 2a or 2b will be hampered for the helices of
2c and 2d (Figure 9).
Extended helix 2B and its dimer 2B1 have few precedents
among synthetic foldamers.29,30 Gaining its stability from in-
ter-strand H-bonding and aromatic stacking interactions, such
a double helical conformation is reminiscent of duplex DNAs
but is based on a much simpler and easily modifiable structur-
al scaffold.
Generalization of the observed folding/assembling be-
havior. The designs of 1 and 2 allow ready structural modifi-
cation. For example, the Gly and βAla residues can be re-
placed with other chiral α- and β-amino acids. To probe the
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(Figure S14). In the energy-minimized right-handed helix of
1
2
3
4
5
6
7
8
2c, the two methyl side chains point to the same direction and
engage in van der Waals interaction that stabilizes the double
helical structure (Figures 10a and S14b); while in the left-
handed helix, the methyl side chains of the two L-Ala residues
face each other in a head-on fashion (Figure S14a), causing
disruption of intermolecular H-bonds and thus destabilizing
the overall structure.
*
*
*
*
Figure 9. Schematic representation of the equilibrium of a pair
of diastereomeric double helices resulting from introducing a
chiral amino acid residue (*) into oligomer 2.
The only noticeable structural difference between the op-
timized double helices of 2d is that, in the left-handed helix,
the methyl side chain of each L-homoAla residue points away
from the adjacent C=O bond of the Ala residues (Figures 10b
and S15a); while in the right-handed helix, each methyl side
chain is positioned next to the C=O bond, resulting in likely
steric hindrance (Figure S15b). In fact, the left-handed helix is
3.7 kcal/mol more stable than the right-handed one. That 2d
exits as its left-handed double helix is experimentally con-
firmed by a strong NOE between the protons of L-homoAla
methyl side chains and protons f on the N-terminal benzene
residues (Figure S13). This NOE cannot be accounted for by
the right-handed helix of 2d in which the distance between the
same methyl and protons f is about 5.5 Å (Figure S15a).
9
Unlike the severely broadened resonances of methylene
protons i, j, and k of the Gly and βAla residues of 2a and 2b at
10
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o
1
25 C (Figures 2b and S1), well-dispersed H NMR peaks are
found for the corresponding methylene protons of 2c and 2d at
this temperature (Figures S11), indicating hampered helix
inversion. While protons i, j, or k of 2a and 2b give a broad,
single peak (Figures 2b and S1), the protons of each corre-
sponding methylene groups of 2c (protons i and j) and 2d
(protons j and k) give two well-separated peaks, a phenome-
o
non observed with 2a and 2b only at -45 C. Thus, bond rota-
tion involving the methylene carbons of 2c and 2d is limited
even at 25 C, suggesting that 2c or 2d exists mainly as the
left- or right-handed helix and the double helices of 2c and 2d
are more stable than those of 2a and 2b.
o
CONCLUSIONS
The relative stabilities of the double helices of 2b, 2c, and
2d were probed by comparing the upfield shifts of the inter-
molecularly H-bonded amide protons c and d from 0 to 45 C.
Protons c and d of 2d give the smallest shifts of 0.29 and 0.24
ppm, respectively, followed by 0.32 and 0.27 ppm for 2c, and
0.36 and 0.28 ppm for 2b (Table S9). These results suggest
that the double helices of 2c and 2d, with their chiral amino
acid residues, have higher stabilities than that of 2b.
Differing only in their central αβ dipeptide sequences, hy-
brid tetrapeptides 1 and 2 exhibit distinct folding and assem-
bling behavior. The turn formed by 1 is an unusual, expanded
β-turn with a hybrid dipeptide sequence consisting of glycine
and β-alanine that gives rise to a highly flexible α/β loop. In
contrast to the 10-atom H-bonded ring typical of natural β-
turns and their mimetics,31 the turns of 1 contains a two-
residue, 11-atom H-bonded ring. Tetrapeptides 2 fold into an
extended helical conformation that further assembles into a
quaternary structure. Such a feature is observed only with pol-
ypeptides and a few longer peptidomimetic oligomers.32,33 The
subtle structural difference between 1 and 2 results in com-
pletely different pre-organization effected by intramolecular
H-bondin patterns that lead to the drastically different folding
behavior of 1 and 2. The generality of the folding and assem-
bling behavior revealed with 1 and 2 is demonstrated by in-
corporating chiral α- and β-amino acids, L-alanine and L-β-
homalanine, into 1 and 2. The resultant derivatives 1a, 1b, 2c,
and 2d follow the same folding and assembling patterns, form-
ing expanded β-turns or extended helices that dimerize into
double helices with enhanced stabilities at elevated tempera-
ture and in polar solvents, controlled chirality, and defined
helical sense. The folding and assembly of these two groups of
very short hybrid peptides have demonstrated the unique ca-
pability and versatility of synthetic foldamers in incorporating
and combining natural and unnatural building blocks. The
unexpected structural modules presented in this paper offer
themselves as valuable scaffolds that allow the incorporation
of a variety of α- and β-amino acid residues, base on which
interaction interfaces and functionalities in supramolecular
biomimetic systems will be designed.
o
That 2c and 2d exist as double helices was confirmed by
numerous strong ROEs in their 2D (ROESY) spectra meas-
ured at 0 C (Figures S12 and S13). ROESY was used for 2c
and 2d because it gave the best results at 0 C while NOESY
failed to reveal NOE cross peaks at this temperature. The de-
tected ROEs can be fully assigned to double helical confor-
mations similar to those of 2a and 2b, indicating that 2c and
2d exist as double helices in which the L-Ala and L-β-
homoAla residues are well accommodated.
o
o
(a)
(b)
Figure 10. Energy-minimized structures of the dominant dou-
ble helices of (a) 2c, in which the two methyl side chains (or-
ange balls) of the Ala residues (highlighted inside dashed pur-
ple circle) are arranged side-by-side and undergo van der
Waals contact and, (b) 2d, in which the methyl side chain (or-
ange ball) of the β-homoAla residue and the C=O bond of the
Ala residue (highlighted inside dashed purple circle) of the
same molecule point away from each other.
The helical sense of the dominant double helix adopted by
2c or 2d was determined. Computational modeling revealed
that 2c favors the right-handed double helix, which is unam-
biguously demonstrated by the large difference in the stabili-
ties of the left- and right-handed helices, with the right-handed
helix being 20 kcal/mol more stable than the left-handed one
ASSOCIATED CONTENT
Supporting Information. Synthetic procedures, experimental
conditions, additional NMR spectra, mass spectra, and description
of theoretical calculations can be found in the Supplementary
Information, which is available free of charge on the ACS Publi-
cations website.
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Journal of the American Chemical Society
(8) (a) Nelson, J. C.; Saven, J. G.; Moore, J. S.; Wolynes, P. G.
AUTHOR INFORMATION
Solvophobically driven folding of nonbiological oligomers. Science
1997, 277, 1793. (b) Jones, T. V.; Blatchly, R. A.; Tew, G. N. Synthe-
sis of Alkoxy-Substituted ortho-Phenylene Ethynylene Oligomers.
Org. Lett. 2003, 5, 3297.
1
2
3
Corresponding Author
*bgong@buffalo.edu
*jinzhu@cioc.ac.cn
4
5
6
7
8
9
(9) (a) Hamuro, Y.; Geib, S. J.; Hamilton, A. D. Novel folding pat-
terns in a family of oligoanthranilamides: non-peptide oligomers that
form extended helical secondary structures. J. Am. Chem. Soc. 1997,
119, 10587. (b) Zhu, J.; Parra, R. D.; Zeng, H. Q.; Skrzypczak-
Jankun, E.; Zeng, X. C.; Gong, B. A New Class of Folding Oligo-
mers: Crescent Oligoamides. J. Am. Chem. Soc. 2000, 122, 4219. (c)
Jiang, H.; Léger, J. M.; Huc, I. Aromatic δ-Peptides. J. Am. Chem.
Soc. 2003, 125, 3448. (d) Yan, Y.; Qin, B.; Shu, Y. L.; Chen, X. Y.;
Yip, Y. K.; Zhang, D. W.; Su, H. B.; Zeng, H. Q. Helical Organiza-
tion in foldable aromatic oligoamides by a continuous hydrogen-
bonding network. Org. Lett. 2009, 11, 1201. (e) Singleton, M. L.;
Pirotte, G.; Kauffmann, B.; Ferrand, Y.; Huc, I. Increasing the size of
an aromatic helical foldamer cavity by strand intercalation. Angew.
Chem. Int. Ed. 2014, 53, 13140. (f) Burslem, G. M.; Kyle, H. F.;
Prabhakaran, P.; Breeze, A. L.; Edwards, T. A.; Nelson, A. S.; War-
riner, S. L.; Wilson, A. J. Synthesis of highly functionalized oligo-
benzamide proteomimetic foldamers by late stage introduction of
sensitive groups. Org. Biomol. Chem. 2016, 14, 3782. (g) Meisel,
JW.; Hu, C. H. T.; Hamilton, A.D. Mimicry of a β-hairpin turn by a
nonpeptidic laterally flexible foldamer. Org. Lett. 2018, 20, 3879. (h)
Urushibara, K.; Ferrand, Y.; Liu, Z. W.; Masu, H.; Pophristic, V.;
Tanatani, A.; Huc, I. Frustrated helicity: Joining the diverging ends of
a stable aromatic amide helix to form a fluxional macrocycle. Angew.
Chem. Int. Ed. 2018, 57, 7888. (i) Liu, C. Z.; Koppireddi, S.; Wang,
H.; Zhang, D. W.; Li, Z. T. Halogen Bonding Directed Supramolecu-
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Foldamers. Angew. Chem. Int. Ed. 2019, 58, 226.
Notes
The authors declare no competing financial interest.
Author contributions
#Y. K. Z. and Y. L. Z. contributed equally.
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ACKNOWLEDGMENT
This work was supported by the US Natural Science Foundation
(CBET-1512164 to BG; MCB-1615570 to TS), the American
Chemical Society – Petroleum Research Fund (PRF# 70641-ND,
to BG), the Natural Science Foundation of China (NSFC
21072187 and 20772123, to JZ), the Silbert Fellowship from the
University at Buffalo, SUNY (to DPM), and the Center of Com-
putational Research, CCR, at the University at Buffalo (to EZ).
We thank Jabin Gong for creating the art work.
REFERENCES
(1) (a) Gellman, S. H. Foldamers: a manifesto. Acc. Chem. Res.
1998, 31, 173. (b) Seebach, D.; Matthews, J. L. β-Peptides: a surprise
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