Can Helical Peptides Unwind One Turn at a Time? - Controlled Conformational Transitions in α,β2,3-Hybrid Peptides

Article abstract of DOI:10.1002/chem.201501198

Unfolding of helical trans-β^2,3-hybrid peptides with (α-β)_nα composition, when executed by increasing solvent polarity or temperature, proceeded in a systematic manner with the turns unwinding sequentially; C-terminal region of these peptides were first to unwind and the process propagated towards N terminus with more and more β residues equilibrating from the gauche to the anti rotameric state across C_α￡C_β. This is evidenced by clear change in their C_βH signal splitting, ³J_CαH-CβH values, and sequential disappearance of i,i+2 NOEs. Helix-strand hybrid: Unfolding of helical trans-β^2,3-hybrid peptides with (α-β)_nα composition when executed by increasing solvent polarity or temperature, proceeded in a systematic manner with the turns unwinding sequentially; the C-terminal region of these peptides were first to unwind and the process propagated towards the N terminus with more and more β residues equilibrating from gauche to anti rotameric states across C_α￡C_β.

Full text of DOI:10.1002/chem.201501198

DOI: 10.1002/chem.201501198
Communication
&
Protein Folding
Can Helical Peptides Unwind One Turn at a Time? - Controlled
Conformational Transitions in a,b2,3-Hybrid Peptides
Dhayalan Balamurugan and Kannoth M. Muraleedharan*[a]
tyric acid (Aib); the latter is a known promoter of helical struc-
Abstract: Unfolding of helical trans-b2,3-hybrid peptides
tures in peptides.[1l,5] Since these building-blocks have an inter-
with (a–b)na composition, when executed by increasing
mediate flexibility compared to pre-organized (e.g., cyclopen-
solvent polarity or temperature, proceeded in a systematic
tane amino carboxylic acid) and a,b-unsubstituted building-
manner with the turns unwinding sequentially; C-terminal
blocks, we expected their peptides to be useful in studying
region of these peptides were first to unwind and the pro-
partially folded structures. Initially, we looked at the conforma-
cess propagated towards N terminus with more and more
tional preferences of oligomers 1–4 with (a–b)na composition
b residues equilibrating from the gauche to the anti rota-
(Figure 1); previous literature reports as well as results from
meric state across CaÀCb. This is evidenced by clear
3
change in their CbH signal splitting, JCaH–CbH values, and
sequential disappearance of i,i+2 NOEs.
Efforts over the past couple of decades have unraveled the
ability of b- and g-amino acids to assume biologically relevant
conformations, such as helix, strand, and turns.[1] Incorporation
of natural a-amino acids in the design has not only increased
Figure 1. Chemical structures of a,b-hybrid peptide benzyl esters 1 and 3
their structural diversity but also has paved way for the devel-
opment of hybrid systems capable of targeting specific biomo-
lecular recognition events.[2] Results accumulated during these
years tend to show many parallels in the folding behavior of
synthetic and natural peptides, which gives confidence in
using them as models to understand how solvation, secondary
interactions, and entropy act in concert during folding and un-
folding processes.[3] Folding can be viewed as a cooperative
process, during which the energy advantage through intramo-
lecular secondary interactions makes corrections for the loss in
entropy.[4] In the case of short helices, folding/unfolding, in
principle, can happen step-wise or as a single event. Because
the time scales in which the transitions happen are fast, it may
be difficult to get a closer look at these processes. However, if
the torsions accessible to the amino acid residues are restrict-
ed through steric and/or electronic factors, one could expect
stabilization of partially folded conformations and a control
over their transitions. Results along these lines from our stud-
ies on a selected group of trans-b2,3-hybrid peptides are dis-
cussed below.
and benzyl amides 2 and 4 chosen for the study.
our own studies have indicated that the availability of an extra
hydrogen bond donor at the C terminus can have dramatic in-
fluence on the folding profile of this class of compounds.[5a,6]
Typically, three intramolecular hydrogen bonds that can stabi-
lize 11-helical conformation are possible in 1 [Boc-C=O···NH(3),
CO(1)···NH(4), and CO(2)···NH(5)], whereas 2, having an addi-
tional NH at the C terminus, could accommodate four such
bonds [Boc-C=O···NH(3), CO(1)···NH(4), CO(2)···NH(5), and
Table 1. Selected backbone torsions of hybrid peptides 1 and 2.
Peptide Torsion angle Residues
Our previous efforts revealed the ability of trans-b2,3-amino
acid residues to choose a gauche conformation across CaÀCb in
response to intramolecular hydrogen bonding, and facilitate
11-helical conformation in their 1:1 hybrids with a-aminoisobu-
Aib(1)
b
2,3-Aa (2) Aib(3)
b
2,3-Aa (4) Aib(5)
1[a]
f
À57.9 À110.1
À55.5 À141.7
À56.4
–
À50.2
67.1
–
q[b]
y
–
85.9
–
176.1
125.5
À36.7
À73.5
À38.7
2
f
À60.9 À110.8
À57.0 À119.3
q[b]
y
–
80.8
–
171.4
134.8
[a] Dr. D. Balamurugan, Dr. K. M. Muraleedharan
Department of Chemistry
À30.7
À69.7
À40.4
28.6
Indian Institute of Technology Madras, Chennai, 600036 (India)
E-mail: mkm@iitm.ac.in
[a] Data representative of one of the two isomorphs present in the unit
cell. [b] Values in boldface indicate gauche conformational preference of
these b residues.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501198.
Chem. Eur. J. 2015, 21, 9332 – 9338
9332
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
CO(3)···NH(Bn)]. However, folding
will happen only if these interac-
tions give impetus for a confor-
mational shift from anti to
gauche across CaÀCb in one or
both of b residues. Peptides 3
and 4 are the next higher oligo-
mers, which can choose to exist
either as a full-length helix or in
a
partially folded form, with
lesser number of turns.
The hybrid peptide benzyl
esters 1 and 3 and their benzyl
amides 2 and 4 were prepared
through solution-phase protocol
and characterized by spectro-
scopic and mass spectrometric
techniques (see the Supporting
Information). Efforts to get infor-
mation on their conformations
in solid and solution states were
then initiated. Out of various at-
tempts, we succeeded in getting
good quality crystals of 1 from
a CHCl3/iPrOH (1:1) solution for
X-ray diffraction analysis.[7] As ex-
pected on the grounds of avail-
able hydrogen bonding interac-
tions, it was found to adopt
a partially folded structure, with
the second b unit in gauche and
the 4th b unit in anti conforma-
tion (Table 1 and Figure 2c).
There were two hydrogen
bonds, involving Boc-C=O···NH(3)
and CO(1)···NH(4), which stabi-
lized a 11-helix in the first half of
the molecule with the remaining
Figure 2. a–d) Chemical- and X-ray crystal structures of peptides 1 and 2. Dotted arrow lines in b) show non-se-
part
extended, making it quential i,i+2 NOEs in its ROESY (CDCl3) spectrum. Solid arrow lines show intramolecular hydrogen bondings in
the crystal structures. e) and f) Organization of molecules in the lattices of 1 and 2 respectively; H-bonding inter-
a hybrid of helix and strand!
(Figure 2c and 2e). In fact, crys-
tal structure of its next higher
homologue with (a–b)3 composi-
actions are shown by dotted lines. Non-relevant hydrogen atoms have been removed for clarity. Co-crystallized
methanol molecules in the crystal structure of 2 are shown as solid-spheres.
tion, that was reported earlier (crystals grown from CHCl3 solu-
Supporting Information). Interestingly, the spectrum recorded
tion) had all the four expected intramolecular H-bonds [Boc-C= in [D8]toluene/CDCl3 mixture (4:1) had well resolved peaks.
O···NH(3), CO(1)···NH(4), CO(2)···NH(5), and CO(3)···NH(6)], which
stabilized a 11-helical conformation.[5a] In comparison with this,
the peptide 1 presented here is shorter by one b residue at
the C terminus. This difference has, interestingly, caused partial
unwinding, with the N-terminal region still holding the first
two sets of H-bonds, which is noteworthy. To know the prefer-
ence in the solution state, the 1H NMR and ROESY spectra
were analyzed simultaneously. Normally, information on CaÀCb
torsions in such systems (and, hence, an indirect information
There were two clear doublets of doublets at d=5.28 and
5.24 ppm, corresponding to 2nd and 4th CbHs, respectively.
J values of 4.0 and 5.5 Hz for their coupling with Ca protons in-
dicated tendency to equilibrate to gauche rotameric states in
these b residues. The 1H NMR spectrum of its higher homo-
logue 3 gave good dispersion when recorded in [D8]toluene/
CDCl3 mixture (4:1) and had three clear doublets of doublets
3
for CbHs. JCaH–CbH values for the CbHs were in the range of 3.5–
5.5 Hz, again indicative of their equilibration to gauche confor-
mation in this solvent mixture. Although these indicated
a drive towards folding in 1 and 3, no supporting NOEs were
seen in their ROESY spectra. Hence, the amide derivatives 2
3
on local folding) can be obtained from JCaH–CbH. However, poor
1H NMR resonance dispersion in CDCl3 made analysis of the
torsions as well as NOE interpretation difficult (Figure S9 in the
Chem. Eur. J. 2015, 21, 9332 – 9338
www.chemeurj.org
9333
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
cleation and lattice develop-
ment. Since our attempts to
crystallize 1 and 2 from CHCl3
were not fruitful, a direct com-
parison of their solid-state struc-
tures with that of hexapeptide
homologue[5a] could not be
made: Nevertheless, the ob-
served difference is attributable
to the difference in the chemical
environment during crystalliza-
tion. Important crystallographic
data of 1 and 2 are presented in
Table 2.
Supramolecular organizations
of peptide helices and strands
individually are abundant in liter-
ature.[1e,9] Having
a hybrid of
these two conformations, 1 and
2 appeared useful to understand
1
new
preferences,
hitherto
Figure 3. a) Relevant regions from the H NMR spectra of pentapeptide benzylamide 2 recorded in varying pro-
unseen. Analysis of their lattices
showed two intermolecular hy-
drogen bonds in each case [C=
portions of [D6]DMSO in CDCl3 (i-v); percentage of [D6]DMSO is indicated on the left hand side. Selected CbH re-
gions showing the temperature effect on 2 in: b) 75% [D6]DMSO/CDCl3, and c) in 100% CDCl3.
and 4, which gave well-dispersed NMR spectra, were chosen
Table 2. Hydrogen bond lengths and angles in the crystal structures of
peptides 1 and 2.
for looking at partially folded structures and conformational
transitions.
The 1H NMR signals of 2 in CDCl3 were well resolved and
peak assignments were made using a combination of 1H,
COSY, TOCSY, and ROESY experiments (Figure 3a(i)). Here, the
2nd CbH appeared as an apparent doublet at d=5.49 ppm
Peptide
Type
H···O [Š] N···O [Š] aN-H···O [8]
1
Boc(C=O)···HN(3) intra 2.197
3.035
2.962
2.969
3.038
3.033
2.919
2.979
2.886
164.78
152.61
162.13
147.89
163.18
163.35
175.74
166.08
C=O(1)···HN(4)
C=O(3)···HN(1)
C=O(5)···HN(5)
2.172
inter 2.138
2.275
3
(3JCaH–CbH very small and JNH–CbH about 10.0 Hz), whereas the
2
Boc(C=O)···HN(3) intra 2.200
4th CbH gave a clear doublet of doublet with J values of 3.5
and 9.5 Hz (Figure S3 in the Supporting Information). Appear-
ance of these peaks suggested 2nd and 4th b residues as
having gauche conformational preference. This, along with two
C=O(1)···HN(4)
C=O(3)···HN(1)
C=O(5)···HN(5)
2.085
inter 2.121
2.044
2
4
4
6
long-range NOEs between CbH! NH and CbH! NH suggest-
ed 11-helical conformation in this solvent (Figure S12 in the
Supporting Information).[8] This was anticipated, because this
peptide (2) has all hydrogen bonding partners, as in the hexa-
peptide with (a–b)3 composition mentioned earlier.[5a] The fact
that a hydrogen bonding solvent can cause partial unwinding
and assist lattice assembly became clear when crystals ob-
tained from a CHCl3/MeOH (1:1) mixture were subjected to X-
ray diffraction analysis.
O(3)···HN(1) and C=O(5)···HN(5)], apart from the intramolecular
hydrogen bonds mentioned above. Of these, the C=
O(3)···HN(1) interaction was between molecules arranged one
above the other, which aligned the helical parts vertically,
whereas C=O(5)···HN(5) interaction was between molecules
positioned horizontally, and gave a sheet-like arrangement typ-
ical of b strands (Figure 2e,f). Such a combination of extreme
conformations in a single lattice, with supramolecular arrange-
ment through segregation of helical and extended regions, is
exceptional and probably the first of its kind.
Unlike the situation in CDCl3, presence of methanol during
crystallization of 2 led to partial unwinding of the helix to give
the hybrid structure shown in Figure 2d; there were two intra-
molecular [Boc(C=O)···HN(3) and C=O(1)···HN(4)] hydrogen
bonds as seen in the solid-state structure of 1. But the lattice
of 2 was distinguished by the presence of methanol as part of
With these preliminary results, we set out to look at the
presence of partially unfolded structures of these peptides in
CDCl3, by using [D6]DMSO as the co-solvent. DMSO titration is
a standard procedure to locate the solvent-exposed NHs. By
using the same logic, we envisaged that partial unfolding in
the presence of this solvent, if at all, will happen in such way
that more labile hydrogen bond(s) are disrupted first, which
2
the lattice, establishing hydrogen bonding to NH of each mol-
ecule, as shown in Figure 2 f. This lattice-bound MeOH may
not be the sole contributor of partial unwinding, and many
such interactions in the C-terminal region might have given
way for intermolecular CO···HN hydrogen bonding during nu-
3
can be monitored by looking at JCaH–CbH values as well as NOEs
along the backbone.
Chem. Eur. J. 2015, 21, 9332 – 9338
www.chemeurj.org
9334
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
1
The H NMR spectrum of peptide 2 in 50% [D6]DMSO/CDCl3
298 and 328 K was carried out. As evident from Figure 3b, the
2
had a well-dispersed amide region along with two broad
peaks for their Cb hydrogens (Figure 3a(iii)). ROESY experiment
at this [D6]DMSO concentration had two long range NOEs
splitting pattern of CbH became more refined and changed to
apparent triplet with equal J value for CaH–CbH and CbH–NH
couplings (8.5 Hz) on increasing the temperature to 323 K.
During this, the signal for 4CbH remained intact, suggesting
that the transition happens mainly in the first half of the mole-
cule (Figure 3b). When a similar experiment was done for
a sample in 100% CDCl3, we could see a refinement of splitting
(2CbH! NH and 4CbH! NH) as in the spectrum recorded in
4
6
CDCl3 (Figure S24 in the Supporting Information). Further in-
4
crease in [D6]DMSO content to 75% changed the CbH splitting
into apparent triplet with equal J values of 9.2 Hz for their cou-
plings with CaH and NH peaks. This indicated the anti confor-
mational preference of 4th Cb unit and an extended structure
in the second half of the molecule. At the same time, 2nd CbH
signal remained broad. Whereas this suggested conformational
exchange between the gauche and the anti forms in the
second residue, the ROESY spectrum recorded in this solvent
composition (75% [D6]DMSO, Figure 4) had the long range
3
pattern without any significant change in JCbH–CaH values,
showing better stability of 11-helical structure under this con-
dition (Figure 3c).
1
The H NMR signals of the higher homologue 4 (Figure 5) re-
corded in CDCl3 were also well resolved. Its 2nd, 4th, and 6th
CbH signals appeared at d=5.55, 5.65, and 5.45 ppm, respec-
tively, as apparent doublets with J values of 10.0, 10.0, and
4
3
2CbH! NH NOE in support of a preference for 11-helical con-
9.0 Hz (for JNH), respectively. This suggests that their coupling
formation in the first half.[8] Thus, the partially unfolded confor-
constants with the adjacent CaHs (3JCbH–CaH) are small (Fig-
ure 6a(i)), which indicates their
gauche conformational prefer-
ence. The ROESY spectrum re-
corded in CDCl3 showed three
non-sequential
i,i+2
NOEs
4
6
(2CbH! NH, 4CbH! NH and
8
6CbH! NH), characteristic of 11-
helical structure (Figure 5). At-
tempts to get good quality crys-
tals of this compound for X-ray
diffraction analysis were not
fruitful.
This peptide (4), having three
pairs of intramolecular hydrogen
bonding, seemed ideal to delve
deeper in to the process of step-
wise unwinding. If such a process
really happens, each of the long
2
4
4
range NOEs, CbH! NH, CbH!
6NH, and CbH! NH, should dis-
appear one by one on increasing
the [D6]DMSO content; this
should also be accompanied by
a concomitant change in the
6
8
3
splitting pattern and JCaH–CbH
values of CbHs from one end to
the other.
Figure 4. Relevant regions from ROESY spectra of 2 recorded in 100% CDCl3 and 75% [D6]DMSO/CDCl3 suggestive
of ‘step-wise’ unwinding; variation in JCaH–CbH values of 2nd and 4th b residues are also shown.
3
Changes in CbH signal splitting
pattern on increasing [D6]DMSO
mation that was seen in the solid state seems to also exist in
solution on appropriate solvation. Further increase in the
[D6]DMSO concentration to 100% caused complete disruption
of all the H-bonds, resulting in an extended structure, which is
3
evidenced by large JCaH–CbH values of second and fourth b resi-
4
dues, and disappearance of NOEs between 2CbH! NH and
6
4CbH! NH.
At this stage, it was important to see whether a small tem-
Figure 5. Chemical structure of peptide 4. Dotted arrow lines show non-se-
quential i,i+2 NOEs in its ROESY spectrum recorded in CDCl3. Intramolecular
hydrogen bonds proposed based on NMR data are shown by solid arrow
lines.
perature jump can change the partially folded state of sample
in 75% [D6]DMSO/CDCl3 to a completely extended structure.
Towards this, a variable temperature (VT) NMR study between
Chem. Eur. J. 2015, 21, 9332 – 9338
www.chemeurj.org
9335
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
a shift towards anti conforma-
tion in these residues and com-
plete unfolding. Overall, use of
50% [D6]DMSO has caused the
peptide to unwind in the C ter-
minus, with the N-terminal
region equilibrating more to-
wards helical form due to larger
proportion of gauche rotameric
states in 2nd b residue. Increas-
ing the [D6]DMSO content to
75% could give a similar effect,
and the outcome is presented in
Figure 6a(iv). Here, both 4th and
6th CbHs were apparent triplets,
showing extended conformation
in that segment, whereas the
2CbH signal was broad. As in the
previous case, increasing tem-
perature caused this also to
transform to apparent triplet as
shown in Figure 6e.
Nucleation of secondary struc-
tures, directed through specific
sets of intramolecular secondary
interactions and facilitated by
appropriate backbone/side-chain
torsions, is central to protein
folding process.[4b,10] The guiding
principles involved seem to be
applicable to oligomers from
1
Figure 6. a) Relevant regions from the H NMR spectra of hepta peptide- benzylamide 4 recorded in varying pro-
portions of [D6]DMSO in CDCl3 (i–v); percentage of [D6]DMSO is indicated on the left hand side. b) Expanded
region of ROESY spectrum recorded in 50% [D6]DMSO/CDCl3 showing 2CbH! NH NOE; Selected CbH regions
4
showing the temperature effect on 4 in c) 100% CDCl3, d) 50% [D6]DMSO/CDCl3 and, e) 75% [D6]DMSO/CDCl3.
synthetic
amino
acids
as
well.[5e,11] Although both secon-
content from 0 to 100% and the effect of temperature are
shown in Figure 6. As evident from Figure 6c, the apparent
doublets observed for all CbHs in CDCl3 and their
3JCaH–CbH values remained largely unaffected on increasing the
temperature from 298 to 323 K, showing good helical prefer-
ence along the entire backbone. Although the dispersion of
the NMR signals was poor at 25% [D6]DMSO, there was im-
dary and super-secondary structures based on peptides from
b- and g-amino acids have been reported,[1k,l] such synthetic
systems have rarely been used for understanding the folding/
unfolding pathways. Previous observations in this area include:
the existence of partially folded structure in oligocarbamate
foldamers belonging to g-peptide superfamily,[12] zig-zag tap-
like structures in the hybrid foldamer of leucine with 8-amino-
2-quinolinecarboxylic acid,[13] inversion of helix handedness in
aromatic oligoamide foldamers based on 8-amino-2-quinoline
carboxylic acid,[14] ‘non-cooperative’ unfolding in b-peptide fol-
damers reported by Gademann et al.,[15] accessibility of both
10- and 14-helical conformations in trans-2-amino-cyclohexa-
necarboxylic acid-based foldamers[16] and a/310-helix dimor-
phism in Na-acylated heptapeptide amide, Ac-[l-(aMe)Val]7-
NHiPr which contain Ca-methyl-l-valine as the building
block.[17] The details presented in this manuscript are part of
our efforts along similar lines. Among the benzyl esters and
amides, NMR signal dispersion was better in the latter and,
hence, they were chosen for monitoring the unfolding process
upon changing the solvent polarity and/or increasing tempera-
ture. Since the splitting pattern of CbH signal was diagnostic of
the q torsion of that residue, it was possible to get information
on the extent of unfolding by looking at this signal from differ-
ent b residues. On increasing the solvent polarity, each of the
6
provement on increasing it to 50%. Notably, the CbH signal
changed its splitting to apparent triplet at this concentration,
2
4
whereas those for CbH and CbH were broad. This suggest anti
conformation across CaÀCb in the former and some degree of
equilibration between gauche and anti forms in the latter two
2
4
residues. Remarkably, the CbH! NH NOE was still present in
the ROESY spectrum, pointing towards helical preference in
the N-terminal region (Figure 6b).[8] Since there was only d=
4
6
0.02 ppm difference in the chemical shifts of NH and NH sig-
4
6
nals, the NOE between CbH! NH was difficult to distinguish
6
(Figure 6b). As expected, based on the splitting of CbH signal,
6
8
the NOE between CbH! NH was absent, suggesting an ex-
tended C-terminal region. Figure 6d shows the effect of tem-
perature on the splitting pattern of CbH signals at this
[D6]DMSO concentration (50%). The 2nd and 4th CbH signals,
which were broad, became more refined and emerged as ap-
parent triplets on rising the temperature to 323 K, suggesting
Chem. Eur. J. 2015, 21, 9332 – 9338
www.chemeurj.org
9336
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1997, 119, 11719; c) D.
Seebach, S. Abele, K. Gademann, G. Guichard, T. Hintermann, B. Jaun,
J. L. Matthews, J. V. Schreiber, L. Oberer, U. Hommel, H. Widmer, Helv.
Chim. Acta 1998, 81, 932; d) S. Hanessian, X. Luo, R. Schaum, S. Mich-
nick, J. Am. Chem. Soc. 1998, 120, 8569; e) D. Seebach, S. Abele, K. Ga-
demann, B. Jaun, Angew. Chem. Int. Ed. 1999, 38, 1595–1597; Angew.
Chem. 1999, 111, 1700–1703; f) R. P. Cheng, S. H. Gellman, W. F. DeGra-
do, Chem. Rev. 2001, 101, 3219; g) X. Daura, K. Gademann, H. Schaefer,
B. Jaun, D. Seebach, W. F. van Gunsteren, J. Am. Chem. Soc. 2001, 123,
2393; h) D. Seebach, M. Brenner, M. Rueping, B. Schweizer, B. Jaun,
Chem. Commun. 2001, 207; i) D. Seebach, M. Brenner, M. Rueping, B.
Jaun, Chem. Eur. J. 2002, 8, 573; j) T. A. Martinek, F. Fülçp, Eur. J. Bio-
chem. 2003, 270, 3657; k) D. Seebach, A. K. Beck, D. J. Bierbaum, Chem.
Biodiversity 2004, 1, 1111; l) P. G. Vasudev, S. Chatterjee, N. Shamala, P.
Balaram, Chem. Rev. 2011, 111, 657.
CbH signal was found to change from apparent doublet to ap-
parent triplet indicating gauche-to-anti shift in a systematic
manner. Increased population of partially folded conformations
was also seen, which is indicated by the relevant NOEs in their
ROESY spectrum.
A comparison of the conformational preferences of peptides
presented here with those of a,b2,3-hybrid peptides with (a–b)n
composition reported previously is also important. The tetra-,
hexa-, and octa-peptides belonging to the latter group had
shown good tendency to adopt 11-helical structures; all their
internal b residues were found to adopt gauche conformation
across CaÀCb to facilitate intramolecular hydrogen bonding,
leaving the terminal one in anti conformation.[5a] The drive to
undergo anti to gauche shift in response to hydrogen bonding
was evident from the conformations of tripeptide benzyl ester
(strand) and the corresponding benzylamide (11-helix) in the
same series. The present work has taken the study to the next
level and has shown that higher homologues with (a–b)na
composition, though largely helical in CDCl3 solution, can exist
in partially unfolded conformation on appropriate solvation
and/or rise in temperature. More importantly, it was possible
to execute the transition from a folded- to completely unfold-
ed state in a systematic manner through stepwise gauche-to-
anti shift of CaÀCb torsions in b residues from C to N terminus,
which manifested as sequential unfolding of helical turns.
To summarize, the present study involving trans-b2,3-hybrid
peptides gives a closer look at folding/unfolding process in re-
sponse to variation in solvent polarity and temperature. X-ray
crystallography and NMR spectroscopy of penta- and hepta-
peptide C-terminal amides from this series show that specific
sets of hydrogen bonds, stabilizing each of the helical folds,
can be disrupted in a sequential manner through incremental
variation in solvent polarity and/or temperature. In CDCl3,
these peptides have an 11-helical conformation, but increase
of [D6]DMSO content from 0–100% caused a concomitant dis-
ruption of hydrogen bonds from C-to N-terminal region, lead-
ing to gradual unwinding of the helical structure. The partially
folded intermediate conformations seem to have extended C-
terminal region, whereas the b residues located towards the N
terminus showed greater tendency to adopt gauche conforma-
tion and retain i,i+3 C=O···HN intramolecular H-bonds.
[2] a) M. A. Schmitt, B. Weisblum, S. H. Gellman, J. Am. Chem. Soc. 2004,
ˇ
126, 6848; b) D. F. Hook, P. Bindschädler, Y. R. Mahajan, R. Sebesta, P.
Kast, D. Seebach, Chem. Biodiversity 2005, 2, 591; c) M. A. Schmitt, B.
Weisblum, S. H. Gellman, J. Am. Chem. Soc. 2007, 129, 417; d) J. D.
Sadowsky, W. D. Fairlie, E. B. Hadley, H.-S. Lee, N. Umezawa, Z. Nikolov-
ska-Coleska, S. Wang, D. C. S. Huang, Y. Tomita, S. H. Gellman, J. Am.
Chem. Soc. 2007, 129, 139; e) W. S. Horne, S. H. Gellman, Acc. Chem. Res.
2008, 41, 1399; f) W. S. Horne, L. M. Johnson, T. J. Ketas, P. J. Klasse, M.
Lu, J. P. Moore, S. H. Gellmana, Proc. Natl. Acad. Sci. USA 2009, 106,
14751; g) L. K. A. Pilsl, O. Reiser, Amino Acids 2011, 41, 709.
[3] a) D. H. Appella, L. A. Christianson, D. A. Klein, M. R. Richards, D. R.
Powell, S. H. Gellman, J. Am. Chem. Soc. 1999, 121, 7574; b) Y. J. Chung,
B. R. Huck, L. A. Christianson, H. E. Stanger, S. Krauthauser, D. R. Powell,
S. H. Gellman, J. Am. Chem. Soc. 2000, 122, 3995; c) S. H. Choi, I. A.
Guzei, L. C. Spencer, S. H. Gellman, J. Am. Chem. Soc. 2008, 130, 6544.
[4] a) G. D. Rose, P. J. Fleming, J. R. Banavar, A. Maritan, Proc. Natl. Acad. Sci.
USA 2006, 103, 16623; b) R. L. Baldwin, J. Mol. Biol. 2007, 371, 283.
[5] a) D. Balamurugan, K. M. Muraleedharan, Chem. Eur. J. 2012, 18, 9516;
b) B. V. Venkataram Prasad, P. Balaram, CRC Crit. Rev. Biochem. 1984, 16,
307; c) I. L. Karle, P. Balaram, Biochemistry 1990, 29, 6747; d) C. Toniolo,
E. Benedetti, Macromolecules 1991, 24, 4004; e) J. Venkatraman, S. C.
Shankaramma, P. Balaram, Chem. Rev. 2001, 101, 3131.
[6] a) P. Maity, M. Zabel, B. Kçnig, J. Org. Chem. 2007, 72, 8046; b) S. H.
Choi, I. A. Guzei, S. H. Gellman, J. Am. Chem. Soc. 2007, 129, 13780; c) A.
Bandyopadhyay, H. N. Gopi, Org. Lett. 2012, 14, 2770.
[7] There were two isomorphs in the unit cell of 1. Both had their 2nd
b residue in gauche and the 4th b residue in anti conformations, and
had overall ‘helix-strand hybrid’ conformation. Crystal data for 1: from
CHCl3/iPrOH, C48H67N5O8; Mr =842.07; crystal size 0.35”0.20”0.15 mm;
colorless needles; monoclinic; space group P21; a=21.291(9), b=
10.585(3), c=23.222(7) Š; a=90, b=90.059(14), g=908; V=5233(3) Š3;
Z=4, T=298 K; MoKa =0.71073; 0.88<q<26.53, 13928 reflections
measured; 9079 unique reflections; R1=0.1100 and wR2=0.2408;
GOF=1.21; Crystal data for 2: from CHCl3/MeOH, C49H71N6O8; Mr =
872.12; 0.25”0.20”0.15 mm; colorless needles; orthorhombic; space
group P212121; a=10.4792(6), b=20.2449(12), c=25.0931(6) Š; a=90,
b=90, g=908; V=5323.5(6) Š3; Z=4; T=298 K; MoKa =0.71073; 1.91<
q<17.55; 8188 reflections measured; 3387 unique reflections; R1=
0.0508 and wR2=0.1293; GOF=0.981. CCDC-945944 (1) and CCDC-
945945 (2) contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
[8] The non-sequential NOEs observed in the present study were either in
the medium or weak range.
[9] a) D. H. Appella, L. A. Christianson, I. L. Karle, D. R. Powell, S. H. Gellman,
J. Am. Chem. Soc. 1999, 121, 6206; b) J. S. Nowick, Acc. Chem. Res. 2008,
41, 1319.
[10] a) J. K. Myers, T. G. Oas, Nat. Struct. Biol. 2001, 8, 552; b) S. E. Miller, A. M.
Watkins, N. R. Kallenbach, P. S. Arora, Proc. Natl. Acad. Sci. USA 2014,
111, 6636.
[11] T. A. Martinek, F. Fülçp, Chem. Soc. Rev. 2012, 41, 687.
[12] Y. R. Nelli, L. Fischer, G. W. Collie, B. Kauffmann, G. Guichard, Biopolymers
2013, 100, 687.
Acknowledgements
Financial support of this work by the Department of Science
and Technology, INDIA (Grants SR/S1/OC-13/2007), instrumen-
tation facility by IIT Madras, and DST-FIST (New Delhi) are
gratefully acknowledged. We thank Mr. V. Ramkumar, Depart-
ment of Chemistry for X-ray diffraction analysis. D. Balamuru-
gan thanks DST and IITM for the research fellowship.
Keywords: alpha/beta-hybrid peptides
·
amino acids
·
foldamers · helix-strand hybrid · protein folding
[13] M. Kudo, V. Maurizot, H. Masu, A. Tanatani, I. Huc, Chem. Commun.
2014, 50, 10090.
[1] a) D. H. Appella, L. A. Christianson, I. L. Karle, D. R. Powell, S. H. Gellman,
J. Am. Chem. Soc. 1996, 118, 13071; b) S. Krauthäuser, L. A. Christianson,
Chem. Eur. J. 2015, 21, 9332 – 9338
www.chemeurj.org
9337
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
[14] N. Delsuc, T. Kawanami, J. Lefeuvre, A. Shundo, H. Ihara, M. Takafuji, I.
Huc, ChemPhysChem 2008, 9, 1882.
[17] M. Crisma, M. Saviano, A. Moretto, Q. B. Broxterman, B. Kaptein, C. To-
niolo, J. Am. Chem. Soc. 2007, 129, 15471.
[15] K. Gademann, B. Jaun, D. Seebach, R. Perozzo, L. Scapozza, G. Folkers,
Helv. Chim. Acta 1999, 82, 1.
Received: March 26, 2015
Published online on May 15, 2015
[16] A. HetØnyi, I. M. Mandity, T. A. Martinek, G. K. Toth, F. Fueloep, J. Am.
Chem. Soc. 2005, 127, 547.
Chem. Eur. J. 2015, 21, 9332 – 9338
www.chemeurj.org
9338
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim