Heterogeneous H-Bonding in a Foldamer Helix

Article abstract of DOI:10.1021/jacs.5b03382

Structural characterization of new α/γ-peptide foldamers containing the cyclically constrained γ-amino acid I is described. Crystallographic and 2D NMR analysis shows that γ residue I promotes the formation of a 12/10-helical secondary structure in α/γ-pe

Full text of DOI:10.1021/jacs.5b03382

Communication
pubs.acs.org/JACS
Heterogeneous H‑Bonding in a Foldamer Helix
Brian F. Fisher, Li Guo,† Brian S. Dolinar, Ilia A. Guzei, and Samuel H. Gellman*
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
S
* Supporting Information
bones. Such differences are inherent for helices formed by
heterogeneous backbones because of subunit diversity. Thus, for
example, an α/β-peptide contains H-bond-accepting groups
(CO) and -donating groups (N−H) from both α and β
residues. Different types of H-bonds are found also in foldameric
helices in which H-bond directionality alternates along the
backbone, whether the backbone is homogeneous (as in the β-
peptide 10/12-helix7 and 18/20-helix8) or heterogeneous (as in
the α/β-peptide 11/9-helix9 and 18/16-helix10). These systems
raise a fundamental question: are the different types of
intrahelical H-bonds comparably favorable? Here we describe a
new type of α/γ-peptide foldamer and provide the first evidence
that distinct types of H-bonds formed within a regular secondary
structure can differ in terms of favorability.
ABSTRACT: Structural characterization of new α/γ-
peptide foldamers containing the cyclically constrained γ-
amino acid I is described. Crystallographic and 2D NMR
analysis shows that γ residue I promotes the formation of a
12/10-helical secondary structure in α/γ-peptides. This
helix contains two different types of internal H-bond, and
the data show that the 12-atom CO(i) → H−N(i+3) H-
bond is more favorable than the 10-atom CO(i) → H−
N(i−1) H-bond. Several foldamer helices featuring
topologically distinct H-bonds have been discovered, but
our findings are the first to show that such H-bonds may
differ in their favorability.
The new foldamers contain ring-constrained γ residues of type
I (Figure 1). Either enantiomer of the necessary γ-amino acid
iology relies on proteins and nucleic acids to carry out
B
diverse functions, most of which require the biopolymer to
adopt a complex and specific folding pattern. The relationship
between sophisticated function and higher-order structure has
inspired many groups to seek nonbiological oligomers that favor
specific conformations (“foldamers”).1 Extrapolation from the
poly-α-amino acid backbone of proteins has led to the study of β-
peptides, γ-peptides, and higher homologues.2 Secondary
structural motifs reminiscent of (but geometrically distinct
from) those well-known in proteins, including helices, sheets,
and reverse turns, have been characterized for these new
backbones, and approaches to tertiary structure have been
reported.3 The folding rules established for β- and γ-peptides
have enabled the development of specific examples that display
biomimetic function.4
Expansion beyond the biopolymer prototypes allows deviation
from particular structural parameters associated with proteins
and nucleic acids. The backbones of these biopolymers, for
example, are homogeneous in that each subunit is drawn from a
single chemical class (e.g., α-amino acids), but heterogeneous
backbones are readily accessed among synthetic oligomers.5 A
variety of discrete secondary structures have been identified
among peptidic foldamers with mixed backbones, including
combinations of α + β residues or α + γ residues. For some
applications, such as functional mimicry of a natural α-helix,
heterogeneous backbones containing both α and β residues have
proven to be superior to the homogeneous β residue backbone.6
Within the regular helices found in proteins, each type of
internal non-covalent contact is topologically equivalent across
all of the subunits involved. In an α-helix, for example, all of the
CO(i) → H−N(i+4) H-bonds should be comparable to one
another, excluding terminal effects, since the CO and H−N
groups are all similar. In contrast, different types of internal H-
bonds occur within many of the helices that have been
documented among peptidic foldamers with unnatural back-
Figure 1. (a) Comparison of cyclic γ-amino acids studied by our group.
(b) Inversion of the Cγ stereocenter of II, forming I, is predicted to
invert the adjacent amide H-bond directionality in the full-length H-
bonded foldamer.
building block can be efficiently prepared.11 Previous character-
ization of oligomers containing the stereoisomeric γ residue of
type II established that this subunit, alone12 or in alternation with
α or β residues,11,13 favors helices defined by CO(i) → H−N(i
+3) H-bonds. The present studies of α/γ-peptides containing I
were intended to test the generality of the “stereochemical
patterning” hypothesis of Martinek, Fulop, and co-workers,14
̈
̈
who proposed a correlation between H-bond directionality
within foldamer helices and the signs of the torsion angles about
the bonds that flank amide linkages (ψ,ϕ pairs from adjacent
residues). If these torsion angles are all of the same sign, then all
of the intrahelical H-bonds should be oriented in the same
direction (as observed, for example in the α-helix). If the torsion
Received: March 31, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b03382
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Journal of the American Chemical Society
Communication
angles alternate between neighboring amides (i.e., if amides that
have positive flanking torsion angles are adjacent to amides that
have negative flanking torsion angles and vice versa), then
intrahelical H-bonds should alternate in directionality relative to
the helix axis.
crystal structures offered hints that the 12-atom H-bonds are
more favorable than the 10-atom H-bonds. Pentamer 2a (α
residues = Val) displayed a 12/10-helical conformation in which
the maximum number of amide-to-amide H-bonds was formed,
two in 12-atom rings and one in a 10-atom ring (Figure 3). All of
The Martinek−Fulop hypothesis led us to predict that altering
̈
̈
only the stereocenter adjacent to the ϕ torsion angle in γ residues
(i.e., II → I) would cause a wholesale change in helix geometry in
α/γ-peptides from a conformation with unidirectional H-bonds
to a conformation with bidirectional H-bonds. This prediction
relied on the fact that α residues can readily adopt conformations
in which the backbone torsion angles ϕ and ψ have the same sign
(α-helix) or different signs (β-sheet). To test this prediction, γ
residues of type I with absolute configuration of (S,S,R) at the
(α,β,γ) carbons had to be paired with L-α-amino acid residues
and (R,R,S)-I with D-α residues.
Our initial efforts were aimed at crystallographic character-
ization of the new α/γ(I)-peptides. Seven structures were
obtained15 for oligomers containing four to six residues, three by
racemic crystallization.16 The structures of tetramers 1a−c,
collectively, were not wholly consistent with our expectations. All
three formed a 12-atom H-bond between the N-terminal Boc
CO and N−H of the α residue closest to the C-terminus
(Figure 2). This CO(i) → H−N(i+3) interaction is
Figure 3. (a) Structures of pentamers 2a and 2b. (b) Crystal structures
of pentamers 2a and 2b. Other colors as in Figure 2.
the H-bonded N···O distances in this structure were relatively
long (>3.00 Å). The two structurally very similar symmetry-
independent conformers of pentamer 2b17 (α residues = Ala)
adopted a 12/10-helix-like conformation in the crystal, but an
ethanol molecule was found to be interpolated between the
CO and H−N groups that would have formed the 10-atom
intrahelical H-bond (Figure 3b). The two 12-atom H-bonds in
this structure featured significantly shorter N···O distances than
were found in the crystal structure of 2a.17
Hexamer rac-3 (α residues = Phe) provided two crystal forms
under slightly different solvent conditions (Figure 4). One crystal
contained a 12/10-helix that seemed to unravel toward the C-
terminus; this structure contained two short 12-atom H-bonds
(2.85/2.86 Å) and a longer 10-atom H-bond (3.02 Å). A second
Figure 2. Structures of tetramers 1a−c depicting H-bonds observed in
the crystal structures. Enantiomers of 1b and 1c are shown. α residues
are shaded in yellow, γ residues in green, and protecting groups in gray.
12-Atom H-bonds are shown in red and 10-atom H-bonds in blue. The
geometric criteria used for all of the H-bond assignments are N···O
distance < 4.0 Å and N−H···O angle > 130°.
characteristic of both the 12/10-helix we expected and the 12-
helix previously documented for α/γ-peptides containing γ
residue II. Only rac-1a (α residues = Phe), however, displayed
the 10-atom CO(i) → H−N(i−1) H-bond characteristic of
the 12/10-helix. In this structure, the 10-atom H-bond was
significantly longer than the 12-atom H-bond (N···O = 3.00 vs
2.83 Å). For 1b (α residues = Ala), a second 12-atom H-bond
formed, i.e., this α/γ-peptide adopted a 12-helix rather than a 12/
10-helix. α/γ-Peptide 1c (α residues = Ala) displayed an
interesting variation on the 10-atom H-bond: the carbonyl of
Ala-3 was linked to N−H(i−1) via an intermolecular H-bond
chain that included a water molecule along with the N-terminal
Ala carbonyl of its lattice neighbor.17
Longer α/γ-peptides containing γ residue I manifested a
stronger preference for the 12/10-helix in the solid state, but the
Figure 4. (a) Structure of hexamer 3. (b) Crystal structures 3a and 3b.
The inserted H2O molecule of 3b is also shown. Colors as in Figure 2.
B
DOI: 10.1021/jacs.5b03382
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Communication
amide−amide 10-atom H-bond would be possible toward the C-
terminus, but in this case the poor geometric parameters (N···O
distance of 4.09 Å and N−H···O angle of 135°) suggest that this
interaction, if present, is substantially weaker than all of the other
H-bonds described here.18 The other crystalline form of rac-3
revealed a 12/10-helix-like conformation in which a water
molecule was interpolated between the CO and H−N groups
that would have formed the C-terminal 10-atom H-bond. The set
of seven crystal structures collectively suggests that the new α/γ-
peptide backbone has a propensity for 12/10-helix-like
conformations but that H-bonding may be more favorable in
the 12-atom rings than in the 10-atom rings.
To gain insight into the folding propensity of α/γ(I)-peptides
in solution, we employed 2D NMR (COSY, TOCSY, and
ROESY) to evaluate the conformations in CDCl3 of five
oligomers containing from five to nine residues. Evidence for
12/10-helical folding was obtained in each case in the form of
inter-residue nuclear Overhauser effects (NOEs) between
protons separated by 7 to 12 covalent bonds. Here we focus
on nonamer 4. Three nonsequential backbone proton NOE
patterns previously reported to be consistent with 12/10-helical
secondary structure19 were observed along most or all of 4
(Figure 5). In addition, we observed a set of strong i, i + 2 NOEs
Figure 6. (a) NMR ensemble generated by CNS using distance
restraints from ROESY data for nonamer 4 in CDCl3. The 10 lowest-
energy structures were collected from 1000 trial structures. (b)
Backbone alignment of the first four residues of the minimized average
of the 10 lowest-energy NMR structures with pentamer crystal structure
2a.
The normalized t1/2 values (Table 1) are generally largest
toward the center of 5, which is consistent with the hypothesis
that this α/γ-peptide adopts a helical conformation featuring
internal H-bonds of alternating directionality. The α residue NH
resonances show larger normalized t1/2 values than do the γ
residue NH resonances. This trend is consistent with the
hypothesis that the H-bonds formed by the α residue NH groups
(12-atom rings) are more favorable than the H-bonds formed by
the γ residue NH groups (10-atom rings). Further support for the
proposed distinction between 12- and 10-atom internal H-bonds
was provided by experiments in which small aliquots of dimethyl
sulfoxide (DMSO) were added to a CDCl3 solution of octamer 5
(“DMSO titrations”).17
Figure 5. (a) Nonsequential ROESY cross-peaks observed in nonamer 4
in CDCl3. Uncertain integrals could not be reliably software-integrated.
(b) 12/10-Helical cross-peak patterns observed in 4.
The crystallographic and NMR data reported here show that
α/γ-peptide secondary structure can be engineered on the basis
of the stereochemical patterning hypothesis of Martinek, Fulop,
̈
̈
and co-workers. Altering just one configuration in the dipeptide
repeating unit of the backbone by replacing γ(II) residues with
γ(I) residues leads to a change in conformational preference
from a helix with unidirectional H-bonds to a helix in which the
H-bond direction alternates. The ability to alter the helix type by
switching a single configuration within the γ residues represents a
significant accomplishment in foldamer design.
from CαH of each Val residue to CβH of the next Val residue,
which is consistent with the 12/10-helical conformation. NMR-
restrained simulated annealing calculations were carried out for
each of the five oligomers with the Crystallography and NMR
System (CNS) software suite (Figure 6). A total of 24 NOE-
derived distance restraints from 4 were used to generate an
ensemble of 10 lowest-energy structures. The N-terminal four-
residue segment in the average generated from these 10
structures overlays well with the crystal structure of pentamer 2a.
We used H/D exchange (HDX) measurements to test the
hypothesis that 12/10-helices formed by the new α/γ(I)-peptide
backbone contain H-bond types with different intrinsic
favorabilities. These studies focused on the octamer Boc-
([Phe][γ(I)])4-OBn (5), for which 2D NMR data provided
evidence of 12/10-helical folding in CDCl3.17 Amino acid residue
identity exerts a large influence on HDX;20 therefore, the results
for octamer 5, expressed as half-life (t1/2) for disappearance of
Exploration of the new α/γ(I) backbone led to the unexpected
discovery that the 12/10-helix formed by these foldamers
contains H-bonds that differ in terms of favorability, with the γ
residue CO(i) → α residue H−N(i+3) H-bonds intrinsically
superior to the α residue CO(i) → γ residue H−N(i−1) H-
bonds. This possibility was initially suggested by the seven crystal
structures described here, in which the α residue CO(i) → γ
residue H−N(i−1) H-bond distances were long or other
molecules were interpolated into these interactions. Such
interpolations have occasionally been observed in proteins21 or
short conventional peptides22 and more recently in α/γ-
peptides,23 but the frequency of this unusual phenomenon in
our structural data set stands out. HDX and DMSO titration
results support the conclusion that there is an energetic
differentiation between the two types of H-bond in the 12/10-
helical conformation formed by the α/γ(I) backbone. We
speculate that the γ(I) residue is not ideally preorganized for the
10-atom H-bonding mode.17 Several foldamer secondary
1
amide H NMR resonances after dissolution in 95:5 CDCl3/
CD3OD, were normalized to the results for model dipeptide 6.
Specifically, the t1/2 value for each α residue NH in 5 was divided
by t1/2 for the α residue NH in 6, and the t1/2 value for each γ
residue in 5 was divided by t1/2 for the γ residue in 6. This
normalization should highlight the impact of folding on HDX at
each site within the octamer.
C
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Journal of the American Chemical Society
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Table 1. Relative HDX Half-Life Data for Octamer 5, Normalized to Dimer 6.
Phe-1
γ(I)-2
Phe-3
γ(I)-4
Phe-5
γ(I)-6
Phe-7
γ(I)-8
a
t
1/2 (h)
2.2
1.7
−
102
13
95
73
12
199
25
121
93
−
−
46
35
12
37
4.6
−
a
normalized t1/2
H-bonded ring size
10
10
12
10
a
Resonance overlapped with phenyl H.
4147. (c) Wu, H.; Qiao, Q.; Hu, Y.; Teng, P.; Gao, W.; Zuo, X.; Wojtas,
L.; Larsen, R. W.; Ma, S.; Cai, J. Chem.Eur. J. 2015, 21, 2501.
(6) (a) Sadowsky, J. D.; Schmitt, M. A.; Lee, H. S.; Umezawa, N.;
Wang, S.; Tomita, Y.; Gellman, S. H. J. Am. Chem. Soc. 2005, 127, 11966.
(b) Sadowsky, J. D.; Fairlie, W. D.; Hadley, E. B.; Lee, H. S.; Umezawa,
N.; Nikolovska-Coleska, Z.; Wang, S.; Huang, D. C.; Tomita, Y.;
Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 139. (c) Johnson, L. M.;
Gellman, S. H. Methods Enzymol. 2013, 523, 407.
structures containing different H-bond types have been
discovered, and our results raise the possibility that topologically
distinct H-bonds will in general be energetically differentiated.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic routes, NMR data, details of NMR structure
calculations for 4 and other foldamers, DMSO titration data,
HDX data, helix and H-bond parameters for crystal structures,
crystallographic data, and zip files containing CIF and PDB files.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b03382.
(7) Seebach, D.; Gademann, K.; Schreiber, J. V.; Matthews, J. L.;
Hintermann, T.; Jaun, B.; Oberer, L.; Hommel, U.; Widmer, H. Helv.
Chim. Acta 1997, 80, 2033.
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(8) Szolnoki, E.; Heten
́
yi, A.; Mandity, I. M.; Fulop, F.; Martinek, T. A.
́
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Eur. J. Org. Chem. 2013, 3555.
(9) Sharma, G. V.; Nagendar, P.; Jayaprakash, P.; Radha Krishna, P.;
Ramakrishna, K. V.; Kunwar, A. C. Angew. Chem., Int. Ed. 2005, 44, 5878.
AUTHOR INFORMATION
Corresponding Author
*gellman@chem.wisc.edu
■
(10) (a) Berlicki, L.; Pilsl, L.; Web
́ ́
er, E.; Mandity, I. M.; Cabrele, C.;
Martinek, T. A.; Fulop, F.; Reiser, O. Angew. Chem., Int. Ed. 2012, 51,
̈
̈
́
2208. (b) Legrand, B.; Andre, C.; Moulat, L.; Wenger, E.; Didierjean, C.;
Aubert, E.; Averlant-Petit, M. C.; Martinez, J.; Calmes, M.; Amblard, M.
Angew. Chem., Int. Ed. 2014, 53, 13131.
Present Address
†L.G.: Halliburton, 3000 N Sam Houston Parkway E, Houston,
TX 77032, United States.
(11) Guo, L.; Chi, Y.; Almeida, A. M.; Guzei, I. A.; Parker, B. K.;
Gellman, S. H. J. Am. Chem. Soc. 2009, 131, 16018.
(12) Guo, L.; Zhang, W.; Reidenbach, A. G.; Giuliano, M. W.; Guzei, I.
A.; Spencer, L. C.; Gellman, S. H. Angew. Chem., Int. Ed. 2011, 50, 5843.
(13) Guo, L.; Almeida, A. M.; Zhang, W.; Reidenbach, A. G.; Choi, S.
H.; Guzei, I. A.; Gellman, S. H. J. Am. Chem. Soc. 2010, 132, 7868.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(14) Man
́ ́
dity, I. M.; Weber, E.; Martinek, T. A.; Olajos, G.; Toth, G. K.;
This work was supported by NSF Grant CHE-1307365. NMR
spectrometers used in this work were purchased with support
from a generous gift by Paul J. Bender and from NIH (1 S10
RR13866-01). The authors thank Dr. Michael W. Giuliano for
helpful discussions regarding CNS calculations, Dr. Young-Hee
Shin for assistance in acquiring 2D NMR spectra, and Dr. Matt
Benning for acquiring single-crystal diffraction data of 2b at the
Bruker AXS facility in Fitchburg, WI.
Vass, E.; Fulop, F. Angew. Chem., Int. Ed. 2009, 48, 2171.
̈
̈
(15) Crystallographic data have been deposited at the Cambridge
Crystallographic Data Centre with accession codes 1056028−1056034
and can be accessed free of charge at www.ccdc.cam.ac.uk/data_
request/cif.
(16) (a) Yeates, T. O.; Kent, S. B. Annu. Rev. Biophys 2012, 41, 41.
(b) Lee, M.; Shim, J.; Kang, P.; Guzei, I. A.; Choi, S. H. Angew. Chem., Int.
Ed. 2013, 52, 12564.
(17) See the Supporting Information.
(18) Jeffrey, G. A. An Introduction to Hydrogen Bonding; Oxford
University Press: New York, 1997.
(19) (a) Sharma, G. V.; Jadhav, V. B.; Ramakrishna, K. V.; Jayaprakash,
P.; Narsimulu, K.; Subash, V.; Kunwar, A. C. J. Am. Chem. Soc. 2006, 128,
14657. (b) Giuliano, M. W.; Maynard, S. J.; Almeida, A. M.; Guo, L.;
Guzei, I. A.; Spencer, L. C.; Gellman, S. H. J. Am. Chem. Soc. 2014, 136,
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