Impact of γ-Amino Acid Residue Preorganization on α/γ-Peptide Foldamer Helicity in Aqueous Solution

Article abstract of DOI:10.1021/jacs.6b06177

α/γ-Peptide foldamers containing either γ⁴-amino acid residues or ring-constrained γ-amino acid residues have been reported to adopt 12-helical secondary structure in nonpolar solvents and in the solid state. These observations have engendered

Full text of DOI:10.1021/jacs.6b06177

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Impact of γ‑Amino Acid Residue Preorganization on α/γ-Peptide
Foldamer Helicity in Aqueous Solution
Brian F. Fisher and Samuel H. Gellman*
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
S
* Supporting Information
evaluate the impact of a specific cyclic constraint on helical
ABSTRACT: α/γ-Peptide foldamers containing either γ4-
secondary structure formed by α/γ-peptides.
amino acid residues or ring-constrained γ-amino acid
residues have been reported to adopt 12-helical secondary
structure in nonpolar solvents and in the solid state. These
observations have engendered speculation that the
seemingly flexible γ4 residues have a high intrinsic helical
propensity and that residue-based preorganization may not
significantly stabilize the 12-helical conformation. How-
ever, the prior studies were conducted in environments
that favor intramolecular H-bond formation. Here, we use
2D-NMR to compare the ability of γ4 residues and cyclic γ
residues to support 12-helix formation in more challenging
environments, methanol and water. Both γ residue types
support 12-helical folding in methanol, but only the
cyclically constrained γ residues promote helicity in water.
These results demonstrate the importance of residue-based
preorganization strategies for achieving stable folding
among short foldamers in aqueous solution.
Peptidic foldamers containing γ residues have been examined
in many laboratories,6 but only a few studies7 have evaluated the
relationships between γ residue structure and conformational
stability. The α/γ-peptide 12-helix is probably the most
extensively studied secondary structure in this foldamer class.
This conformation, formed by oligomers with sequentially
alternating α and γ residues, is defined by 12-atom CO(i) →
H−N(i+3) H-bonds between backbone amide groups. The α/γ-
peptide 12-helix was initially observed by Balaram et al. in crystal
structures of achiral oligomers containing gabapentin (Gpn) and
α-aminoisobutyric acid (Aib) residues.8,9 Guo et al. developed an
efficient asymmetric synthesis leading to trisubstituted γ-amino
acid residue I (Figure 1) and showed that α/γ-peptides
he study of unnatural oligomers that display biopolymer-
Tlike folding behavior offers a framework for interrogating
relationships between covalent and noncovalent structure
(constitution and conformation) in a way that transcends the
deep understanding that has emerged from analysis of proteins
and nucleic acids. For example, examination of “foldamers” that
contain β-amino acid residues instead of or in addition to α
residues (i.e., β-peptides or α/β-peptides) has revealed the
strong influence of small-ring constraints on the identity and
stability of H-bond-mediated secondary structure.1 This issue is
not pertinent to the folding of conventional polypeptides,
containing exclusively α residues, because imposition of a cyclic
constraint necessarily abolishes backbone H-bond potential and
disrupts common secondary structures, as seen with proline
residues.2
Insights gained from experimental correlations among residue
identity, secondary structure, and conformational stability have
proven invaluable in the development of functional foldamers
containing β residues. Stabilization of the β-peptide 14-helix by
trans-cyclohexyl residues, for example, enabled development of a
foldamer catalyst3 and fundamental single-molecule studies of
hydrophobic interactions.4 Stabilization of α/β-peptide helices
by trans-cyclopentyl residues has allowed the refinement of
protein−protein interaction antagonists.5 These functional
outcomes with β-containing foldamers highlight the importance
of elucidating relationships between residue structure and
conformational stability in other foldamer classes. Here we
Figure 1. Structures of γ residues.
containing I form the 12-helix in chloroform (based on NOE
analysis) and in the crystalline state.10 The cyclic constraint and
substitution pattern of I should limit conformational freedom. In
particular, the Cα-Cβ and Cβ-Cγ bonds are predicted to favor a
g+,g+ torsion angle sequence, as required for the 12-helix; this
prediction is consistent with the many crystal structures of
foldamers containing residue I.11
The groups of Gopi12 and Balaram13 have reported a large set
of crystal structures showing that α/γ-peptides containing
exclusively γ4 residues (Figure 1) (4- to 16-mers) can adopt
the 12-helix. In these structures the Cα-Cβ and Cβ-Cγ torsion
angles favor a g+,g+ torsion sequence similar to that observed in
12-helices containing residue I. Balaram et al. further
demonstrated via 2D NMR that 12-helicity is maintained by a
16-mer α/γ-peptide in chloroform.13b These findings are striking
because one would predict γ4 residues to be much more flexible
than is γ residue I. As Balaram et al. noted, the extensive structural
results with α/γ4-peptides lead one to question whether the
constraint inherent in I stabilizes the 12-helical conformation.
Received: June 15, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.6b06177
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society
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This uncertainty must be resolved because constrained residues
generally require greater synthetic effort than do flexible residues.
To address this question, we moved to a new family of α/γ-
peptide octamers (1−4; Figure 2) tailored for aqueous solubility.
which we assume to be monomeric, does not vary in this range. In
general, larger number of constrained γ residues correlated with
improved chemical shift dispersion. In order to detect 12-helical
folding, we focused on three types of backbone-to-backbone i,i+2
NOE that are known to be characteristic of this secondary
structure (Table 1).10,13b
a
Table 1. Summary of Crosspeaks Observed in CD3OH
H-atom
pair
12-helix distance
NOE
pairs
b
(Å)
1
2
3
4
a
b
c
2.72
2.70
2.37
2···4
4···6
6···8
2···4
4···6
6···8
1···3
3···5
5···7
W
?
S
?
S
M
S
S
S
M
M
S
M
?
c
2.60
W
?
S
c
2.69
W
?
M
W
W
W
W
S
−
?
S
3.42
3.64
3.40
W
VW
VW
W
N.D.
VW
W
W
W
a
VW = very weak; W = weak; M = medium; S = strong; ? =
b
ambiguous; N.D. = not detected. From crystal structure of P3 in ref
11c. Distance to pseudoatom between diastereotopic H nuclei.
c
For fully flexible α/γ-peptide 1 in CD3OH, partial chemical
shift overlap precluded precise integration of several expected 12-
helical crosspeaks in the 2D-ROESY spectrum. Nevertheless,
unambiguous observation of six characteristic NOEs, uniformly
distributed across 1, supported the conclusion that this α/γ-
peptide is at least partially 12-helical in methanol (Table 1).
Figure 2. Structures of water-soluble α/γ-peptides.
Water is the least favorable among common solvents for H-
bonded conformations such as the α-helix or β-sheets formed by
conventional peptides or helices formed by β- or α/β-peptide
foldamers.14 Presumably the strong H-bond competition
provided by water molecules diminishes the ability of intra-
molecular H-bonds to stabilize secondary structure. Admixture
of an alcohol cosolvent, such as methanol, enhances the
population of internally H-bonded secondary structures for
which there is an intrinsic propensity,14,15 but aqueous-alcohol
mixtures do not guarantee folding. In contrast, chloroform allows
intramolecular H-bonds to serve as a strong driving force for
secondary structure formation. Qualitative comparisons based
on data obtained in chloroform or crystal structures cannot
decisively address the relative 12-helical propensities of I vs a γ4
residue.
1
Improved H signal dispersion enabled unambiguous assign-
ment of almost all expected 12-helix NOEs for α/γ-peptide 2 in
CD3OH. The two 12-helical NOEs involving the cyclic γ residue
(types a and b in Table 1) were more intense than those
involving γ4 residues. The 12-helical NOEs involving the α
residues (type c) were generally not very intense; consistent with
this trend, protons that would give rise to type c NOEs are
further apart in available 12-helix crystal structures than are
protons that would give rise to type a or b NOEs.12c α/γ-Peptide
3, containing two cyclic γ residues, showed excellent chemical
shift dispersion, and numerous 12-helical NOEs were detected.
As seen for 2, crosspeak intensity was lower for γ-to-γ NOEs
involving exclusively γ4 residues relative to such NOEs involving
at least one cyclic γ residue.
α/γ-Peptide 4, containing three cyclic γ residues, showed the
most intense 12-helical crosspeaks among the four oligomers.
Furthermore, numerous long-range NOEs between protons on
side chains were detected that were not observed for 1−3.
Collectively, the ROESY spectra for 1−4 in CD3OH
demonstrate a tendency of all α/γ-peptides to adopt the 12-
helix conformation, but the trend of increasing crosspeak
intensity and number with increasing number of γ4 → II
replacements suggests that the helical state becomes more stable
as cyclic constraints are added.
α/γ-Peptide series 1−4 features both γ4 residues and γ residue
II (Figure 1), which is an analogue of I bearing a side chain that
should be cationic at acidic pH. α/γ-Peptide 1 contains
exclusively γ4 residues, which are progressively replaced with
II, from N- to C-terminus, in analogues 2−4. The γ residue side
chain complement varies at one position across the α/γ-peptide
series: 1 and 2 have a glutamate-like side chain at residue 4, but 3
and 4 have a lysine-like side chain at this position. This variation
1
was intended to promote H NMR resonance dispersion. The
four α residues are invariant among 1−4. Two aromatic side
chains (Tyr-5 and Phe-7) were included to enhance dispersion of
1H NMR signals.
Initial 2D NMR studies were conducted in CD3OH, in which
1−4 were highly soluble. 1H resonances were unchanged
between 0.1 and 3 mM, suggesting that aggregation state,
We next characterized the folding behavior of α/γ-peptides 1−
4 in water. All four α/γ-peptides were highly soluble (>3 mM),
and chemical shifts did not vary between 0.1 and 3 mM. The data
obtained in this competitive environment support our
B
DOI: 10.1021/jacs.6b06177
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society
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conclusion that the cyclic constraint of residue II stabilizes the
12-helix.
For the fully flexible α/γ-peptide 1, only two very weak i,i+2
ROESY crosspeaks consistent with the 12-helix conformation
could be distinguished, with most others impossible to assign
unambiguously because of poor resonance dispersion. For all
ambiguous i,i+2 crosspeaks, the low intensity outside the
overlapping region means that, at most, these NOEs were very
weak. Three of the anticipated 12-helical i,i+2 crosspeaks would
have occurred in relatively uncrowded regions of the 2D-ROESY
spectrum but were not detected, which suggests that any
tendency toward 12-helical folding by α/γ-peptide 1 is low in
water. Three of the five nonsequential crosspeaks that could be
unambiguously assigned were inconsistent with the 12-helix
conformation (Figure 3). The i,i+2 NOEs of 1 in water fail to
converge to a single mean structure and thus reflect a disordered
conformational ensemble.16
Figure 4. Excerpt of the 600 MHz ROESY spectrum of 3 mM α/γ-
peptide 2 in 9:1 H2O:D2O, 100 mM acetate, pH 3.8 at 5 °C. i,i+2 NOEs
indicated in red. Missing type a NOE shown with dashed line; observed
type a NOE shown with unbroken line.
Results of NOE distance-restrained simulated annealing
calculations17 suggest an increasingly well-defined helical
conformation in methanol as γ4 residues are replaced with
constrained residues (Figure 5a). The calculations in water
Figure 3. Summary of detected ROESY crosspeaks between protons on
nonadjacent residues in aqueous solution.
Figure 5. Superposition of 10 lowest energy among 100 trial structures
from simulated annealing calculations of 1−4. Mean pairwise backbone
RMSD is shown below each structure. Sidechains omitted for clarity. (a)
Calculated ensembles using distance restraints derived from (a)
CD3OH ROESY and (b) aqueous ROESY data.
The aqueous 2D-ROESY spectra of α/γ-peptides 2-4 traced
the formation of helicity with increasing cyclic constraint
1
content. As seen in CD3OH, the H resonances became more
dispersed with increasing content of cyclic γ residue II,
permitting unambiguous detection of an increasing fraction of
the expected 12-helical ROESY crosspeaks as the content of II
grows. All observed i,i+2 ROESY crosspeaks for 2−4 involved at
least one residue II or occurred between protons from α residues
that are separated by a residue II. α/γ-Peptide 2, with a single
residue II, illustrates the difference between cyclic and acyclic γ
residues in terms of supporting local folding (Figure 4). The
crosspeak of type a between II-2 and γ4Glu-4 was very intense,
whereas the possible a crosspeak between γ4Glu-4 and γ4Lys-6
was not detected, even though this crosspeak would have
occurred in an uncrowded region of the 2D spectrum. This trend
continued for α/γ-peptides 3 and 4.
suggest an even more pronounced disorder-to-order transition
across the series 1−4 than is evident in methanol (Figure 5b vs
5a). The non-12-helical NOEs of 1 in water result in a variety of
simulated conformations that do not correspond to a regular
helix. For α/γ-peptides 2 and 3 in water, partial ordering only at
the segments containing cyclic γ residues is observed, which is
expected since there was a consistent lack of characteristic i,i+2
ROESY crosspeaks for the segments containing γ4 residues. α/γ-
Peptide 4 in water displays a relatively tight cluster of structures,
each with the maximum number of 12-helix H-bonds. Increasing
C
DOI: 10.1021/jacs.6b06177
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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the number of γ4 → II replacements across the series 1−4
improves the overlay of the NMR-derived structures with 12-
helical conformations observed for crystalline α/γ-peptides,
whether the oligomer crystallized contains constrained γ residue
I or unconstrained γ4 residues; this trend is illustrated for a
specific α/γ4-peptide crystal structure in Figure 6.
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18
1
well as other H NMR data, support the hypothesis that the
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b06177.
Experimental details, including synthetic route to II; NMR
spectra; HDX, VT-NMR, and J-coupling analysis; ROESY
crosspeak assignments; CD data; comparison of I and γ4
structure; and NMR calculation parameters (PDF)
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Raghothama, S.; Shamala, N.; Balaram, P. Org. Lett. 2014, 16, 1656.
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AUTHOR INFORMATION
Corresponding Author
*gellman@chem.wisc.edu
■
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Notes
The authors declare no competing financial interest.
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(18) See Supporting Information for discussion.
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ACKNOWLEDGMENTS
■
This work was supported by NSF grant CHE-1307365. NMR
spectrometers were purchased with support from a generous gift
by Paul J. Bender and from NIH (S10 OD012245).
D
DOI: 10.1021/jacs.6b06177
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX