Crystallographic characterization of 12-helical secondary structure in β-peptides containing side chain groups

Article abstract of DOI:10.1021/ja1062532

Helices are the most extensively studied secondary structures formed by β-peptide foldamers. Among the five known β-peptide helices, the 12-helix is particularly interesting because the internal hydrogen bond orientation and macrodipole are analogous to those of α-peptide helices (α-helix and 3₁₀-helix). The β-peptide 12-helix is defined by i, i+3 C - O???H-N backbone hydrogen bonds and promoted by β-residues with a five-membered ring constraint. The 12-helical scaffold has been used to generate β-peptides with specific biological functions, for which diverse side chains must be properly placed along the backbone and, upon folding, properly arranged in space. Only two crystal structures of 12-helical β-peptides have previously been reported, both for homooligomers of trans-2-aminocyclopentanecarboxylic acid (ACPC). Here we report five additional crystal structures of 12-helical β-peptides, all containing residues that bear side chains. Four of the crystallized β-peptides include trans-4,4-dimethyl-2-aminocyclopentanecarboxylic acid (dm-ACPC) residues, and the fifth contains a β³-hPhe residue. These five β-peptides adopt fully folded 12-helical conformations in the solid state. The new crystal structures, along with previously reported data, allow a detailed characterization of the 12-helical conformation; average backbone torsion angles of β-residues and helical parameters are derived. These structural parameters are found to be similar to those for i, i+3 C = O...H-N hydrogen-bonded helices formed by other peptide backbones generated from α- and/or β-amino acids. The similarity between the conformational behavior of dm-ACPC and ACPC is consistent with previous NMR-based conclusions that 4,4-disubstituted ACPC derivatives are compatible with 12-helical folding. In addition, our data show how a β³-residue is accommodated in the 12-helix, thus enhancing understanding of the diverse conformational behavior of this flexible class of β-amino acids.

Full text of DOI:10.1021/ja1062532

Published on Web 09/09/2010
Crystallographic Characterization of 12-Helical Secondary
Structure in ꢀ-Peptides Containing Side Chain Groups
Soo Hyuk Choi,† Ilia A. Guzei, Lara C. Spencer, and Samuel H. Gellman*
Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
Received July 14, 2010; E-mail: gellman@chem.wisc.edu
Abstract: Helices are the most extensively studied secondary structures formed by ꢀ-peptide foldamers.
Among the five known ꢀ-peptide helices, the 12-helix is particularly interesting because the internal hydrogen
bond orientation and macrodipole are analogous to those of R-peptide helices (R-helix and 310-helix). The
ꢀ-peptide 12-helix is defined by i, i+3 CdO· · ·H-N backbone hydrogen bonds and promoted by ꢀ-residues
with a five-membered ring constraint. The 12-helical scaffold has been used to generate ꢀ-peptides with
specific biological functions, for which diverse side chains must be properly placed along the backbone
and, upon folding, properly arranged in space. Only two crystal structures of 12-helical ꢀ-peptides have
previously been reported, both for homooligomers of trans-2-aminocyclopentanecarboxylic acid (ACPC).
Here we report five additional crystal structures of 12-helical ꢀ-peptides, all containing residues that bear
side chains. Four of the crystallized ꢀ-peptides include trans-4,4-dimethyl-2-aminocyclopentanecarboxylic
acid (dm-ACPC) residues, and the fifth contains a ꢀ3-hPhe residue. These five ꢀ-peptides adopt fully folded
12-helical conformations in the solid state. The new crystal structures, along with previously reported data,
allow a detailed characterization of the 12-helical conformation; average backbone torsion angles of
ꢀ-residues and helical parameters are derived. These structural parameters are found to be similar to
those for i, i+3 CdO· · ·H-N hydrogen-bonded helices formed by other peptide backbones generated from
R- and/or ꢀ-amino acids. The similarity between the conformational behavior of dm-ACPC and ACPC is
consistent with previous NMR-based conclusions that 4,4-disubstituted ACPC derivatives are compatible
with 12-helical folding. In addition, our data show how a ꢀ3-residue is accommodated in the 12-helix, thus
enhancing understanding of the diverse conformational behavior of this flexible class of ꢀ-amino acids.
R-helix (i, i+4 CdO· · ·H-N hydrogen bonds).3 In contrast,
the hydrogen bond orientations of other ꢀ-peptide helices (e.g.,
Introduction
ꢀ-Peptides are oligomers of ꢀ-amino acids and constitute
perhaps the most extensively studied class of unnatural
foldamers.1,2 Many examples of ꢀ-peptides that display folded
conformations analogous to secondary structures of R-peptides
and proteins have been identified, including examples that form
helices, turns, or extended conformations. ꢀ-Peptide helices have
drawn considerable attention because they may be used to mimic
helical substructures within proteins.1 Five ꢀ-peptide helices
(14-, 12-, 10/12-, 10-, and 8-helix) have been reported to date.1,2
The nomenclature of these helices is based on the number of
atoms in the characteristic hydrogen bonds. For example, a
ꢀ-peptide 12-helix is defined by i, i+3 CdO· · ·H-N hydrogen
bonds that contain 12 atoms. This particular helix is interesting
from the perspective of mimicking R-helices among conven-
tional peptides and proteins. The internal hydrogen bond
orientation and macrodipole of the ꢀ-peptide 12-helix are
analogous to those for the helices most commonly observed in
proteins: 310-helix (i, i+3 CdO· · ·H-N hydrogen bonds) and
i, i-2 CdO· · ·H-N hydrogen bonds for the 14-helix and
alternating i, i-1 CdO· · ·H-N and i, i+3 CdO· · ·H-N
hydrogen bonds for the 10/12-helix) are not observed among
R-peptide helices.2a
Experimental and computational studies suggest that the 12-
helix is not intrinsically favorable for acyclic ꢀ-residues, such
as the commonly used ꢀ3-homoamino acids (side chain adjacent
to nitrogen), but that this secondary structure is promoted by
cyclic ꢀ-residues with a five-membered ring constraint.4 We
have shown that ꢀ-peptide oligomers that contain residues
derived from trans-2-aminocyclopentanecarboxylic acid (ACPC)
and/or trans-4-aminopyrrolidine-3-carboxylic acid (APC) adopt
12-helical conformations.5,6a In complementary work, Martinek
et al. have recently shown that 12-helical folding is promoted
by ꢀ-amino acid residues with a bicyclo[3.3.1]heptane
skeleton.6b Even more recently, Aitken et al. have shown that
trans-2-aminocyclobutanecarboxylic acid residues favor 12-
helical folding.6c The 12-helical scaffold has served as a basis
† Present address: Department of Chemistry, Northwestern University,
Evanston, IL 60208.
(1) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173.
(2) (a) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001,
101, 3219. (b) Seebach, D.; Hook, D. F.; Glattli, A. Biopolymers 2006,
84, 23.
(3) Crisma, M.; Formaggio, F.; Moretto, A.; Toniolo, C. Biopolymers 2006,
84, 3.
(4) (a) Wu, Y. D.; Wang, D. P. J. Am. Chem. Soc. 1999, 121, 9352. (b)
Gunther, R.; Hofmann, H. J.; Kuczera, K. J. Phys. Chem. B 2001,
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2006, 27, 20.
9
10.1021/ja1062532  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 13879–13885 13879

A R T I C L E S
Choi et al.
Chart 1. ꢀ-Peptides for Which Crystal Structures Are Reported
for developing ꢀ-peptides that display antimicrobial activity,7
inhibit protein-protein interactions,8 and project carbohydrates
in specific three-dimensional patterns.9 For these biological
applications, functional groups must be introduced at precise
locations along the 12-helical backbone, which can be achieved
by incorporation of acyclic ꢀ-amino acid residues5g,i or cyclic
ꢀ-amino acid residues that bear side chains.5e,f,h,j Data obtained
in solution suggest that a few ꢀ2- or ꢀ3-residues can be
incorporated into a 12-helix if most other residues have the five-
membered ring constraint.5g,i Use of ꢀ-residues with function-
alized five-membered rings, however, provides functional
diversity in 12-helical scaffolds without loss of conformational
stability. Several approaches to five-membered ring function-
alization have been reported: sulfonylation of the pyrrolidine
nitrogen atom of APC,5e,f 3-substitution,5h 4,4-disubstitution5j
or 3,4-disubstitution of ACPC,10a N-functionalization of a
pyrrolidinone-based ꢀ-amino acid,10b and generation of ꢀ-amino
acid building blocks from AZT.10c,11
Only two crystal structures of 12-helical ꢀ-peptides have been
reported to date, and both have been obtained with ACPC
homooligomers.5c Here we provide five additional crystal
structures that display 12-helical conformations. Four of the new
structures contain trans-4,4-dimethyl-2-aminocyclopentanecar-
boxylic acid (dm-ACPC) residues, and the fifth structure contains
a ꢀ3-homophenylalanine (ꢀ3-hPhe) residue embedded in an
ACPC-rich sequence. This structural information enables us to
identify characteristic structural parameters for the 12-helical
secondary structure and to make comparisons with other well-
characterized helices.
(5) (a) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.;
Huang, X. L.; Barchi, J. J.; Gellman, S. H. Nature 1997, 387, 381.
(b) Applequist, J.; Bode, K. A.; Appella, D. H.; Christianson, L. A.;
Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 4891. (c) Appella, D. H.;
Christianson, L. A.; Klein, D. A.; Richards, M. R.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 7574. (d) Barchi, J. J.;
Huang, X. L.; Appella, D. H.; Christianson, L. A.; Durell, S. R.;
Gellman, S. H. J. Am. Chem. Soc. 2000, 122, 2711. (e) Wang, X. F.;
Espinosa, J. F.; Gellman, S. H. J. Am. Chem. Soc. 2000, 122, 4821.
(f) Lee, H. S.; Syud, F. A.; Wang, X. F.; Gellman, S. H. J. Am. Chem.
Soc. 2001, 123, 7721. (g) LePlae, P. R.; Fisk, J. D.; Porter, E. A.;
Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2002, 124, 6820.
(h) Woll, M. G.; Fisk, J. D.; LePlae, P. R.; Gellman, S. H. J. Am.
Chem. Soc. 2002, 124, 12447. (i) Park, J. S.; Lee, H. S.; Lai, J. R.;
Kim, B. M.; Gellman, S. H. J. Am. Chem. Soc. 2003, 125, 8539. (j)
Peelen, T. J.; Chi, Y. G.; English, E. P.; Gellman, S. H. Org. Lett.
2004, 6, 4411.
(6) (a) Hete´nyi, A.; To´th, G. K.; Somlai, C.; Vass, E.; Martinek, T. A.;
Fu¨lo¨p, F. Chem.sEur. J. 2009, 15, 10736. (b) Hete´nyi, A.; Szakonyi,
´
Z.; Ma´ndity, I. M.; Szolnoki, E.; To´th, G. K.; Martinek, T. A.; Fu¨lo¨p,
F. Chem. Commun. 2009, 177. (c) While our paper was under review,
a very thorough characterization of 12-helix formation by oligomers
of trans-2-aminocyclobutanecarboxylic acid appeared: Fernandes, C.;
Faure, S. Pereira, E.; The´ry, V.; Declerck, V.; Guillot, R.; Aitken,
D. J. Org. Lett. 2010, 12, 3606-3609. The crystal structure reported
by Aitken et al. is not included in our structural analysis. (d) See
also: Winkler, J. D.; Piatnitski, E. L.; Mehlmann, J.; Kasparec, J.;
Axelsen, P. H. Angew. Chem. Int. Ed. 2001, 40, 743.
Results and Discussion
Design and Synthesis. Chart 1 shows the five ꢀ-peptides for
which crystal structures are reported. These molecules include
a homooligomer of dm-ACPC (1) and three oligomers that
contain both ACPC and dm-ACPC residues (2-4). Heptamer
5 contains a ꢀ3-hPhe residue along with five ACPC residues;
the crystal structure of 5 represents the first high-resolution data
for a 12-helical ꢀ-peptide containing an acyclic residue. Oli-
gomer 5 contains an R-residue, R-aminoisobutyric acid (Aib),
at the C-terminus, which was included to enhance solubility
and crystallization.12 This R-residue does not participate in the
(7) (a) Porter, E. A.; Wang, X. F.; Lee, H. S.; Weisblum, B.; Gellman,
S. H. Nature 2000, 404, 565. (b) Porter, E. A.; Weisblum, B.; Gellman,
S. H. J. Am. Chem. Soc. 2002, 124, 7324.
(8) (a) English, E. P.; Chumanov, R. S.; Gellman, S. H.; Compton, T.
J. Biol. Chem. 2006, 281, 2661. (b) Imamura, Y.; Watanabe, N.;
Umezawa, N.; Iwatsubo, T.; Kato, N.; Tomita, T.; Higuchi, T. J. Am.
Chem. Soc. 2009, 131, 7353.
(9) Simpson, G. L.; Gordon, A. H.; Lindsay, D. M.; Promsawan, N.;
Crump, M. P.; Mulholland, K.; Hayter, B. R.; Gallagher, T. J. Am.
Chem. Soc. 2006, 128, 10638.
(10) (a) Kiss, L.; Kazi, B.; Forro´, E.; Fu¨lo¨p, F. Tetrahedron Lett. 2008,
49, 339. (b) Menegazzo, I.; Fries, A.; Mammi, S.; Galeazzi, R.;
Martelli, G.; Orena, M.; Rinaldi, S. Chem. Commun. 2006, 4915. (c)
Chandrasekhar, S.; Reddy, G. P. K.; Kiran, M. U.; Nagesh, C.;
Jagadeesh, B. Tetrahedron Lett. 2008, 49, 2969.
(11) An alternative secondary structure has been observed for an AZT-
derived subunits in a different solvent: Threlfall, R.; Davies, A.;
Howarth, N. M.; Fisher, J.; Cosstick, R. Chem. Commun. 2008, 585.
9
13880 J. AM. CHEM. SOC. VOL. 132, NO. 39, 2010

12-Helical Secondary Structure in ꢀ-Peptides
A R T I C L E S
Figure 1. Crystal structures of tetramers: (a) 1, (b) 2, (c) overlay of the two structures. The top views are perpendicular to the helical axis, and the bottom
views are along the helical axis. Dashed lines indicate hydrogen bonds. A cocrystallized chloroform molecule for 2 and most hydrogen atoms for both 1 and
2 are omitted for clarity.
helical hydrogen bonding pattern because there is no hydrogen
bond donor at the C-terminus.
ꢀ-Peptides 1-5 were synthesized via conventional solution-
phase methods involving carbodiimide-mediated coupling.12b-d
ACPC13 and dm-ACPC5j building blocks were prepared by
reported procedures. Each of the three shortest ꢀ-peptides (1-3)
was crystallized by solvent diffusion of n-pentane into a diethyl
ether/chloroform solution of the oligomer. Crystals of octamer
4 were grown by solvent diffusion of n-heptane into a 1,2-
dichloroethane solution of the oligomer. Heptamer 5 was
crystallized in two ways, by slow evaporation of either a
methanol-water mixture or an isopropyl alcohol solution.
Crystal Structures of ꢀ-Peptides Containing dm-ACPC
Residues. All of the four dm-ACPC-containing ꢀ-peptides
display fully folded 12-helical conformations with the maximum
number of i, i+3 CdO· · ·H-N hydrogen bonds in the crystal-
line state (Figures 1-3). Pentamer 3 displays a left-handed 12-
helical conformation in the solid state because 3 contains
(1R,2R)-forms of ACPC and dm-ACPC residues (Figure 2). The
other ꢀ-peptides discussed here contain (1S,2S)-forms of ACPC
and dm-ACPC residues and therefore display right-handed
helical conformations. In the crystal structure of octamer 4
Figure 2. Crystal structure of pentamer 3 (stereoview). The top view is
perpendicular to the helical axis, and the bottom view is along the helical
axis. Dashed lines indicate hydrogen bonds. A cocrystallized ether molecule,
a disordered carbon atom in ACPC(1), and most hydrogen atoms are omitted
for clarity.
(12) (a) Moretto, V.; Crisma, M.; Bonora, G. M.; Toniolo, C.; Balaram,
H.; Balaram, P. Macromolecules 1989, 22, 2939. (b) Choi, S. H.;
Guzei, I. A.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 13780. (c)
Choi, S. H.; Guzei, I. A.; Spencer, L. C.; Gellman, S. H. J. Am. Chem.
Soc. 2008, 130, 6544. (d) Choi, S. H.; Guzei, I. A.; Spencer, L. C.;
Gellman, S. H. J. Am. Chem. Soc. 2009, 131, 2917.
(Figure 3), the C-terminal benzyl group and some cyclopentane
ring puckers are disordered over two positions (9:1 to 6:4 ratio).
Interestingly, ring pucker disorder is limited to ACPC residues;
no disordered carbons are found among the four dm-ACPC
residues. The sequence of 4 is analogous to that of a previously
reported homooligomer, Boc-[(1R,2R)-ACPC]8-OBn.5c The
(13) (a) Lee, H. S.; LePlae, P. R.; Porter, E. A.; Gellman, S. H. J. Org.
Chem. 2001, 66, 3597. (b) LePlae, P. R.; Umezawa, N.; Lee, H.-S.;
Gellman, S. H. J. Org. Chem. 2001, 66, 5629.
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A R T I C L E S
Choi et al.
Figure 3. (a) Crystal structure of octamer 4. Alternative positions of disordered atoms are indicated with thin lines (stereoview). (b) Overlay of the structure
of 4 (gray lines) and the mirror image of the structure of Boc-[(1R,2R)-ACPC]8-OBn (black lines; ref 5c) (stereoview). The top view is perpendicular to the
helical axis, and the bottom view is along the helical axis. Dashed lines indicate hydrogen bonds. Most hydrogen atoms are omitted for clarity.
principal differences between 4 and Boc-[(1R,2R)-ACPC]8-OBn
are the gem-dimethyl substituents of the dm-ACPC residues in
4 and the opposite handedness of the two helices. In Figure 3b,
the right-handed 12-helical conformation observed for 4 in the
solid state is superimposed on the mirror image of the
left-handed 12-helical conformation observed for Boc-[(1R,2R)-
ACPC]8-OBn. The backbones of these two 12-helices are
virtually identical except for the C-terminal residue in each
ꢀ-peptide, the ester of which cannot serve as a hydrogen bond
donor. These results suggest that the gem-dimethyl substituents
of the dm-ACPC residues do not cause any distortion of the
12-helical conformation, a conclusion that is consistent with
the results of two-dimensional NMR studies of analogous
ꢀ-peptides in solution.5j
interactions may provide guidance for future efforts to design
12-helical segments that participate in discrete tertiary or
quaternary structures.5c There is space near the R-carbon of the
ꢀ3-hPhe residue at the interface shown in Figure 4b because no
substituent projects outward from this carbon (in contrast to
ACPC and dm-ACPC residues). This space is occupied by an
isopropyl alcohol molecule, which minimizes lateral contact
between these two ꢀ-peptide molecules. In contrast, the interface
highlighted in Figure 4c involves extensive intermolecular
contact between cyclopentane rings of ACPC residues. This
interaction is very similar to a lateral packing interaction
previously observed in the crystal of an ACPC hexamer.5c
A second crystal form of heptamer 5 was obtained from
methanol-water solution (see Supporting Information). These
crystals did not provide diffraction data of high quality;
nevertheless, it was possible to determine that this crystal form
contained two symmetry-independent molecules, both of which
displayed 12-helical conformations similar to those seen in the
crystal form from isopropyl alcohol, though the crystal packing
is different from the packing seen in Figure 4b,c. Overall, these
crystallization results suggest that adoption of the 12-helical
conformation by 5 is not determined by crystal packing forces
but is instead intrinsically favorable for the ꢀ-peptide portion
of this oligomer, despite the presence of a flexible ꢀ3-residue.
Relationship between Interproton Distances and NOE
Patterns. Medium-range nuclear Overhauser enhancements
(NOEs) between protons on residues that are not adjacent in a
Crystal Structure of a ꢀ-Peptide Containing a ꢀ3-hPhe
Residue. Figure 4 shows the two symmetry-independent con-
formations in the crystal of heptamer 5 grown from isopropyl
alcohol solution. These two independent conformations are very
similar to one another (Figure 4a); they are fully 12-helical,
without any significant distortion around the ꢀ3-hPhe residue.
The C-terminal Aib residue curls away from the helical axis in
each molecule and does not affect the 12-helical conformation.
There are two types of lateral interactions between adjacent
molecules: close packing between two symmetry-independent
conformations (Figure 4b) and close packing between two
identical molecules (Figure 4c). In both cases, the interacting
molecules are parallel to one another. These crystal packing
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12-Helical Secondary Structure in ꢀ-Peptides
A R T I C L E S
Figure 4. Crystal structure of heptamer 5, based on a crystal grown from isopropyl alcohol: (a) overlay of two symmetry-independent molecules in the
crystal, viewed perpendicular to the helical axis (top) and along the helical axis (bottom); (b) close packing between the two symmetry-independent molecules,
viewed along the crystallographic a axis (top) and b axis (bottom); (c) close packing between two identical molecules, viewed along the crystallographic c
axis (top) and b axis (bottom). Solvent molecules (isopropyl alcohol) are highlighted in cyan. Dashed lines indicate hydrogen bonds. Disordered atoms and
most hydrogen atoms are omitted for clarity. Molecular surfaces for the two helical conformations are rendered as shaded areas.
Table 1. Average Interproton Distances (Å) Corresponding to
Medium-Range NOE Patterns Observed for 12-Helical ꢀ-Peptides
does not reveal any other type of medium-range NOE that would
be expected between backbone proton pairs from nonadjacent
ꢀ-amino acid residues within a 12-helical conformation.
We have previously reported that an unexpected NOE pattern,
CꢀH(i)-CRH(i+3), was observed for a dm-ACPC-containing
ꢀ-peptide hexamer, in addition to the three NOE types in Table
1.5j This unexpected NOE pattern is not consistent with a 12-
helical conformation because the corresponding interproton
distances are >5 Å in the available crystal structures. Interest-
ingly, comparable NOE patterns have been observed for
NOE type
distancea
oligomers containing both R- and ꢀ-amino acid residues (“R/
ꢀ-peptides”) in either a 2:1 and a 1:2 ratio, in which all
ꢀ-residues are ACPC.12d Numerous crystal structures for these
types of R/ꢀ-peptides have revealed i, i+3 CdO· · ·H-N
hydrogen-bonded helical conformations, which are not consistent
with the CꢀH(i)-CRH(i+3) NOE pattern. We have proposed
that the CꢀH(i)-CRH(i+3) NOEs observed for the 2:1 and 1:2
R/ꢀ-peptides can be explained by alternative helical conforma-
tions involving i, i+4 CdO· · ·H-N hydrogen bonds. Indeed,
this alternative helix has been documented crystallographically
I
II
III
CꢀH(i)-NH(i+2)
CꢀH(i)-CRH(i+2)
CꢀH(i)-NH(i+3)
3.5(4) [25]
2.9(4) [21]
3.6(5) [21]
a The number in brackets indicates the number of measured and
averaged distances.
peptide sequence can develop if the corresponding interproton
distance is within 5 Å. Table 1 lists three types of medium-
range NOEs between backbone protons that have been associ-
ated with the ꢀ-peptide 12-helix in solution5a,j and corresponding
average interproton distances derived from the seven available
12-helical ꢀ-peptide crystal structures (the five structures in this
paper and the two previously reported ACPC homooligomer
structures5c). The three average distances for each of these types
of proton pairing is <4 Å. This correlation confirms that the
three NOE patterns in Table 1 are excellent indicators of 12-
helical folding in solution. Inspection of the crystal structures
for
a
33-residue 2:1 R/ꢀ-peptide.14 By analogy, the
CꢀH(i)-CRH(i+3) NOE pattern observed for the dm-ACPC-
containing ꢀ-peptide hexamer could arise from an i, i+4
CdO · · · H-N hydrogen-bonded helical conformation, which
would be designated a “16-helix”.5j Although this alternative
(14) Horne, W. S.; Price, J. L.; Gellman, S. H. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 9151.
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A R T I C L E S
Choi et al.
Table 2. Average ꢀ-Residue Torsion Angles (deg) for the 12- and
it is possible that the 16-helical conformation will become
dominant when the length of an ACPC-rich ꢀ-peptide is >10
residues.
the 14-Helices
12-helixa e
dm-ACPC
ꢀ3-hPheb
-100(15) [25] -101(20) [9] -93
14-helixa e
,
,
ACPC
overallc
ACHCd
Backbone Torsion Angle Analysis. The set of available crystal
structures (five from this study and two from ref 5c) enables
the identification of characteristic backbone torsion angle
averages for the ꢀ-peptide 12-helix (Table 2). Figure 5 presents
the crystallographically determined torsion angles for 12-helical
ꢀ-peptides in Ramachandran-type plots.17 For comparison, these
plots contain also backbone torsion angles derived from the two
14-helical crystal structures of homooligomers of 2-aminocy-
clohexanecarboxylic acid (ACHC).18 Torsion angles for left-
handed helical structures (pentamer 3, the two ACPC
homooligomers,5c and one of the 14-helical structures18) were
inverted for this comparison. For heptamer 5 and the two 14-
helical ACHC homooligomers, torsion angles of only one
independent molecule in the crystal are included. In all three
Ramachandran-type plots, the ACPC and dm-ACPC residues
fall in the same region, which is distinct from the region that is
characteristic of the 14-helix. The ꢀ3-hPhe residue in the
structure of 5 falls into the region for the 12-helix instead of
the region for the 14-helix, even though acyclic ꢀ3-amino acid
residues are generally understood to prefer 14-helical secondary
structure relative to 12-helical secondary structure.2 Table 2
shows that the local conformation of ꢀ3-hPhe in 5 is adjusted
to the 12-helical environment; for example, the θ angle is 87°,
which is substantially larger than θ angles common for ꢀ3-amino
acid residues in a 14-helical environment (∼60°)2a and close
to the average θ observed for residues with a five-membered
ring constraint in a 12-helical environment (94°). The ability
of the lone ꢀ3-hPhe in 5 to conform to the 12-helical environ-
ment is consistent with the view that this type of ꢀ-amino acid
φ
ψ
θ
-100(16) [35] -141(13) [10]
-102(14) [25] -101(14) [9] -111 -102(14) [35] -126(6) [9]
94(11) [25] 96(11) [9] 87 94(10) [35] 57(3) [10]
a The number in brackets indicates the number of measured and
averaged distances. b Only one set of torsion angles was used. c Torsion
angle averages identified from ACPC, dm-ACPC, and ꢀ3-hPhe residues.
d From ref 18; ACHC ) (1S,2S)-2-aminocyclohexanecarboxylic acid.
e For heptamer
5 and the two ACHC homooligomers, only one
independent molecule was used.
helical conformation is not observed in the crystal structures of
dm-ACPC-containing oligomers 1-4, which are fully 12-helical,
it is possible that both the 12- and 16-helical conformations
are populated in solution. Previous 2D NMR analysis of 1:1
R/ꢀ-peptides led us to conclude that the analogous pair of helical
conformations (designated the 11- and 14/15-helices, respec-
tively) is populated in solution for examples containing <10
residues,15 and subsequent crystallographic studies have verified
the occurrence of distinct helical conformations of 1:1 R/ꢀ-
peptides that contain either i, i+3 CdO· · ·H-N or i, i+4
CdO· · ·H-N hydrogen bonds.12b,c Comparable behavior is
well-known among R-peptides, involving the 310-helix (i, i+3
CdO · · · H-N hydrogen bonds) and the R-helix (i, i+4
CdO· · ·H-N hydrogen bonds).16 Among R- and R/ꢀ-peptides,
the available data suggest that increasing the number of amino
acid residues causes a preference for i, i+4 CdO· · ·H-N
hydrogen-bonded helices relative to i, i+3 CdO· · ·H-N
hydrogen-bonded helices. If this trend holds for ꢀ-peptides that
are rich in residues with a five-membered ring constraint, then
Figure 5. Ramachandran-type plots for right-handed helical ꢀ-peptides. Torsion angles for the 14-helix were measured from the crystal structures in ref 18.
Torsion angles for left-handed helical structures have been inverted. For heptamer 5 and the two 14-helical ACHC homooligomers, torsion angles of only
one independent molecule in the crystal are shown. ACHC ) (1S,2S)-2-aminocyclohexanecarboxylic acid.
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13884 J. AM. CHEM. SOC. VOL. 132, NO. 39, 2010

12-Helical Secondary Structure in ꢀ-Peptides
A R T I C L E S
Table 3. Average Parameters for i, i+3 CdO· · ·H-N
Hydrogen-Bonded Helices for R-, ꢀ-, and R/ꢀ-Peptides
The radii and the rise-per-residue values for these five helices
are quite similar to one another. The rise-per-turn (p) becomes
slightly smaller as the proportion of ꢀ-residues increases,
suggesting that fine-tuning of the helical pitch can be achieved
by changing the proportion of ꢀ-residues. This correlation should
be useful in design efforts that require specific arrangements of
side chain groups along the helical surface.
peptide backbone
(helix type)
res/turn
n
rise/turn
p (Å)
rise/res
d (Å)
radius
r (Å)
ꢀ-
residue %
ꢀ-peptide
(12-helix)
1:2 R/ꢀ-
2.7(1)
2.9
5.4(1)
5.5
2.0(1)
1.9
2.1(1)
2.1
100
67
50
33
0
a
peptide
1:1 R/ꢀ-peptide
2.8
5.6
2.0
2.1
b
(11-helix)
Conclusions
2:1 R/ꢀ-
peptide
2.9
5.8
2.0
2.0
a
The five crystal structures disclosed here, along with two
previously reported structures of ACPC homooligomers, have
allowed us to identify canonical structural characteristics of the
ꢀ-peptide 12-helix. The conformational behavior of dm-ACPC
is seen to be very similar to that of ACPC, implying that
functional groups could be placed at C4 of the five-membered
ring without interfering with 12-helical propensity. Analysis of
torsion angles and helical parameters suggests that the ꢀ-peptide
12-helix is comparable to the i, i+3 CdO· · ·H-N hydrogen-
bonded helices formed by R-peptides and by 1:1, 1:2, and 2:1
R/ꢀ-peptides. The structural insight that emerges from this study
should facilitate the design of 12-helical ꢀ-peptides intended
to perform specific functions.
R-peptide
(310-helix)
3.2
5.8
1.8
2.0
c
a From ref 12d. b From ref 12c. c From ref 20.
residue is intrinsically flexible and can adapt to a variety of
conformational contexts.
Helical Parameter Analysis. Average structural parameters
for the ꢀ-peptide 12-helix were derived from five of the available
crystal structures (oligomers containing five or more ꢀ-residues).
Each helical parameter was calculated from sets of four
consecutive R-carbons by reported methods.19 Nonhelical
residues at C-termini were excluded from these calculations.
This approach prevented the use of structures for tetramers 1
and 2 in the analysis. For heptamer 5, only one independent
conformation was used because all four independent conforma-
tions of 5 in the two crystal structures are quite similar.
The calculated parameters reveal the characteristic features
of the ꢀ-peptide 12-helix. The rise-per-turn of the ꢀ-peptide 12-
helix is 5.4 Å, which is the same as the pitch of the R-helix
and a bit smaller than that of the 310-helix (5.8 Å). The radius
of ꢀ-peptide 12-helix is 2.1 Å, which is slightly smaller than
that of the R-helix (2.3 Å) and comparable to that of the 310-
helix (2.0 Å).20 Table 3 lists the parameters for five i, i+3
CdO· · ·H-N hydrogen-bonded helices adopted by ꢀ-peptides
(the 12-helix), R-peptides (the 310-helix),20 or R/ꢀ-peptides.12c,d
Acknowledgment. We thank Dr. T. Peelen for providing
synthetic materials. This work was supported in part by NSF Grant
CHE-0848847 and NIH Grant GM-56414. S.H.C. was supported
in part by a fellowship from the Samsung Scholarship Foundation.
X-ray equipment purchase was supported in part by grants from
the NSF.
Note Added after ASAP Publication. Chart 1 contained an
error in the version published asap September 9, 2010; the correct
version and an updated SI file were reposted September 15, 2010.
Supporting Information Available: Experimental details,
characterization data, crystallographic information, full table of
helical parameters, and alternative crystal structure of heptamer
5. This material is available free of charge via the Internet at
http://pubs.acs.org.
(15) Hayen, A.; Schmitt, M. A.; Ngassa, F. N.; Thomasson, K. A.; Gellman,
S. H. Angew. Chem., Int. Ed. 2004, 43, 505.
(16) (a) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747. (b) Toniolo,
C.; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350.
(17) Ramachandran, G. N.; Sasisekharan, V. AdV. Protein Chem. 1968,
23, 283.
Note Added in Proof. Very recently, Lee et al. have reported
extraordinary self-assembly behavior of an ACPC heptamer: Kwon,
S.; Jeon, A.; Yoo, S. H.; Chung, I. S.; Lee, H.-S. Angew. Chem.
Int. Ed. 2010 Early View (DOI: 10.1002/anie.201003302).
(18) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 6206.
(19) (a) Sugeta, H.; Miyazawa, T. Biopolymers 1967, 5, 673. (b) Kahn,
P. C. Comput. Chem. 1989, 13, 185.
(20) Barlow, D. J.; Thornton, J. M. J. Mol. Biol. 1988, 201, 601.
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