Synthesis and structural characterization of helix-forming β-peptides: Trans-2-aminocyclopentanecarboxylic acid oligomers

Article abstract of DOI:10.1021/ja991185g

Synthetic protocols and circular dichroism (CD) spectra are reported for a series of oligomers of (R,R)-trans-2-aminocyclopentanecarboxylic acid (trans-ACPC). The two longest oligomers, a hexamer and an octamer, have also been examined crystallographicall

Full text of DOI:10.1021/ja991185g

7574
J. Am. Chem. Soc. 1999, 121, 7574-7581
Synthesis and Structural Characterization of Helix-Forming
â-Peptides: trans-2-Aminocyclopentanecarboxylic Acid Oligomers
Daniel H. Appella, Laurie A. Christianson, Daniel A. Klein, Michele R. Richards,
Douglas R. Powell, and Samuel H. Gellman*
Contribution from the Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706-1396
ReceiVed April 14, 1999
Abstract: Synthetic protocols and circular dichroism (CD) spectra are reported for a series of oligomers of
(R,R)-trans-2-aminocyclopentanecarboxylic acid (trans-ACPC). The two longest oligomers, a hexamer and
an octamer, have also been examined crystallographically. Both crystal structures show that the â-peptide
backbone adopts a regular helix that is defined by a series of interwoven 12-membered ring hydrogen bonds
(“12-helix”). Each hydrogen bond links a carbonyl oxygen to an amide proton three residues toward the
C-terminus. CD data suggest that the conformational preference of trans-ACPC oligomers in methanol is
strongly length-dependent, which implies that 12-helix formation is a cooperative process, as seen for the
R-helix formed by conventional peptides. Previous work has established that oligomers and polymers of â-amino
acids can adopt helical conformations, but the 12-helix is an unprecedented â-peptide secondary structure.
Unnatural oligomers that adopt well-defined conformations,
adoption of specific shapes by flexible molecules. In addition
to this fundamental motivation, foldamer research has practical
significance because oligomers with predictable conformations
should provide new strategies for mimicry of biomolecular
function. For example, foldamers that adopt robust helical or
sheet secondary structures may allow one to mimic helices or
sheets displayed on the surfaces of folded proteins. A foldamer
that mimics a helix or sheet involved in a protein-protein
recognition site could inhibit the recognition process.19 Alter-
similar to those displayed by proteins and RNA, are a subject
of increasing interest to chemists.1-18 We use the term “fol-
damers” to describe this subset of oligomers, because most
oligomers do not have strong conformational preferences.
Creation of new foldamers tests and refines our ability to
understand how networks of noncovalent interactions promote
* To whom correspondence should be addressed.
(1) Review: Gellman, S. H. Acc. Chem. Res. 1998, 31, 173.
(2) â-Peptide folding: (a) Seebach, D.; Overhand, M.; Ku¨hnle, F. N.
M.; Martinoni, B.; Oberer, L.; Hommel, U.; Widmer, H. HelV. Chim. Acta
1996, 79, 913. (b) Seebach, D.; Ciceri, P.; Overhand, M.; Juan, B.; Rigo,
D.; Oberer, L.; Hommel, U.; Amstutz, R.; Widmer, H. HelV. Chim. Acta
1996, 79, 2043. (c) Seebach, D.; Matthews, J. L. J. Chem. Soc., Chem.
Commun. 1997, 2015-2022. (d) Seebach, D.; Abele, S.; Gademann, K.;
Guichard, G.; Hintermann, T.; Jaun, B.; Matthews, J. L.; Schreiber, J.;
Oberer, L.; Hommel, U.; Widmer, H. HelV. Chim. Acta 1998, 81, 932. (e)
Seebach, D.; Abele, S.; Sifferlen, T.; Ha¨nggi, M.; Gruner, S.; Seiler, P.
HelV. Chim. Acta 1998, 81, 2218.
(3) â-Peptide folding: (a) Appella, D. H.; Christianson, L. A.; Karle, I.
L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071. (b)
Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.; Huang,
X.; Barchi, J. J.; Gellman, S. H. Nature 1997, 387, 381. (c) Krautha¨user,
S.; Christianson, L. A.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc.
1997, 119, 11719. (d) Chung, Y. J.; Christianson, L. A.; Stanger, H. E.;
Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 10555. (e)
Appella, D. H.; Barchi, J. J.; Durell, S.; Gellman, S. H. J. Am. Chem. Soc.
1999, 121, 2309. (f) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell,
D. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 6206.
(4) â-Peptide folding: (a) Diederichsen, U.; Schmitt, H. W. Eur. J. Org.
Chem. 1998, 827. (b) Gung, B. W.; Zou, D.; Stalcup, A. M.; Cottrell, C. E.
J. Org. Chem. 1999, 64, 2176.
(5) Vinylogous peptide folding: Hagihara, M.; Anthony, N. J.; Stout,
T. J.; Clardy, J.; Schreiber, S. L. J. Am. Chem. Soc. 1992, 114, 6568.
(6) Nucleic acid analogue folding: Beier, M.; Reck, F.; Wagner, T.;
Krishnamurthy, R.; Eschenmoser, A. Science 1999, 283, 699 and references
therein.
(7) Aedamer folding: (a) Lokey, R. S.; Iverson, B. L. Nature 1995, 375,
303. (b) Nguyen, J. Q.; Iverson, B. L. J. Am. Chem. Soc. 1999, 121, 2639.
(8) Sulfonamide oligomer folding: Gennari, C.; Salom, B.; Potenza, D.;
Longari, C.; Fioravanzo, E.; Carugo, O.; Sardone, N. Chem. Eur. J. 1996,
2, 644 and references therein.
(10) δ-Peptide folding: (a) Szabo, L.; Smith, B. L.; McReynolds, K.
D.; Parrill, A. L.; Morris, E. R.; Gervay, J. J. Org. Chem. 1998, 63, 1074.
(b) Smith, M. D.; Claridge, T. D. W.; Tranter, G. E.; Sansom, M. S. P.;
Fleet, G. W. J. J. Chem. Soc., Chem. Commun. 1998, 2041. (c) Long, D.
D.; Hungerford, N. L.; Smith, M. D.; Brittain, D. E. A.; Marquess, D. G.;
Claridge, T. D. W.; Fleet, G. W. J. Tetrahedron Lett. 1999, 40, 2195. (d)
Claridge, T. D. W.; Long, D. D.; Hungerford, N. L.; Aplin, R. T.; Smith,
M. D.; Marquess, D. G.; Fleet, G. W. J. Tetrahedron Lett. 1999, 40, 2199.
(11) Oligourea folding: Nowick, J. S.; Mahrus, S.; Smith, E. M.; Ziller,
J. W. J. Am. Chem. Soc. 1996, 118, 1066.
(12) Anthranilic acid oligomer folding: Hamuro, Y.; Geib, S. J.;
Hamilton, A. D. J. Am. Chem. Soc. 1997, 119, 10587 and references therein.
(13) Peptoid folding: (a) Armand, P.; Kirshenbaum, K.; Goldsmith, R.
A.; Farr-Jones, S.; Barron, A. E.; Truong, K. T.; Dill, K. A.; Mierke, D. F.;
Cohen, F. E.; Zuckermann, R. N.; Bradley, E. K. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 4309. (b) Kirshenbaum, K.; Barron, A. E.; Goldsmith, R.
A.; Armand, P.; Bradley, E. K.; Truong, K. T.; Dill, K. A.; Cohen, F. E.;
Zuckermann, R. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303.
(14) m-Phenylacetylene oligomer folding: (a) Nelson, J. C.; Saven, J.
G.; Moore, J. S.; Wolynes, P. G. Science 1997, 277, 1793. (b) Prince, R.
B.; Okada, T.; Moore, J. S. Angew. Chem., Int. Ed. 1999, 38, 233. (c) Gin,
M. S.; Yokozawa, T.; Prince, R. B.; Moore, J. S. J. Am. Chem. Soc. 1999,
121, 2643.
(15) R-Aminooxy acid oligomer folding: Yang, D.; Qu, J.; Li, B.; Ng,
F.; Wang, X.; Cheung, K.; Wang, D.; Wu, Y. J. Am. Chem. Soc. 1999,
121, 589 and references therein.
(16) Guanidine oligomer folding: Tanatani, A.; Yamaguchi, K.; Azumaya,
I.; Fukutomi, R.; Shudo, K.; Kagechika, H. J. Am. Chem. Soc. 1998, 120,
6433 and references therein.
(17) Oligopyrrolinone conformations: Smith, A. B.; Guzman, M. C.;
Sprengler, P. A.; Keenan, T. P.; Holcomb, R. C.; Wood, J. L.; Carroll, P.
J.; Hirschmann, R. J. Am. Chem. Soc. 1994, 116, 9947.
(9) γ-Peptide folding: (a) Hintermann, T.; Gademann, K.; Jaun, B.;
Seebach, D. HelV. Chim. Acta 1998, 81, 983. (b) Hanessian, S.; Luo, X.;
Schaum, R.; Michnick, S. J. Am. Chem. Soc. 1998, 120, 8569.
(18) Pyridine/pyrimidine oligomer folding: (a) Bassani, D. M.; Lehn,
J.-M.; Baum, G.; Fenske, D. Angew. Chem., Int. Ed. Engl. 1997, 36, 1845.
(b) Bassani, D. M.; Lehn, J.-M. Bull. Soc. Chim. Fr. 1997, 134, 897.
10.1021/ja991185g CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/06/1999

trans-2-Aminocyclopentanecarboxylic Acid Oligomers
J. Am. Chem. Soc., Vol. 121, No. 33, 1999 7575
We have shown that different â-peptide helical conformations
can be induced by use of cycloalkyl restraints. The trans-2-
aminocyclohexanecarboxylic acid (trans-ACHC) residue leads
to 14-helix formation,3a,e,f,21 while, as reported in a preliminary
account,3b the trans-2-aminocyclopentanecarboxylic acid (trans-
ACPC) residue leads to 12-helix formation. The hydrogen bonds
that define these two helices (Figure 1) are oriented in opposite
directions relative to the backbone; in contrast, the hydrogen
bonds that define the two helices observed in proteins (R-helix
and 310-helix) are oriented in the same direction. Here we
describe the synthesis and structural characterization (crystal-
lography and circular dichroism (CD)) of trans-ACPC â-pep-
tides. The solid-state structures suggest strategies for design of
helical bundle tertiary structures.
Figure 1. Hydrogen bonds associated with helical â-peptide conforma-
tions discussed in the text.
natively, foldamers with well-defined tertiary structures might
lead to creation of enzyme-like catalysts. There are several
examples of foldamers that adopt discrete secondary structures,1-18
but no unnatural tertiary structure has yet been reported.
â-Amino acid oligomers (“â-peptides”) are among the most
thoroughly studied foldamers at present.1-4 Reports from
Seebach et al.2 and from our laboratory3 have shown that careful
choice of â-amino acid residues and â-peptide sequence can
produce several different helices. We have identified â-amino
acid residues with high propensities for sheet or reverse turn
secondary structure,3c,d thus demonstrating that each of the three
classes of regular secondary structure observed in proteins can
be reproduced in â-peptides. These â-peptides offer greater
opportunities to control the secondary structural propensities
of individual residues than do conventional peptides (composed
of R-amino acids), because the residues we have used have two
sp3 carbons between the carboxyl and amino groups. Bonds
between sp3-hybridized carbon atoms tend to have more clearly
defined torsional preferences than do bonds between an sp3-
hybridized carbon and an sp2-hybridized atom.20 Further, the
two sp3 atoms of each â-amino acid residue can be incorporated
into a ring, thereby reducing residue flexibility, without destroy-
ing backbone hydrogen-bonding sites.3 In contrast, incorporating
rings into the R-amino acid backbone will eliminate hydrogen
bond donor sites (e.g., proline).
The work of Seebach et al. provides an excellent illustration
of residue-based control of â-peptide conformation.2 These
workers have shown that â-peptides containing residues bearing
a single â-substituent (all with identical configuration) adopt a
helix defined by a series of 14-membered ring hydrogen bonds
between backbone amide groups (Figure 1).2a-c We refer to this
secondary structure as “14-helix”, and Seebach et al. designate
it “31-helix”. (For comparison, the familiar R-helix is defined
by a series of 13-membered ring hydrogen bonds between
backbone amides.) The 14-helix also results if all residues bear
a single R-substituent (again, with identical configuation).
However, certain “mixed” sequences of R- and â-monosubsti-
tuted residues induce formation of a very different helix, defined
by alternating 10- and 12-membered ring hydrogen bonds.2d â,â-
Disubstituted residues disrupt 14-helix formation.2c
Results and Discussion
Synthesis. All â-peptides were prepared from (R,R)-trans-
ACPC. Synthesis of the optically pure, doubly protected
monomer, tert-butyloxycarbonyl-trans-ACPC ethyl ester (Boc-
trans-ACPC-OEt), has been reported.22 This route begins with
ethyl (1R,2S)-2-hydroxycyclopentanecarboxylate, which we
generated via the reported baker’s yeast reduction of racemic
ethyl 2-oxocyclopentanecarboxylate.22a The hydroxyl group is
stereospecifically converted to an amino group via Mitsunobu
reaction with HN3, followed by reduction.22b We were unable
to purify the intermediate azide from the alkene byproduct of
the Mitsunobu reaction, so reduction was carried out with the
mixture, followed by Boc protection of the amino group. Boc-
ACPC-OEt was then purified by chromatography. After depro-
tection, amide bond formation between Boc-ACPC-OH and
H2N-ACPC-OEt was accomplished using (N,N-dimethylamino)-
propyl-3-ethylcarbodiimide (as the hydrochloride salt (EDCI‚
HCl)) and 4-(N,N-dimethylamino)pyridine (DMAP), in DMF
solution, to yield Boc-ACPC-ACPC-OEt. At this point, the ethyl
ester was replaced by a benzyl ester, and the synthesis of longer
â-peptides was accomplished by coupling dimer or longer
fragments as described above. The benzyl esters were more
crystalline than analogous ethyl esters.
Circular Dichroism. Far-UV circular dichroism (CD) data
are commonly used to analyze the secondary structure of
conventional peptides.23 In this spectral region, CD signals arise
largely from the amide groups in the peptide backbone. Regular
secondary structures (helices, sheets, reverse turns) give rise to
characteristic far-UV CD signatures.
Our work provides additional examples of residue-based
control of â-peptide secondary structure. We have shown that
â-amino acid residues bearing one R-substituent and one
â-substituent, with appropriate relative stereochemistry, have a
high propensity to adopt sheet secondary structure in which the
CR-Câ bond of each residue is anti and all carbonyls are
oriented in roughly the same direction.3c,d In contrast, â-sub-
stituted residues or unsubstituted residues (â-alanine) have a
Figure 2 shows far-UV CD data for a homologous series of
trans-ACPC â-peptides in methanol solution, including dimer
2, trimer 3, tetramer 4, and hexamer 5 (6 was insufficiently
soluble in methanol to be analyzed). The data in Figure 2 have
much lower propensity to adopt this sheet conformation.3c
A
heterochiral dinipecotic acid segment forms a reverse turn
(21) Independent prediction of 14-helix formation by trans-ACHC
oligomers: Bode, K. A.; Applequist, J. Macromolecules 1997, 30, 2144.
(22) (a) Herrado´n, B.; Seebach, D. HelV. Chim. Acta 1989, 72, 690-
714. (b) Tilley, J. W.; Danho, W.; Shiuey, S.; Kulesha, I.; Swistok, J.;
Makofske, R.; Michalewsky, J.; Triscari, J.; Nelson, D.; Weatherford, S.;
Madison, V.; Fry, D.; Cook, C. J. Med. Chem. 1992, 35, 3774.
(23) Woody, R. W. Circular Dichroism: Principles and Applications;
VCH Publishers: New York, 1994; pp 24-38, 473-496.
secondary structure.3d
(19) Fairlie, D. P.; West, M. L.; Wong, A. K. Curr. Med. Chem. 1998,
5, 29.
(20) (a) Page, M. I.; Jencks, W. P. Proc. Natl. Acad. Sci. U.S.A. 1971,
68, 1678. (b) Mammen, M.; Shakhnovich, E. I.; Whitesides, G. M. J. Org.
Chem. 1998, 63, 3168.

7576 J. Am. Chem. Soc., Vol. 121, No. 33, 1999
Appella et al.
Figure 2. Circular dichroism data in CH3OH; the molar ellipticity [Θ]
values for dimer 2, trimer 3, tetramer 4, and hexamer 5 have been
normalized for oligomer concentration and the number of backbone
amide groups (one, two, three, and five, respectively).
been normalized for both â-peptide concentration and the
number of amide chromophores in each molecule, as is common
for conventional peptide CD data. Thus, the vertical axis
represents molar ellipticity per residue.
The CD signatures vary significantly across the series 2-5.
Since there is no isodichroic point, it is likely that at least three
conformational states contribute to these CD spectra. Dimer 2
shows little ellipticity between 215 and 250 nm; below 215 nm,
the ellipticity becomes positive. Similar behavior is seen for
trimer 3, except that there is a shallow minimum at ca. 218
nm, and the rise at lower wavelengths is sharper. Tetramer 4
displays a shallow minimum at ca. 220 nm and a maximum at
ca. 202 nm. Hexamer 5 has a pattern similar to that of 4, except
that both the minimum and maximum are slightly red-shifted
(222 and 207 nm) and more intense for 5 relative to those for
4. The pattern observed for 5 has been shown to agree with
theoretical predictions for the 12-helix CD spectrum.24 The 12-
helical CD signature observed for 5 is quite distinct from the
CD signatures of 14-helical â-peptides reported by us3e,f and
by Seebach et al.2a-c
The data for 2-5 suggest that the 12-helical conformation
becomes more highly populated, i.e., more stable, with additional
trans-ACPC residues. This trend suggests cooperativity in 12-
helix formation, which parallels the well-established cooperat-
ivity in R-helix formation by conventional peptides.25 The 12-
helical conformation becomes increasingly stable relative to
other conformations as the trans-ACPC oligomer grows longer,
presumably because the favorable energy of adding residues to
the 12-helix out-weighs the energetic cost of helix initiation.
Solid-State Structures. Crystal structures were obtained of
both hexamer 5 and octamer 6. In each crystal, there is only
one molecule in the asymmetric unit. Both oligomers display
12-helical conformations in the solid state (Figure 3), with the
maximum number of 12-membered ring hydrogen bonds in each
case (four for 5 and six for 6). One molecule of methanol per
â-peptide was included in the crystal of 5. Both methanol and
water were included in the crystal of 6.
Figure 3. Solid-state structures (stereoviews) of trans-ACPC octamer
6 from two perspectives: (a) perpendicular to the helix axis and (b)
along the helix axis. Hydrogen atoms other than those attached to
nitrogen have been omitted for clarity. Dotted lines indicate hydrogen
bonds. Hexamer 5 displays a similar conformation; stereoviews for 5
may be found in the Supporting Information.
illustrates the two types of side-to-side interactions in the crystal
of 5. Interface 1 involves relatively little contact between
cyclopentane rings (Figure 6); much of the contact involves the
terminal tert-butyl and phenyl groups interacting with cyclo-
pentane rings. Interface 2 has extensive intermolecular contact
between cyclopentyl rings (Figure 7). Particularly intriguing is
the way in which the cyclopentyl ring of residue 3 on one
molecule interdigitates between the cyclopentyl rings of residues
2 and 4 on the neighbor (Figure 7a). Each hexamer engages in
this type of tight interaction with two neighboring molecules,
on opposite sides, which results in infinite strips of tightly
packed helices in the crystal.
A spacing-filling view of interface 2, perpendicular to the
12-helix axis (Figure 7b), highlights the extensive and intimate
contact between the cyclopentyl rings on neighboring â-peptides.
This pairing causes burial of 371 Å2 of molecular surface from
solvent exposure, as estimated using the program MacroModel
6.0 and a probe radius of 1.4 Å (approximating a water
molecule; an isolated molecule of 5 in the solid-state conforma-
tion displays a solvent-accessible surface area of 1090 Å2).26
Such extensive burial of predominantly nonpolar surface could
The molecules of hexamer 5 are all parallel to one another
in the crystal (i.e., the helix axes are parallel, and all molecules
display the same orientation of N- and C-termini). This â-peptide
is organized into intermolecularly hydrogen-bonded columns
in the solid state (Figure 4a). The included methanol molecule
participates in this hydrogen-bonding pattern.
Lateral interactions between â-peptide helices are of interest
with regard to creating â-peptide tertiary structure. Figure 5
(24) Applequist, J.; Bode, K.; Appella, D. H.; Christianson, L. A.;
Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 4891.
(25) Hong, Q.; Schellman, J. A. J. Phys. Chem. 1992, 96, 3987.

trans-2-Aminocyclopentanecarboxylic Acid Oligomers
J. Am. Chem. Soc., Vol. 121, No. 33, 1999 7577
Figure 4. Schematic view of the intra- and intermolecular hydrogen-bonding patterns in trans-ACPC oligomer crystals. (a) Hexamer 5, showing
the involvement of a methanol molecule in hydrogen bonds at the helix termini. (b) Octamer 6, showing that the termini of each molecule form
hydrogen bonds to four other molecules.
Figure 5. Solid-state packing pattern of hexamer 5, viewed along the
12-helical axes. Lines indicate bonds between non-hydrogen atoms.
See text for detailed discussion.
provide a significant hydrophobic driving force for helix-helix
interaction in aqueous solution. If the helices were part of a
single â-peptide, then the helix-helix interaction would con-
stitute tertiary structure formation.
Figure 6. Ball-and-stick view of the lateral interaction between adjacent
molecules of hexamer 5 in the solid state (interface 1 in Figure 5).
In the crystal of octamer 6, there are both parallel and
antiparallel neighbor relationships (i.e., all helix axes are parallel,
but neighbors display both identical and opposite orientations
of N- and C-termini), in contrast to the exclusively parallel
packing of hexamer 5. Intermolecular hydrogen bonding in 6
occurs only between â-peptide molecules with parallel orienta-
tion. The end-to-end intermolecular hydrogen-bonding pattern
for octamer 6 is illustrated in Figure 4b. The solvent molecules
included in the crystal of 6 do not appear to hydrogen bond to
The packing pattern of octamer 6 is more complex than the
packing pattern of hexamer 5. Figure 8 provides a schematic
representation of the octamer packing pattern viewed along the
12-helix axis. Individual octamer molecules are represented by
squares with N- and C-termini labeled. Disordered solvent
molecules (water and methanol) lie along one side of each
octamer; the solvent molecules and the C-terminal benzyl groups
occupy the apparent gaps in Figure 8.
the â-peptide.
In the crystalline form of octamer 6 there are two types of
side-to-side interactions between â-peptide helices that involve
extensive contact between cyclopentyl rings. The tighter of these
two lateral interactions occurs between antiparallel neighbors
(26) Mohamdi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton,
M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem.
1990, 11, 440.

7578 J. Am. Chem. Soc., Vol. 121, No. 33, 1999
Appella et al.
Figure 8. Solid-state packing pattern of octamer 6 viewed along the
12-helix axis, which corresponds approximately to the crystallographic
b axis. N and C represent the amino and carboxyl termini of the
â-peptide molecules. See text for detailed discussion.
The interface is defined by residues 1, 3, and 6 on one molecule
and residues 2, 5, and 7 on the other. This lateral interaction is
analogous to but looser than the parallel lateral interaction
between hexamers in crystalline 5 (Figure 7), although the
relative orientations of the helices are not identical. This parallel
pairing of molecules of 6 causes burial of 325 Å2 (1.4 Å probe
radius).
Conclusions. The crystallographic and CD data presented
here show that short â-peptides comprised of (R,R)-trans-ACPC
residues adopt a well-defined helical secondary structure. This
helix, involving a series of interlocking 12-membered ring
hydrogen bonds between backbone amide groups, is distinct
from other secondary structures that have been observed for
short â-peptides. Seebach et al.2a-c and we3a,e,f,21 have previously
reported different types of â-peptide oligomers that adopt a 14-
helical conformation. Poly[R-isobutyl-L-aspartate] and related
â-peptide homopolymers have been proposed to adopt the 14-
helix; interestingly, 16-, 18-, and 20-helical conformations, but
not the 12-helix, have also been proposed for these polymers.27
A major long-range goal of our â-peptide studies is to create
protein-like tertiary structures. The solid-state packing patterns
observed for hexamer 5 and octamer 6 suggest that it will be
possible to achieve this goal with trans-ACPC-based â-peptides.
The helical bundle is one of the simplest tertiary packing motifs
in proteins: R-helical segments pack side-to-side, with short
loops connecting the helices.28 The helix surfaces that contact
one another are typically dominated by nonpolar residues, and
burial of the hydrophobic surface provides a driving force for
tertiary folding in aqueous solution. Relatively short conven-
tional peptides designed to adopt amphiphilic R-helical con-
formations (i.e., R-helices that display discrete hydrophobic and
hydrophilic surfaces) have been shown to self-assemble into
helical bundles in solution and in the solid state.29 Incorporation
of several amphiphilic R-helical segments into a longer polypep-
tide allows de novo creation of protein tertiary structures.30 This
Figure 7. Lateral interactions between adjacent molecules of hexamer
5 in the solid state (interface 2 in Figure 5). (a) Ball-and-stick view,
perpendicular to the 12-helix axis. (b) Space-filling view, perpendicular
to the 12-helix axis (same perspective as in panel a). These and other
space-filling views were generated with the program Molscript.31 (These
are not stereoviews.)
(27) (a) Ferna´ndez-Santin, J. M.; Aymam´ı, J.; Rodr´ıguez-Gala´n, A.;
Mun˜oz-Guerra, S.; Subirana, J. A. Nature 1984, 311, 53. (b) Ferna´ndez-
Santin, J. M.; Mun˜oz-Guerra, S.; Rodr´ıguez-Gala´n, A.; Aymam´ı, J.;
Lloveras, J.; Subirana, J. A.; Giralt, E.; Ptak, M. Macromolecules 1987,
20, 62. (c) Bella, J.; Alema´n, C.; Ferna´ndez-Santin, J. M.; Alegre, C.;
Subirana, J. A. Macromolecules 1992, 25, 5225. (d) Lo´pez-Carrasquero,
F.; Alema´n, C.; Mun˜oz-Guerra, S. Biopolymers 1995, 36, 263.
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J. W.; Betz, S. F.; Lu, H. S.; Suich, D. J.; Zhou, H. X.; O’Neil, K. T.;
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Bryson, J. W.; Roder, H.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A.
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(interface 1 in Figure 8). This interaction is illustrated in detail
in Figure 9. The interface is comprised largely of the cyclopentyl
rings of residues 3, 5, and 8 and the tert-butyl group of the
N-terminal Boc group. This antiparallel pairing causes burial
of 652 Å2 of molecular surface from solvent exposure (1.4 Å
probe radius; an isolated molecule of 6 in the solid-state con-
formation displays a solvent-accessible surface area of 1330 Å2).
The looser of the two types of side-to-side interactions
between â-peptide helices in crystalline 6 occurs between
parallel neighbors (interface 2 in Figure 8). This interaction is
illustrated in detail in Figure 10. Each octamer forms this type
of interface with two neighboring molecules, on opposite sides.
(30) Sheldrick, G. M. SHELXTL Version 5 Reference Manual; Bruker-
AXS: 6300 Enterprise Dr., Madison, WI 53719-1173, 1994.
(31) Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 946.

trans-2-Aminocyclopentanecarboxylic Acid Oligomers
J. Am. Chem. Soc., Vol. 121, No. 33, 1999 7579
Figure 10. Close packing between parallel pairs of octamer 6 in the
solid state (interface 2 in Figure 8). (a) Ball-and-stick view, perpen-
dicular to the 12-helix axis. (b) Space-filling view, perpendicular to
the 12-helix axis (same perspective as in panel a). (These are not
stereoviews.)
strategy should be transferable to â-peptides if we can prepare
polar analogues of trans-ACPC. The lateral packing patterns
observed in the crystals of 5 and 6 suggest that properly spaced
trans-ACPC residues can be used to create complementary
hydrophobic surfaces on 12-helical â-peptides; we are currently
testing this hypothesis.
Figure 9. Close packing between antiparallel pairs of octamer 6
in the solid state (interface 1 in Figure 8). (a) Ball-and-stick
view, perpendicular to the 12-helix axis. (b) Space-filling
view, perpendicular to the 12-helix axis (same perspective as in panel
a).

7580 J. Am. Chem. Soc., Vol. 121, No. 33, 1999
Appella et al.
The solution was stirred 18 h. Di-tert-butyl dicarbonate (3.1 g, 14 mmol)
was then added, and the resulting solution was stirred 48 h. The solvent
was removed on a rotary evaporator, and the product was purified by
SiO2 column chromatography, eluting with 5:1 hexane/ethyl acetate,
to afford 0.66 g (18% yield) of Boc-trans-ACPC-OEt as a white solid.
Crystals were obtained by vapor diffusion of n-heptane into a solution
Experimental Section
General Procedures. Melting points (mp) were obtained on a
Thomas-Hoover capillary melting point apparatus and are uncorrected.
Infrared spectra (IR) were obtained on a Mattson Polaris FT-IR
spectrophotometer or a Nicolet 740 FT-IR spectrometer. Absorption
maxima are reported in wavenumbers (cm-1) and are standardized to
the 1601 cm-1 reference peak of polystyrene. Proton nuclear magnetic
resonance (1H NMR) spectra were recorded in deuterated solvents on
a Bruker AC-300 (300 MHz) or Bruker AMX (500 MHz) spectrometer.
Chemical shifts are reported in parts per million (ppm, δ) relative to
tetramethylsilane (δ 0.00). If tetramethylsilane was not present in the
of Boc-trans-ACPC-OEt in 1,2-dichloroethane: mp 74 °C; [R]D
)
23
-47.1 (c 0.68, CHCl3); IR (KBr) 3342 (N-H), 3045-2703 (broad),
1717 (CdO), 1700, 1526, 1364, 1309, 1251, 1160, 1029, 863, 801
cm-1; H NMR (CDCl3, 300 MHz) δ 4.56 (br, 1H, NH), 4.18-4.09
1
(m, 3H, CH3CH2 and t-BuOCONHCH), 2.56 (q, J ) 7 Hz, 1H,
EtOOCCH), 2.18-1.63 (m, 4H), 1.53-1.38 (m, 11H), 1.43 (s, CH3),
1.26 (t, J ) 7 Hz, 3H, CH3CH2); 13C NMR (CDCl3, 75.4 MHz) δ 174.7
(C), 155.2 (C), 79.2 (C), 60.5 (CH2), 56.0 (CH), 50.9 (CH), 33.0 (CH2),
28.3 (CH3), 22.8 (CH2), 14.1 (CH3).
1
deuterated solvent, the residual protio solvent is referenced. H NMR
splitting patterns are designated as singlet (s), doublet (d), triplet (t),
or quartet (q). All first-order splitting patterns are assigned on the basis
of the appearance of the multiplet. Splitting patterns that could not be
interpreted or easily visualized are designated as multiplet (m) or broad
(br). Coupling constants are reported in hertz (Hz). Carbon nuclear
magnetic resonance (13C NMR) spectra were recorded on a Bruker AC-
300 spectrometer. Chemical shifts are reported in ppm (δ) relative to
the central line of the CDCl3 triplet (δ 77.0) or the CD3OD septet (δ
49.0). Carbon resonances were assigned using distortionless enhance-
ment by polarization transfer (DEPT) spectra obtained with a phase
angle of 135°: (C) not observed; (CH) positive; (CH2) negative; (CH3)
positive. Mass spectra (MS) were obtained using a Kratos MS-80 mass
spectrometer with a magnetic sector and direct probe when electron
impact (EI, 70 eV) was the ionization method. Fast-atom bombardment
(FAB) mass spectra were obtained on a Micromass AutoSpec (Cs ion
gun) with magnetic sector and direct probe using 3-nitrobenzyl alcohol
as the ionization matrix. DMF was distilled from ninhydrin under
vacuum at 25 °C and stored over activated 3-Å molecular sieves, under
N2, at 4 °C. Unless otherwise noted, all other commercially available
reagents and solvents were purchased from Aldrich and used without
further purification, except for 4 N HCl in dioxane, which was
purchased from Pierce. Analytical thin-layer chromatography (TLC)
was carried out on Whatman TLC plates precoated with silica gel 60
(250 µm layer thickness). Visualization was accomplished using either
a UV lamp, potassium permanganate stain (2 g of KMnO4, 13.3 g of
K2CO3, 3.3 mL of 5% (w/w) NaOH, 200 mL of H2O), or ninhydrin
stain (0.5 g of ninhydrin, 150 mL of n-butanol, 5 mL of glacial acetic
acid). Column chromatography was performed on EM Science silica
gel 60 (230-400 mesh). Solvent mixtures used for TLC and column
chromatography are reported in v/v ratios.
Boc-(trans-ACPC)2-OEt. (R,R)-Boc-trans-ACPC-OEt (0.35 g, 1.4
mmol) was dissolved in methanol (20 mL) and water (7 mL). LiOH‚
H2O (0.57 g, 13.6 mmol) was added. The resulting solution was cooled
to 0 °C, and H2O2 (0.75 mL of a 30.8% aqueous H2O2 solution, 6.8
mmol) was added. The reaction was placed in a cold room at 5 °C and
stirred 20 h. While the reaction was still cold, aqueous Na2SO3 (2.7 g
in 12 mL of water, 21.4 mmol) was added. The solution was moved
out of the cold room, and the methanol was removed on a rotary
evaporator. The resulting solution was adjusted to pH 2 with 3 N HCl,
and the aqueous solution was then extracted with CH2Cl2 (3×, 75 mL
total). The organic extracts were dried over Na2SO4, concentrated, and
put under vacuum to yield 0.21 g (67% yield) of white solid (Boc-
trans-ACPC-OH). Boc-trans-ACPC-OEt (0.23 g, 0.9 mmol) was
dissolved in 4 N HCl/dioxane (2 mL). The solution was stirred for 1
h, and then the solvent was removed under a stream of N2 and the
residue further dried under vacuum to give H2N-trans-ACPC-OEt. Boc-
trans-ACPC-OH was dissolved in DMF (3 mL) and transferred via
cannula to the flask containing H2N-trans-ACPC-OEt. DMAP (0.13
g, 1.1 mmol) was added, followed by EDCI‚HCl (0.38 g, 1.98 mmol).
The reaction was stirred for 36 h under N2. The solvent was removed
under a stream of N2, and the residue was further dried under vacuum.
To this residue was added 1 N HCl (a few milliliters). The resulting
white solid was collected by suction filtration and dried under vacuum
to afford 0.3 g (91% yield) of Boc-(trans-ACPC)2-OEt. Fluffy crystals
were obtained by vapor diffusion of n-heptane into a solution of Boc-
(trans-ACPC)2-OEt in 1,2-dichloroethane: mp 180-181 °C dec; IR
(KBr) 3279 (N-H), 2963, 2872, 2771, 1734 (CdO), 1692, 1653, 1558,
1365, 1311, 1181, 1152, 1022, 678 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 7.72 (br, 1H, NH), 4.66 (br, 1H, NH), 4.41 (quintet, J ) 7 Hz, 1H,
CONHCH), 4.13 (ABX3, JAX ) 7 Hz, JAB ) 11 Hz, 1H, CH3CH2),
4.12 (ABX3, JBX ) 7 Hz, JAB ) 11 Hz, 1H, CH3CH2), 3.95 (quintet, J
) 7 Hz, 1H, CONHCH), 2.68 (dt, J ) 8, 7 Hz, 1H, NHCOCH), 2.58
(m, 1H, NHCOCH), 2.15-1.34 (m, 21H), 1.45 (s, CH3), 1.24 (t, J )
7 Hz, 3H, CH3CH2); 13C NMR (CDCl3, 75.4 MHz) δ 174.9 (C), 173.0
(C), 156.4 (C), 80.0 (C), 60.4 (CH2), 56.3 (CH), 54.6 (CH), 53.2 (CH),
50.7 (CH), 33.5 (CH2), 32.7 (CH2), 28.4 (CH3), 27.4 (CH2), 24.1 (CH2),
23.1 (CH2), 14.2 (CH3); EI-MS m/z (M+) calcd for C19H32N2O5
368.2311, obsd 368.2296; FAB-MS m/z 369.3 (M + H+), 313.2 (M+
- t-Bu), 267.2 (M+ - Boc).
Boc-(trans-ACPC)2-OBn (2). Boc-(trans-ACPC)2-OEt (0.26 g, 0.7
mmol) was dissolved in methanol (10 mL) and water (4 mL). LiOH‚
H2O (0.3 g, 7 mmol) was added, the resulting solution was cooled to
0 °C, and H2O2 (0.35 mL of a 30.8% aqueous H2O2 solution, 3.5 mmol)
was added. The reaction was placed in a cold room at 5 °C and stirred
23 h. While the reaction was still cold, aqueous Na2SO3 (1.6 g in 12
mL of water, 13 mmol) was added. The solution was moved out of the
cold room, and the methanol was removed on a rotary evaporator. The
resulting solution was adjusted to pH 2 with 3 N HCl, and the aqueous
solution was then extracted with CH2Cl2 (3×, 100 mL total). The
combined organic extracts were dried over Na2SO4, concentrated, and
dried under vacuum to yield 0.2 g (81% yield) of white solid Boc-
(trans-ACPC)2-OH. This acid was dissolved in DMF (3 mL), and Cs2-
CO3 (0.2 g, 0.7 mmol) was added, followed by benzyl bromide (0.09
mL, 0.8 mmol). The resulting solution was stirred under N2 for 18 h,
and then the solvent was removed under a stream of N2. The residue
was dissolved in 15 mL of water and extracted with CHCl3 (3×, 40
Boc-trans-ACPC-OEt was prepared by a procedure similar to that
of Tilley and co-workers.22b (CAUTION: HN3 is Very toxic and a
Volatile gas!) NaN3 (4 g, 0.06 mol) was suspended in water (4 mL)
and benzene (25 mL). This solution was cooled to 10 °C in an ice
bath, and concentrated H2SO4 (3.5 mL) was added dropwise to the
solution in a fume hood while the temperature was maintained at 10
°C. After addition was complete, the solution was stirred an additional
15 min, and the layers were then separated. The organic layer was dried
over Na2SO4 to yield a solution of HN3 in benzene. The concentration
of acid in the benzene solution was determined by taking 1 mL of the
solution, diluting with water (10 mL), adding 1 drop of phenolphthalein
indicator, and titrating with 0.14 N NaOH. An HN3 concentration of
1.0-1.7 M was typically obtained. Unused HN3 solution was im-
mediately destroyed by addition of excess NaOH. Ethyl (1R,2S)-2-
hydroxycyclopentanecarboxylate22a (2.2 g, 14 mmol) was dissolved in
dry benzene (60 mL), and triphenylphosphine (5.5 g, 21 mmol) was
added, followed by HN3 solution (13 mL, 1.61 M, 21 mmol). Diethyl
azodicarboxylate (2.7 mL, 17 mmol) was dissolved in dry benzene (10
mL), and the solution was added dropwise over 1 h to the solution of
alcohol, triphenylphosphine, and HN3 so that the reaction temperature
did not go above room temperature. During the addition, white solid
formed. After addition was complete, the mixture was stirred an
additional 8 h. The solvent was removed on a rotary evaporator, and
the products were purified by SiO2 column chromatography, eluting
with CH2Cl2, to afford 1.3 g of yellow liquid that was a mixture of
ethyl (1R,2R)-azidocyclopentanecarboxylate and ethyl 1-cyclopenten-
ecarboxylate. This mixture was dissolved in THF (15 mL), and water
(0.3 mL) was added, followed by triphenylphosphine (3.67 g, 14 mmol).

trans-2-Aminocyclopentanecarboxylic Acid Oligomers
J. Am. Chem. Soc., Vol. 121, No. 33, 1999 7581
mL total). The organic extracts were dried over MgSO4, concentrated,
and dried under vacuum. The product was purified by SiO2 column
chromatography, eluting with 20:1 CHCl3/acetone to give 0.21 g (85%
yield) of Boc-(trans-ACPC)2-OBn as a white solid. Fluffy crystals were
obtained by vapor diffusion of n-heptane into a solution of Boc-(trans-
ACPC)2-OBn in 1,2-dichloroethane: mp 142 °C dec; 1H NMR (CDCl3,
300 MHz) δ 7.79 (br, 1H, NH), 7.38-7.28 (m, 4H, ArH), 5.16 (AB
quartet, J ) 12 Hz, 1H, ArCH2), 5.08 (AB quartet, J ) 12 Hz, 1H,
ArCH2), 4.59 (br, 1H, NH), 4.44 (quintet, J ) 7 Hz, 1H, CONHCH),
3.85 (quintet, J ) 7 Hz, 1H, CONHCH), 2.75 (q, J ) 7 Hz, 1H,
NHCOCH), 2.56 (m, 1H, NHCOCH), 2.16-1.26 (m, 21H), 1.44 (s,
CH3); FAB-MS m/z 453.4 (M+).
Boc-(trans-ACPC)3-OBn (3), Boc-(trans-ACPC)4-OBn (4), Boc-
(trans-ACPC)6-OBn (5), and Boc-(trans-ACPC)8-OBn (6) were
prepared by coupling smaller fragments, using procedures analgous to
those described for the synthesis of dimer 2. Characterization data for
these oligomers may be found in the Supporting Information.
Crystallography. The X-ray diffraction data were measured using
a Bruker SMART CCD area detector on a four-circle P4 diffractometer
equipped with Mo KR radiation (λ ) 0.710 73 Å). Data for both
compounds were collected as a series of φ scan frames, each with a
width of 0.3°/frame. The exposure times were 30 s/frame for 5 and 60
s/frame for 6. Empirical absorption corrections for both compounds
were based on equivalent measured intensities. Both structures were
solved by direct methods and refined by full-matrix least-squares,
minimizing ∆F2 (ref 30). For both compounds, the non-hydrogen atoms
were refined with anisotropic displacement parameters. The positions
for hydrogen atoms were calculated from model geometry. For
compound 6, disorder was observed in two of the five-membered rings
(via ring flips), the phenyl end of the peptide, and the two methanol
sites. Restraints for the positional and displacement parameters of the
disordered atoms were required in order to achieve convergence.
Circular Dichroism. Dry peptide samples were weighed on a Cahn
microanalytical balance and dissolved in an appropriate amount of
HPLC grade methanol. Sample cells of 1- and 5-mm path length were
used. Data were collected on a Jasco J-715 spectrometer at 20 °C.
Baseline spectra were recorded with only solvent in the cell. Baseline
spectra were subtracted from the raw data. Data were converted to
ellipticity (deg cm2 dmol-1) according to the following equation:
[Θ] ) ψMr/100lc
where ψ is the CD signal in degrees, Mr is the molecular weight divided
by the number of chromophores, l is the path length in decimeters,
and c is the concentration in grams per milliliter.
Acknowledgment. This research was supported by the NIH
(GM-56414). D.H.A. was supported in part by a Chemistry-
Biology Training Grant from NIGMS and by a fellowship from
Procter & Gamble. The X-ray instrument and computers used
in this study were provided by funds from NSF (CHE-9310428)
and the University of Wisconsin.
Supporting Information Available: Tables of crystal data,
structure solution and refinement, atomic coordinates, bond
lengths and angles, torsion angles, and hydrogen bond param-
eters for 5 and 6 (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
JA991185G