Synthesis and characterization of trans-2-aminocyclohexanecarboxylic acid oligomers: An unnatural helical secondary structure and implications for β-peptide tertiary structure

Article abstract of DOI:10.1021/ja990748l

The preparation, crystal structures, and circular dichroism (CD) spectra of two oligomers of optically active trans-2-aminocyclohexanecarboxylic acid are reported. In the solid state, both the tetramer and the hexamer of this β-amino acid display a helica

Full text of DOI:10.1021/ja990748l

6206
J. Am. Chem. Soc. 1999, 121, 6206-6212
Synthesis and Characterization of
trans-2-Aminocyclohexanecarboxylic Acid Oligomers: An Unnatural
Helical Secondary Structure and Implications for â-Peptide Tertiary
Structure
Daniel H. Appella,† Laurie A. Christianson,† Isabella L. Karle,*,‡ Douglas R. Powell,† and
Samuel H. Gellman*,†
Contribution from the Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706-1396,
and Laboratory for the Structure of Matter, NaVal Research Laboratory, Washington, D.C. 20375-5341
ReceiVed March 8, 1999
Abstract: The preparation, crystal structures, and circular dichroism (CD) spectra of two oligomers of optically
active trans-2-aminocyclohexanecarboxylic acid are reported. In the solid state, both the tetramer and the
hexamer of this â-amino acid display a helical conformation that involves 14-membered-ring hydrogen bonds
between a carbonyl oxygen and the amide proton of the second residue toward the N-terminus. (For comparison,
the familiar R-helix observed in conventional peptides is associated with a 13-membered-ring hydrogen bond
between a carbonyl oxygen and the amide proton of the fourth residue toward the C-terminus.) These
crystallographic data, along with CD data obtained in methanol, suggest that the 14-helix constitutes a stable
secondary structure for â-amino acid oligomers (“â-peptides”). In addition, the crystal packing pattern observed
for the hexamer offers a blueprint for the design of â-peptides that might adopt a helical bundle tertiary structure.
Introduction
including helices, sheets, and reverse turns, depending upon
residue substitution pattern, in organic solvents and in the solid
state. Very recently, the conformational behavior of water-
soluble â-peptides has been described by several groups.8 In
addition, Seebach et al.9 and Hanessian et al.10 have shown that
γ-peptides can adopt helical conformations, Gervay et al.11 have
suggested helix formation by a set of δ-peptides derived from
sialic acid, Zuckermann et al.12 have shown that peptoid
oligomers can adopt helical conformations, Fleet et al.13 have
reported helix formation by δ-peptides constructed from fura-
nose-derived subunits, Moore et al.14 have demonstrated coop-
erative folding in metal-binding phenylacetylene oligomers, and
Biological systems carry out a wide variety of sophisticated
operations at the molecular level. Proteins are responsible for
most of these complex functions, including catalysis, electron
shuttling, and nucleation of inorganic phase crystallization;
RNA, too, can catalyze reactions. Although proteins and RNA
differ from one another considerably in terms of molecular
structure, they share the ability to adopt compact and specific
three-dimensional shapes, and this conformational behavior is
crucial to activity. Based on the uniquely sophisticated activities
displayed by proteins and RNA, one is tempted to speculate
that new polymers with analogous folding properties might be
engineered to display analogous activities.1
Unnatural polymers with well-defined folding propensities
(“foldamers”) have recently attracted a great deal of attention.
For example, results from Seebach et al.2-5 and from our
laboratory6,7 have demonstrated that oligomers of â-amino acids
(“â-peptides”) can adopt a variety of secondary structures,
(7) (a) Krautha¨user, S.; Christianson, L. A.; Powell, D. R.; Gellman, S.
H. J. Am. Chem. Soc. 1997, 119, 11719. (b) Chung, Y. J.; Christianson, L.
A.; Stanger, H. E.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1998,
120, 10555.
(8) (a) High-resolution NMR data for a â-peptide that displays a large
14-helix population in aqueous solution: Appella, D. H.; Durell, S. R.;
Barchi, J. J.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309. (b) CD
data suggesting partial 14-helix formation in aqueous solution: Abele, S.;
Guichard, G.; Seebach, D. HelV. Chim. Acta 1998, 81, 2141. Gung, B. W.;
Zou, D.; Stalcup, A. M.; Cottrell, C. E. J. Org. Chem. 1999, 64, 2176.
(9) Hintermann, T.; Gademann, K.; Jaun, B.; Seebach, D. HelV. Chim.
Acta 1998, 81, 983.
(10) Hanessian, S.; Luo, X.; Schaum, R.; Michnick, S. J. Am. Chem.
Soc. 1998, 120, 8569.
(11) Szabo, L.; Smith, B. L.; McReynolds, K. D.; Parrill, A. L.; Morris,
E. R.; Gervay, J. J. Org. Chem. 1998, 63, 1074.
(12) (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.
* To whom correspondence should be addressed.
† University of Wisconsin.
‡ Naval Research Laboratory.
(1) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173.
(2) (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.
(3) Seebach, D.; Matthews, J. L. J. Chem. Soc., Chem. Commun. 1997,
2015-2022.
(4) 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.
(5) Seebach, D.; Abele, S.; Sifferlen, T.; Ha¨nggi, M.; Gruner, S.; Seiler,
P. HelV. Chim. Acta 1998, 81, 2218.
(6) (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.
(13) 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.
(14) Prince, R. B.; Okada, T.; Moore, J. S. Angew. Chem., Int. Ed. Engl.
1999, 38, 233.
10.1021/ja990748l CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/10/1999

trans-2-Aminocyclohexanecarboxylic Acid Oligomers
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6207
Yang et al.15 have described helix formation by R-aminoxy acid
oligomers. Earlier contributions in this area have been reviewed.1
Here we describe the synthesis and structural characterization
of â-peptide oligomers constructed from trans-2-aminocyclo-
hexanecarboxylic acid (trans-ACHC). In the solid state, tetramer
1 and hexamer 2 both adopt helical conformations defined by
the 14-membered-ring hydrogen bond that can form between a
backbone carbonyl oxygen and the backbone NH two residues
toward the N-terminus (“14-helix”); a preliminary description
of the structure of 2 has appeared.6a Our choice of trans-ACHC
was based on computational predictions.16 Bode and Applequist
independently made the same prediction.17
Figure 1. Stereoview of tetramer 1 in the solid state. Only one of the
two independent molecules is shown; the two independent molecules
display very similar conformations. Hydrogen atoms other than those
attached to nitrogen have been omitted for clarity. Dotted lines indicate
hydrogen bonds.
are used to seed a solution containing (()-3 and (()-2-
hydroxypropylamine, was more reliable.21
For both proteins and RNA, well-defined tertiary structure
appears to be a minimum requirement for complex function.
Although a number of foldamers that display well-defined
secondary structure have been described,1-15 there is no example
yet of an unnatural oligomer that displays a discrete tertiary
structure. The crystallographic data presented here provide
insight on side-to-side packing preferences of trans-ACHC-
based 14-helices, and this insight should guide the design of
larger â-peptides that adopt helical bundle tertiary structures.
Oligomer Synthesis. As in conventional peptide synthesis,
orthogonal amino and carboxyl protecting groups are required
for â-peptide synthesis. We used tert-butoxycarbonyl (Boc) for
the amino group and benzyl ester (Bn) for the carboxyl group.
trans-ACHC dimer 5 was prepared from the monoprotected
trans-ACHC derivatives by using the hydrochloride salt of (N,N-
dimethylamino)propyl-3-ethylcarbodiimide (EDCI‚HCl) in the
presence of 4-(N,N-dimethylamino)pyridine (DMAP). Dimer 5
could be isolated in high purity after washing the crude product
with aqueous 1 N HCl. Further purification was achieved via
silica gel column chromatography. Trimer 6, tetramer 1, and
hexamer 2 were prepared similarly, by coupling smaller
fragments. Oligomers were constructed in both the (R,R) and
(S,S) series.
Results
Preparation of Optically Pure trans-ACHC. trans-ACHC
oligomers can adopt a 14-helical conformation only if all
residues have the same absolute configuration. Our initial efforts
to prepare optically pure trans-ACHC focused on a reported
procedure for resolving N-benzoyl-trans-ACHC (3).18 We
synthesized (()-3 by following published methods,18,19 with a
few modifications to maximize purity (details may be found in
the Supporting Information). One published procedure18 for the
resolution of (()-3 calls for repeated recrystallization of the
quinine salt of 3, which we designate (-,()-4 (the first symbol
in the parentheses refers to the absolute configuration of quinine,
and the second symbol refers to the absolute configuration of
acid 3), to obtain a salt containing only quinine and (-)-(R,R)-
Dimer 5 is soluble in pure chloroform, but trimer 6, tetramer
1, and hexamer 2 require small proportions of methanol in
chloroform to dissolve. All four trans-ACHC oligomers are
soluble in methanol. Hexamer 2 was difficult to purify by
chromatography, because this molecule would not elute from
silica gel unless large amounts of methanol were present;
therefore, 2 was purified on a silicic acid column. Replacing
either protecting group with a terminal amide significantly
decreased the solubility of the trans-ACHC oligomers in all
solvents.
Solid-State Structures. Tetramer 1 adopts a 14-helical
conformation in the solid state (Figure 1). Both of the possible
14-membered-ring hydrogen bonds form (the intramolecular and
intermolecular hydrogen-bonding pattern in crystalline 1 is
illustrated in Figure 2). Two molecules are present in the
N-benzoyl-trans-ACHC [(-,-)-4], with [R]20 -112° (c 2.5,
D
ethanol). This procedure was capricious in our hands and
provided only small amounts of pure (-,-)-4 and (-,+)-4
(details may be found in the Supporting Information). Structural
analysis of (-,+)-4 confirmed that (+)-3 has the (S,S) config-
uration. A modified crystallization procedure,20 in which enan-
tiopure crystals of 3 (obtained initially from quinine resolutions)
(15) Yang, D.; Qu, J.; Li, B.; Ng, F.; Wang, X.; Cheung, K.; Wang, D.;
Wu, Y. J. Am. Chem. Soc. 1999, 121, 589.
(16) Christianson, L. A.; Gellman, S. H., manuscript in preparation.
(17) Bode, K. A.; Applequist, J. Macromolecules 1997, 30, 2144.
(18) Nohira, H.; Ehara, K.; Miyashita, A. Bull. Chem. Soc. Jpn. 1970,
43, 2230.
(19) Prout, F. S.; Beaucaire, V. D.; Dyrkacz, G. R.; Koppes, W. M.;
Kuznicki, R. E.; Marlewski, T. A.; Pienkowski, J. J.; Puda, J. M. J. Org.
Chem. 1973, 38, 1512.
(21) For other synthetic routes that may provide optically enriched trans-
ACHC derivatives, see: (a) Kobayashi, S.; Kamiyama, K.; Ohno, M. Chem.
Pharm. Bull. 1990, 38, 350. (b) Schultz, A. G. Acc. Chem. Res. 1990, 23,
207. (c) Xu, D.; Prasad, K.; Repic, O.; Blacklock, T. J. Tetrahedron:
Asymmetry 1997, 8, 1445.
(20) Nohira, H.; Miura, H. Nippon Kagaku Kaishi 1975, 1122.

6208 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
Appella et al.
Figure 2. Schematic view of the intra- and intermolecular hydrogen-bonding pattern in the crystal of tetramer 1.
Figure 4. Schematic view of the intramolecular hydrogen-bonding
pattern in the crystal of hexamer 2.
Figure 5. Solid-state packing pattern of hexamer 2, viewed along the
14-helical axes. As discussed in the text, there are two types of three-
way interfaces in this packing pattern, one “tight” and the other “loose”.
The tight three-way interface, involving extensive cyclohexyl-cyclo-
hexyl contacts, is displayed by the three molecules in the upper left
corner of the image. The loose three-way interface, with benzyl groups
and solvent at the center, is displayed by the three molecules in the
lower left corner of the image.
Thus, all hydrogen-bonding sites on each â-peptide are satisfied
either intra- or intermolecularly. ClCH2CH2Cl molecules from
the crystallization solvent (slightly less than one per â-peptide)
are present between these columns. There are very few side-
to-side intermolecular contacts between the cyclohexyl rings
in neighboring columns.
Hexamer 2 is also 14-helical in the solid state (Figure 3).
Three hexamers are present in the asymmetric unit; the three
independent conformations are very similar to one another (the
conformation of the C-terminal benzyl group varies among these
molecules, and in one case this group is disordered). All four
of the possible 14-membered hydrogen-bonded rings are formed
in each molecule (Figure 4), generating two turns of 14-helix.6a
The three hexamers in the asymmetric unit are aligned end-to-
end, with the “dangling” pair of CdO and N-H moieties of
each hexamer forming hydrogen bonds to the nearest neighbor.
Thus, as observed in the crystal of tetramer 1, molecules of
hexamer 2 are aligned in continuously hydrogen-bonded col-
umns, and all hydrogen-bonding sites are satisfied either intra-
or intermolecularly.
Figure 3. Solid-state structure of hexamer 2. Hydrogen atoms other
than those attached to nitrogen have been omitted for clarity. Dotted
lines indicate hydrogen bonds. (a) Stereoview of one of the three
molecules in the asymmetric unit (the three independent molecules adopt
very similar conformations). (b) Stereoview of one molecule along the
helix axis. (c) Stereoview of the three molecules in the asymmetric
unit.
asymmetric unit of the crystal, and these two conformations
are very similar. The two molecules in the asymmetric unit are
aligned end-to-end such that the carbonyls of the N-terminal
carbamate and residue 1 of one tetramer are hydrogen bonded
to the amide protons of residues 3 and 4 of the neighbor (Figure
2). Each pair of molecules (one asymmetric unit) packs end-
to-end with neighboring pairs, via the same set of intermolecular
hydrogen bonds, to create infinite hydrogen-bonded columns.
There are extensive lateral interactions between hydrogen-
bonded columns of hexamer 2, in contrast to the packing pattern
observed for 1. Figure 5 illustrates the packing pattern of 2, as
viewed along the 14-helix axis; the columns are packed in a
hexagonal array. There are two types of helix interfaces in this

trans-2-Aminocyclohexanecarboxylic Acid Oligomers
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6209
Figure 7. Circular dichroism data in CH3OH for dimer 5, trimer 6,
tetramer 1, and hexamer 2 (bottom to top at 215 nm). In each case, the
molar ellipticity [θ] has been normalized for oligomer concentration
and the number of backbone amide groups (1, 2, 3, and 5, respectively).
Hexamer 2 was prepared from (S,S)-trans-ACHC, while the shorter
oligomers were prepared from (R,R)-trans-ACHC; to aid comparison,
the curve shown for 2 is the mirror image of the actual curve. Data
were obtained on an 62A-DS instrument at 25 °C.
At present, there is no simple way to correlate far-UV CD
spectra of non-R-amino acid oligomers with specific conforma-
tions, but CD data for new polyamides can, nevertheless, provide
useful information if correlations with other structural data are
available.
CD spectra for dimer 5, trimer 6, tetramer 1, and hexamer 2,
0.3-1.0 mM in methanol, are presented in Figure 7 (1, 5, and
6 were prepared from (R,R)-trans-ACHC; 2 was prepared from
(S,S)-trans-ACHC, but the data are inverted for ease of
comparison). For each â-peptide, the spectrum has been
normalized for concentration and for the number of amide
chromophores, in keeping with conventional peptide practice.
The monomer Boc-(R,R)-trans-ACHC-OBn, which has no
amide chromophores, did not show an appreciable CD signal
in the far-UV region (not shown).
Figure 6. Space-filling view of six molecules of 2 involved in the
tight three-way interface shown in Figure 5, viewed from a perpen-
dicular perspective. The six molecules are distinguished by differential
shading. All six molecules have the same orientation (C-terminus at
the bottom); the third column of molecules involved in this tight
interface (which is omitted for clarity) is antiparallel to those shown.
See text for further discussion.
Each oligomer displays a maximum at ca. 217 nm in Figure
7, and the intensity of this maximum increases with â-peptide
length. We tentatively assign this maximum to the 14-helix,
since both 1 and 2 adopt the 14-helix in the solid state. Further
support for this assignment comes from previously reported
amide H/D exchange data, which suggest that hexamer 2
strongly favors an intramolecularly hydrogen-bonded conforma-
tion, presumably the 14-helix, in methanol solution.6a The weak
maximum at 217 nm displayed by dimer 5, which cannot form
a complete 14-helical turn, implies that the trans-ACHC
backbone is highly preorganized for this secondary structure.
The steady increase in intensity at 217 nm with additional trans-
ACHC residues indicates that the population of the 14-helical
state increases as â-peptide length increases. This trend suggests
that 14-helix formation is cooperative. Analogous cooperativity
is well-established in R-helix formation by conventional pep-
tides.24 In R-helical peptides, cooperativity arises because
propagating an existing helix is favorable, but initiating a helix
is unfavorable.
array, each with approximate three-fold symmetry. One interface
is “tight”. The center of this interface is defined by intimate
interdigitation of three sets of cyclohexyl rings, each set from
a different column. The interdigitation primarily involves
contacts at C4 and C5 of the cyclohexane rings, and we refer
to this arrangement as a “cyclohexane zipper”. One of the
â-peptide helices is antiparallel to the other two in this tight
interface. The second three-fold interface is “loose”. The center
of this interface is filled with solvent molecules (CH3OH and
ClCH2CH2Cl; both used in the crystallization protocol) and
phenyl rings from the C-terminal benzyl ester.
Figure 6 offers a side-on view of the cyclohexane zipper
interaction (six molecules, each with a different shade). Only
two of the three columns that participate in this interaction are
shown; these two columns are parallel, but the antiparallel
contacts are very similar. The space-filling representation in
Figure 6 highlights the intimate interweaving of cyclohexane
rings from neighboring columns of 2.
Circular Dichroism. Circular dichroism (CD) spectroscopy
is extensively used to analyze the secondary structure of
R-amino acid polypeptides.22,23 Each type of regular R-amino
acid secondary structure gives rise to a characteristic CD
spectrum in the far-UV region (190-240 nm). CD in this
spectral region arises from the backbone amide groups and is,
therefore, comparable among peptides with different sequences.
Our CD data for trans-ACHC oligomers are somewhat
different from the CD data Seebach et al. have reported for
â-peptides constructed from â-substituted residues (e.g., 7),2-4
even after accounting for the fact that our data are reported for
the (R,R) series, while the Seebach â-peptides have the opposite
configuration at the â-position. Two-dimensional NMR data
(22) Woody, R. W. Circular Dichroism: Principles and Applications;
VCH Publishers: New York, 1994; pp 24-38, 473-496.
(23) Applequist, J.; Bode, K.; Appella, D. H.; Christianson, L. A.;
Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 4891.

6210 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
Appella et al.
establish that the Seebach â-peptides adopt the 14-helix (which
Seebach et al. refer to as the 31-helix) in organic solvents. CD
data reported for 7 and related â-peptides show a minimum at
214 nm, a zero-point crossing at 206 nm, and a maximum at
198 nm.2-4 The minimum at 214 nm is presumably related to
the maximum we observe at 217 nm, but the differences at lower
wavelengths are difficult to explain at present. It is likely that
the Seebach â-peptides and our trans-ACHC oligomers equili-
brate between the 14-helix and other conformations in solution;
therefore, the CD data represent an average of the intercon-
verting folding patterns. The variations between our CD results
and those of Seebach et al. could indicate that different non-
14-helical conformations are populated by the two classes of
â-peptide.
in this crystalline form.33,34 A second solid-state form of poly-
(R-isobutyl-L-aspartate) was obtained by varying the preparation
conditions. A 20-helical conformation was originally assigned
to this material,32 but reexamination led the original workers
subsequently to propose an 18-helix for this crystalline form of
poly(R-isobutyl-L-aspartate).33
Several groups have examined â-amino acid oligomers of
defined length and composition. Narita et al. have concluded
that relatively short â-alanine oligomers adopt sheetlike packing
patterns in the solid state.27 Yuki et al. have concluded that the
octamer and decamer of R-isobutyl-L-aspartate aggregate to form
sheets in organic solvents.35 As discussed above, Seebach et
al. have shown definitively that relatively short â-peptides
constructed from â-substituted residues adopt 14-helical con-
formations in solution.2,3 This conformation is also observed
for short oligomers of R-substituted residues and for oligomers
containing R,â-disubstituted residues, if the relative stereo-
chemistry is appropriate.3
Discussion
Comparison with Other â-Peptide Structural Information.
Our crystallographic results for trans-ACHC oligomers 1 and
2 represent the first high-resolution evidence for helix formation
by â-peptides. The conformations of molecules containing
â-amino acid residues have been of interest from a variety of
perspectives; we briefly summarize relevant studies below.
â-Amino acid homopolymers are members of the nylon-3
family, and these materials have received scrutiny since the
1960s.25 No high-resolution structural data are available for these
polymers, but considerable effort has been devoted to deducing
structural information from low-resolution data. Poly-â-alanine,
[NHCH2CH2C(dO)]n, has been proposed to adopt a sheet
structure in the solid state but to be disordered in aqueous
solution.26,27 A number of homopolymers of optically active
â-substituted â-amino acids have been examined. Applequist
et al. examined poly-â-L-aspartatic acid in aqueous solution by
a number of methods and concluded that this polymer adopts
some sort of secondary structure under acidic conditions.28 The
secondary structure could not be identified, but these workers
noted, based on physical model building, that the 14-helix
appeared to be a favorable conformation. Goodman et al.
proposed sheet structure for poly[(S)-â-aminobutyric acid].29
Poly(R-isobutyl-L-aspartate) has received the most attention
among â-peptide polymers. Yuki et al. concluded that this
polymer aggregates to form sheets in organic solvents and in
the solid state.30 Subsequent examination via X-ray diffraction
of this material by Spanish workers, however, led them to
conclude that the solid-state conformation of poly(R-isobutyl-
L-aspartate) is actually helical.31-34 These workers proposed a
helix defined by 16-membered hydrogen-bonded rings between
backbone carbonyls and amide protons (“16-helix”, in our
nomenclature).31,32 Reexamination of this material led to a
revised proposal that the â-peptide polymer adopts the 14-helix
Recent work has shown that short â-peptide oligomers can
adopt conformations other than the 14-helix if residue substitu-
tion patterns and sequence are carefully chosen. We have shown
that oligomers of optically pure trans-2-aminocyclopentanecar-
boxylic acid adopt a helix defined by 12-membered-ring
hydrogen bonds (“12-helix”) in the solid state and organic
solvents.6b Seebach et al. have discovered that short â-peptides
with alternating R- and â-substituted residues adopt a helix
containing both 10- and 12-membered-ring hydrogen bonds in
organic solvents.4 The crystal structure of a tri-â-peptide
composed of â-substituted residues revealed an intermolecular
parallel sheet-type hydrogen-bonding interaction;2a however,
it is not clear that this crystal packing pattern provides insight
on sheet formation in solution, since short conventional peptides
are highly prone to adopt sheet packing patterns, even if the
residues have low â-sheet propensities. We have recently shown
that â-peptide residues containing one R- and one â-substituent,
with appropriate relative configuration, adopt antiparallel sheet
secondary structure in solution and in the solid state.7
A
heterochiral dinipecotic acid segment forms a reverse turn,8b
the â-peptide equivalent of the familiar â-turn from conventional
peptides. Seebach et al. have recently observed a reverse turn
in a tri-â-peptide crystal structure;5 it will be interesting to see
whether this segment displays a turn-forming propensity in
solution.
There has been considerable interest in the conformational
effects of placing â-amino acid residues, especially â-alanine,
into peptides containing mostly R-amino acid residues. Much
of this work has focused on cyclic peptides. An early example
is a cyclic tetramer containing two R-amino acid residues and
two â-alanine residues, for which a crystal structure was
determined.36 Several additional crystal structures, along with
structural data from NMR, have been reported for small cyclic
peptides containing one or two â-alanine residues.37 Both
Seebach et al.38 and Ghadiri et al.39 have described cyclic
tetrapeptides containing exclusively â-amino acid residues. Two
recent studies have focused on mimicry of standard secondary
(24) Hong, Q.; Schellman, J. A. J. Phys. Chem. 1992, 96, 3987.
(25) Bestian, H. Angew. Chem., Int. Ed. Engl. 1968, 7, 278.
(26) Glickson, J. D.; Applequist, J. J. Am. Chem. Soc. 1971, 93, 3276.
(27) Narita, M.; Doi, M.; Kudo, K.; Terauchi, Y. Bull. Chem. Soc. Jpn.
1986, 59, 3553.
(28) Kovacs, J.; Ballina, R.; Rodin, R. L.; Balasubramanian, D.;
Applequist, J. J. Am. Chem. Soc. 1965, 87, 119.
(29) Chen, F.; Lepore, G.; Goodman, M. Macromolecules 1974, 7, 779.
(30) Yuki, H.; Okamoto, Y.; Taketani, Y.; Tsubota, T.; Marubayashi,
Y. J. Polym. Sci. Polym. Chem. Ed. 1978, 16, 2237.
(31) Ferna´ndez-Santin, J. M.; Aymam´ı, J.; Rodr´ıguez-Gala´n, A.; Mun˜oz-
Guerra, S.; Subirana, J. A. Nature 1984, 311, 53.
(32) 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. Macromol-
ecules 1987, 20, 62.
(35) Yuki, H.; Okamoto, Y.; Doi, Y. J. Polym. Sci. Polym. Chem. Ed.
1979, 17, 1911.
(36) Karle, I. L.; Handa, B. K.; Hassall, C. H. Acta Crystallogr. 1975,
B31, 555.
(37) Lombardi, A.; Saviano, M.; Nastri, F.; Maglio, O.; Mazzeo, M.;
Isernia, C.; Paolillo, L.; Pavone, V. Biopolymers 1996, 37, 693 and
references therein.
(33) Bella, J.; Alema´n, C.; Ferna´ndez-Santin, J. M.; Alegre, C.; Subirana,
J. A. Macromolecules 1992, 25, 5225.
(34) Lo´pez-Carrasquero, F.; Alema´n, C.; Mun˜oz-Guerra, S. Biopolymers
1995, 36, 263.
(38) Seebach, D.; Matthews, J. L.; Meden, A.; Wessels, T.; Baerlocher,
C.; McCusker, L. B. HelV. Chim. Acta 1997, 80, 173.
(39) Clark, T. D.; Buehler, L. K.; Ghadiri, M. R. J. Am. Chem. Soc.
1998, 120, 651.

trans-2-Aminocyclohexanecarboxylic Acid Oligomers
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6211
Experimental Section
structures by â-amino acid insertions into acyclic peptides.
Karle, Balaram, and co-workers have shown that a â-alanyl-
â-aminobutyryl segment, which mimics a triglycine segment,
can participate in helix formation.40 Hanessian and Yang have
observed that a segment containing a disubstituted â-amino acid
residue followed by proline can induce reverse turn formation.41
Gung et al. have examined the folding of small oligoamides
containing â-amino acid residues in organic solvents.42
General Procedures. Melting points (mp) were obtained on a
Thomas-Hoover capillary melting point apparatus and are uncorrected.
Optical rotations were measured on a Perkin-Elmer 241 digital
polarimeter using sodium light (D line, 589.3 nm) and are reported in
degrees; concentrations (c) are reported in g/100 mL. 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. 1H NMR spectra were recorded in deuterated
solvents on a Bruker AC-300 (300 MHz) spectrometer. 1H 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). 13C NMR spectra were recorded on a Bruker AC-300 spectrometer.
Carbon resonances were assigned using distortionless enhancement by
polarization transfer (DEPT) spectra obtained with a phase angle of
135°: (C) not observed; (CH) positive; (CH2) negative; (CH3) positive.
Mass spectra 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.
Implications for â-Peptide Tertiary Structure. The packing
pattern displayed by hexamer 2 suggests a strategy for creating
â-peptides that adopt a well-defined tertiary structure, because
there are extensive cyclohexyl-cyclohexyl contacts between
adjacent molecules. (In contrast, the packing of tetramer 1 cannot
provide insight on tertiary packing of 14-helical â-peptides
because the side-to-side contacts occur between cyclohexane
rings and terminal phenyl groups or solvent.) The “tight” three-
fold cyclohexane zipper interaction (Figures 5 and 6) seems
likely to be a significant source of lattice stability for solid 2,
although we acknowledge that it is impossible to identify
definitively the origin of any packing pattern’s stability by
simple inspection. It should be possible to use this trimeric
cyclohexane zipper packing motif to design â-peptides that adopt
a three-helix bundle tertiary structure in aqueous solution, since
the cyclohexane-cyclohexane contacts should be promoted by
the hydrophobic effect. In crystalline 2, there are three inde-
pendent molecules, and there are three slightly different versions
of the tight three-fold interaction. The amount of water-
accessible surface area buried in each of these trimolecular
clusters was estimated with the program MacroModel 6.0, using
a spherical probe with 1.4-Å radius (approximating a water
molecule). The water-accessible surfaces of isolated molecules
of 2 in the solid-state conformations fall between 1103 and 1142
Å2. Assembly of the isolated molecules into the trimolecular
clusters observed in crystalline 2 buries between 562 and 743
Å2 (the variation arises in part from differences in benzyl group
orientation).
Isoamyl alcohol was distilled prior to use and stored over 4-Å
molecular sieves. Dimethyl formamide (DMF) was distilled from
ninhydrin under vacuum at 25 °C and stored over activated 3-Å
molecular sieves, under N2, at 4 °C. Benzene was distilled from sodium/
benzophenone ketyl. Hexanes were distilled at atmospheric pressure.
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). Column chromatography was performed with EM Science
silica gel 60 (230-400 mesh). Solvent mixtures used for TLC and
column chromatography are reported in v/v ratios.
(+)-(S,S)- and (-)-(R,R)-N-benzoyl-trans-2-aminocyclohexane-
carboxylic acid ((+)-3 and (-)-3) were prepared by seed-induced
enantioselective crystallization, following a reported procedure.20 From
4 g of (()-N-benzoyl-trans-2-aminocyclohexanecarboxylic acid,18,19
Creating water-soluble â-peptides will require incorporation
of hydrophilic residues to complement the hydrophobic trans-
ACHC residues. Because the 14-helix has approximately three
residues per turn, â-peptides that contain a trans-ACHC residue
at every third position and hydrophilic residues at the other
positions should adopt helical conformations with a hydrophobic
“stripe” running along one side. Among conventional peptides
(R-amino acid residues), helices with well-defined hydrophilic
and hydrophobic surface patches are referred to as “am-
phiphilic”. Relatively short amphiphilic R-helices self-assemble
into discrete helical bundle aggregates, and this intermolecular
assembly has been parlayed into helical bundle tertiary structure
formation, an intramolecular process, by connecting amphiphilic
R-helical segments with short loops.43-46 An analogous design
strategy could lead to a â-peptide three-helix bundle tertiary
structure built around a cyclohexyl hydrophobic core.
1.63 g (41% yield) of the (-)-(R,R) enantiomer was obtained, [R]23
D
) -45° (c 0.2, EtOH), and 0.395 g (10% yield) of the (+)-(S,S)
enantiomer was obtained, [R]23 ) +45° (c 0.2, EtOH).
D
Boc-trans-2-aminocyclohexanecarboxylic acid (Boc-trans-ACHC-
OH). Crystals of (-)-N-benzoyl-trans-2-aminocyclohexanecarboxylic
acid (0.5 g, 2.02 mmol) were suspended in 12 N HCl (55 mL). The
solution was refluxed 48 h, and the crystals eventually dissolved. After
the solution cooled to room temperature, the solution was cooled to 0
°C for 1 h in an ice bath, and then precipitated benzoic acid was
collected by suction filtration. The filtrate was concentrated on a rotary
evaporator to a few milliliters, and then 2:1 dioxane/H2O (30 mL) was
added, followed by K2CO3 (2.8 g, 20 mmol) and Boc2O (0.441 g, 2.02
mmol). The reaction was stirred for 5 h. The pH of the reaction was
adjusted to 2 with 12 N HCl, and the resulting solution was then
extracted with EtOAc (3×, 150 mL total). The organic extracts were
dried over MgSO4, concentrated, and dried under vacuum to afford
0.41 g (84% yield) of the desired product as a white solid. Crystals
were grown by slow evaporation of a solution of the product in 1,2-
(40) Karle, I. L.; Pramanik, A.; Banerjee, A.; Bhattacharjya, S.; Balaram,
P. J. Am. Chem. Soc. 1997, 119, 9087.
dichloroethane: mp 154-155 °C; [R]23 ) -22.8 (c 0.325, CHCl3);
D
(41) Hanessian, S.; Yang, H. Tetrahedron Lett. 1997, 38, 3155.
(42) (a) Gung, B. W.; Zhu, Z. J. Org. Chem. 1997, 62, 6100. (b) Gung,
B. W.; MacKay, J. A.; Zou, D. J. Org. Chem. 1999, 64, 700.
(43) Regan, L.; DeGrado, W. F. Science 1988, 241, 976.
(44) Mutter, M.; Vuilleumier, S. Angew. Chem., Int. Ed. Engl. 1989,
28, 535.
IR (KBr) 3315 (N-H), 3045-2703 (broad), 2937, 2859, 1738 (Cd
1
O), 1657, 1545, 1348, 1284, 1163, 1124, 1051, 862, 690 cm-1; H
NMR (CDCl3, 300 MHz) δ 10.15 (br, 1H, COOH), 4.68 (br, 1H, NH),
3.64 (br m, 1H), 2.26 (td, J ) 11, 3 Hz, HOOCCH), 2.12-1.9 (m,
2H), 1.8-1.11 (m, 6H), 1.43 (s, 9H, CH3); 13C NMR (CDCl3, 75.4
MHz) δ 178.9 (C), 155.1 (C), 79.4 (C), 51.0 (CH), 49.6 (CH), 32.8
(CH2), 28.6 (CH2), 28.3 (CH2), 24.6 (CH2), 24.4 (CH2); EI-MS m/z
(M+) calcd for C12H21NO4 243.1471, obsd 243.1475.
(45) Bryson, J. W.; Betz, S. F.; Lu, H. S.; Suich, D. J.; Zhou, H. X.;
O’Neil, K. T.; DeGrado, W. F. Science 1995, 270, 935.
(46) Bryson, J. W.; Desjarlais, J. R.; Handel, T. M.; DeGrado, W. F.
Protein Sci. 1998, 7, 1404.

6212 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
Appella et al.
Boc-trans-ACHC-OBn. Boc-trans-ACHC-OH (0.2 g, 0.82 mmol)
was dissolved in dry benzene (20 mL). Benzyl bromide (0.1 mL, 0.82
mmol) and 1,4-diazabicycloundecene (0.12 mL, 0.82 mmol) were
added, and the solution was refluxed under N2 for 6 h. White solid
precipitated from the solution during this time. The mixture cooled to
room temperature. The solution was then filtered through a fritted
funnel, and the filtrate was concentrated to obtain a tan solid. The
product was purified by SiO2 column chromatography, eluting with
70:1 CHCl3/EtOAc (Rf ) 0.31), to afford 0.228 g (83% yield) of Boc-
trans-ACHC-OBn as a white solid that was recrystallized from
n-heptane: mp 103 °C; IR (KBr) 3375 (N-H), 2929, 2854, 1732 (Cd
clinic space group P21, with a ) 15.3932(3) Å, b ) 21.7643(4) Å, c
) 27.8692(2) Å, â ) 101.457(2)°, V ) 9150.8(3) Å3, Z ) 6, and
calculated F ) 1.142 g/cm3. Empirical absorption corrections for both
compounds were based on equivalent measured intensities. For
compound 1, the 35 619 measured data out to 26.053° in θ were merged
to give 15 248 unique data with Rint ) 0.0744. The 28 218 measured
data out to 22.5° in θ for compound 2 were merged to give 21 585
unique data with Rint ) 0.0279.
Compound 1 was solved by direct methods. Compound 2 was solved
using the PATSEE program47 and a 34 atom model of the backbone
from 1. The PATSEE program first performs a three-dimensional
rotational fit of the model atom vectors to a vector (Patterson) map
calculated from the intensity data and then performs a three-dimensional
translation search of the oriented fragment with a subset of the intensity
data. The phases of the subset data for this new model are extended to
other data,48 and this expanded data set is used to compute an E-map49
(a modified electron density map) that should show more or all of the
atoms.
1
O), 1684, 1527, 1369, 1315, 1271, 1176, 1014, 727 cm-1; H NMR
(CDCl3, 300 MHz) δ 7.38-7.29 (m, 5H, ArH), 5.11 (s, 2H, ArCH2),
4.55 (br, 1H, NH), 3.69 (br td, J ) 10.5, 9 Hz, 1H, BocHNCH), 2.30
(td, J ) 12, 3 Hz, 1H, BnOCOCH), 2.06 (br dq, J ) 12, 4 Hz, 1H),
1.98-1.88 (m, 1H), 1.79-1.53 (m, 3H), 1.39 (s, 9H, CH3), 1.36 (m,
1H), 1.27-1.08 (m, 2H); 13C NMR (CDCl3, 75.4 MHz) δ 173.8 (C),
154.9 (C), 136.0 (C), 128.5 (CH), 128.0 (CH), 79.2 (C), 66.3 (CH2),
51.3 (CH), 50.0 (CH), 33.0 (CH2), 28.6 (CH2), 28.3 (CH3), 24.6 (CH2),
24.4 (CH2); EI-MS m/z (M + H+) calcd for C19H28NO4 334.2018, obsd
334.2000.
Both compounds were refined by full-matrix least-squares methods,
minimizing ∆F2 (ref 50). For both structures, the non-hydrogen atoms
were refined with anisotropic displacement parameters. The positions
for hydrogen atoms were calculated from model geometry. The 1018
variables of compound 1 were refined against 85 restraints and 15 234
data to give wR(F2) ) 21.17% with a goodness-of-fit, S, ) 1.184. For
the 10 942 observed [F > 4σ(F)] data, the final R(F) was 8.49%. For
compound 2, a total of 2059 variables were refined against 638 restraints
and 21 585 data to produce wR(F2) ) 28.00% with S ) 1.137, and
R(F) ) 10.91% for 14 406 observed [F > 4σ(F)] data. Restraints were
applied only to disordered regions of the structures.
Boc-(trans-ACHC)2-OBn (5). Boc-trans-ACHC-OBn (0.047 g, 0.14
mmol) was dissolved in 4 N HCl/dioxane (0.5 mL) and stirred for 1 h.
The solvent was then removed under a stream of N2, and the residue
was dried under vacuum. Boc-trans-ACHC-OH (0.034 g, 0.14 mmol)
and DMAP (0.023 g, 0.19 mmol) were added to the flask, followed by
DMF (1 mL). EDCI‚HCl (0.059 g, 0.31 mmol) was added, and the
reaction was stirred for 48 h under N2. Solvent was removed under a
stream of N2, and the residue was further dried under vacuum. To this
residue was added 1 N HCl (approximately 3 mL), and the solid that
did not dissolve was isolated by suction filtration and washed with
additional 1 N HCl. The solid was dried under vacuum to afford 0.051
g (79% yield) of Boc-(trans-ACHC)2-OBn as a white solid. Fluffy
crystals were grown by vapor diffusion of n-heptane into a solution of
Boc-(trans-ACHC)2-OBn in 1,2-dichloroethane: mp 195 °C, IR (KBr)
CD Experiments. Dry peptide samples were weighed and dissolved
in an appropriate amount of HPLC grade methanol. Sample cells of
1-mm path length were typically used. Data were collected on an Aviv
62A-DS spectrometer at 25 °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:
2773, 1744 (CdO), 1685, 1651, 1540, 1176, 1027 cm-1 1H NMR
;
(CDCl3, 300 MHz) δ 7.40-7.27 (m, 5H, ArH), 6.07 (br d, J ) 6 Hz,
1H, NH), 5.11 (AB quartet, J ) 12 Hz, 1H, ArCH2), 5.05 (AB quartet,
J ) 12 Hz, 1H, ArCH2), 4.55 (br d, J ) 9 Hz, 1H, NH), 4.05 (tdd, J
) 11, 9, 4 Hz, 1H, BocHNCH), 3.45 (tdd, J ) 11, 9, 4 Hz, 1H,
CONHCH), 2.36 (td, J ) 11, 4 Hz, 1H, HNCOCH), 2.14-1.81 (m,
5H), 1.78-1.53 (m, 14H), 1.41 (s, CH3), 1.38-1.04 (m, 6H); 1H NMR
(CD3OH, 300 MHz) δ 7.63 (br, 1H, NH), 7.41-7.23 (m, 5H, ArH),
6.17 (br, 1H, NH), 3.94 (m, 1H, CONHCH), 3.52 (m, 1H, CONHCH),
2.44 (td, J ) 11, 3 Hz, HNCOCH), 2.06-1.78 (m, 4H), 1.77-1.45
(m, 5 H), 1.44-1.04 (m, 16H), 1.35 (s, CH3); 13C NMR (CDCl3, 75.4
MHz) δ 173.6 (C), 173.1 (C), 155.6 (C), 136.0 (C), 128.4 (CH), 128.1
(CH), 128.0 (CH), 79.3 (C), 66.2 (CH2), 52.6 (CH), 50.8 (CH), 49.6
(CH), 49.5 (CH), 33.7 (CH2), 32.2 (CH2), 30.6 (CH2), 28.3 (CH3), 25.0
(CH2), 24.4 (CH2); FAB-MS m/z 917.6 (2M + H+), 459.3 (M + H+),
359.2 (M + H+ - Boc).
[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. We are grateful to H. Nohira for provid-
ing a translation of the enantioselective crystallization procedure
in ref 20, and to M. Richards for assistance with graphics. This
research was supported by the NIH (GM56414; S.H.G.). D.H.A.
was supported in part by a Chemistry-Biology Interface
Training Grant from NIGMS, and by a fellowship from Procter
& Gamble. Funds for the purchase of X-ray instruments and
computers were provided by the NSF (CHE-9310428) and the
University of Wisconsin.
Boc-(trans-ACHC)3-OBn (6), Boc-(trans-ACHC)4-OBn (1), and
Boc-(trans-ACHC)6-OBn (2) were prepared by coupling smaller
fragments, using procedures analogous to those described for the
synthesis of 5. In each oligomer, all trans-ACHC units were of the
same absolute configuration. Characterization data for these oligomers
may be found in the Supporting Information.
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 1, 2, and (-,+)-4, and experimental protocols (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
Crystallization and X-ray Analysis. X-ray quality crystals of
tetramer 1 and hexamer 2 were grown by vapor diffusion of a nonpolar
solvent (distilled hexanes or HPLC grade heptane) into a solution of
the molecule in 1,2-dichloroethane (distilled) and HPLC grade metha-
nol. The X-ray diffraction data were measured at -140 °C using a
Bruker SMART CCD area detector on a four-circle 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 1 and 40
s/frame for 2. Crystals of 1 [C40H60N4O7‚0.94(C2H4Cl2), FW ) 801.93]
form in the orthorhombic space group P212121, with a ) 11.6175(3)
Å, b ) 23.1923(5) Å, c ) 33.2538(9) Å, V ) 8959.8(4) Å3, Z ) 8,
JA990748L
(47) Egert, E.; Sheldrick, G. M. Acta Crystallogr. 1985, A41, 262.
(48) Karle, J. Acta Crystallogr. 1968, B24, 182.
(49) Karle, I.; Hauptman, H.; Karle, J.; Wing, A. B. Acta Crystallogr.
1958, 11, 257.
(50) Sheldrick, G. M. SHELXTL Version 5 Reference Manual; Bruker-
AXS, 6300 Enterprise Dr., Madison, WI 53719-1173, 1994.
and calculated
0.33(CH3OH)‚0.80(C2H4Cl2), FW ) 1049.24] crystallizes in the mono-
F ) 2 [C54H82N6O9‚
1.189 g/cm3. Compound