A rationally designed aldolase foldamer

Article abstract of DOI:10.1002/anie.200804996

(Chemical Equation Presented) Neatly folded: A decameric β-peptide shows enzyme-like catalytic properties. The foldamer, which bears a terminal heptanoyl unit and displays a thermostable helical structure with an array of ammonium-group side chains, accel

Full text of DOI:10.1002/anie.200804996

Communications
DOI: 10.1002/anie.200804996
Catalytic Foldamers
A Rationally Designed Aldolase Foldamer**
Manuel M. Mꢀller, Matthew A. Windsor, William C. Pomerantz, Samuel H. Gellman,* and
Donald Hilvert*
Current strategies for creating enzyme-like catalysts range
from rational[1] and computational design[2] to evolutionary
searches of large molecular libraries.[3] Sequence-specific
polymers are particularly attractive starting points for these
approaches because of their ability to adopt three-dimen-
sional structures that preorganize functional groups for
catalysis. Although natural enzymes are constructed from a-
amino acids, many other backbone structures can give rise to
well-defined secondary and tertiary structures. Such non-
natural oligomers, often referred to as “foldamers”, have the
potential to display properties akin to those of proteins.[4–8]
Scheme 1. Retroaldol reaction of sodium 4-phenyl-4-hydroxy-2-oxobuty-
rate (1) and the mechanism of amine catalysis.
b-Peptides are interesting in this context because they
adopt a variety of stable secondary structures, including
helices, sheets, and turns;[4,9] quaternary helix-bundle assem-
blies have also been generated.[10,11] Their predictable struc-
tures have been exploited to inhibit microbial growth,[12]
disrupt protein–protein interactions,[13] and for other applica-
tions.[4,5] Herein we report that b-peptides presenting arrays
of discrete side-chain functional groups can also act as
effective catalysts.
As a model reaction, we examined the retroaldol cleavage
of b-hydroxyketone 1 to give benzaldehyde and pyruvate.
This reaction is subject to amine catalysis (Scheme 1). The
catalytic cycle is initiated by nucleophilic attack of an amine
on the carbonyl group of the substrate. The resulting iminium
Since imine formation is partially limiting in an aqueous
environment,[17] the challenge in the design of catalysts for
such retroaldol reactions is to provide a nucleophilic amine
under physiological conditions. Studies of enzymes that
exploit imine or enamine formation have shown that amine
nucleophilicity can be significantly enhanced through cou-
lombic interactions with nearby cations, which increase the
population of the unprotonated state.[18] This strategy, which
has been successfully utilized in the design of helical a-
peptide catalysts,[15,17,19] can be extended to the construction
of helical b-peptide catalysts.
One of the best-understood b-peptide secondary struc-
tures is the 14-helix, which is characterized by 14-membered
À
ion activates the substrate for C C bond scission, which
À
occurs with concomitant deprotonation of the hydroxyl group
to release benzaldehyde. Tautomerization of the enamine and
subsequent hydrolysis produces pyruvate and regenerates the
catalyst. This mechanism is exploited by natural type-I
aldolases,[14] and has been mimicked by lysine-rich amphi-
philic a-helical peptides,[15] catalytic antibodies,[16] and most
recently by a computationally designed enzyme.[2a]
ring hydrogen bonds between the N H unit of residue i and
=
C O unit of residue i + 2.[4,9] This helix is favored by b3-amino
acid residues, which bear a side chain on the backbone carbon
atom adjacent to the nitrogen atom. To stabilize such
structures in aqueous solution, electrostatic interactions
between the side chains[20] or conformationally restricted
building blocks[21] can be employed. For example, the cycli-
cally constrained trans-2-aminocyclohexanecarboxylic acid
(ACHC), which has a very high propensity to adopt a helical
conformation, has been used to construct short b-peptides
that reliably adopt the 14-helical conformation in water,
regardless of temperature, pH, or concentration.[21,22] A b-
peptide sequence that contains multiple ACHC residues
along with b3-homolysine (b3-hLys) residues in an i, i + 3, i + 6
array should lead to alignment of the amine-containing side
chains along one side of a 14-helix (Scheme 2). We anticipated
that this clustering of b3-hLys residues would cause a decrease
in side-chain ammonium pKa values and thus facilitate amine
catalysis of the retroaldol reaction of 1.
[*] M. A. Windsor, Dr. W. C. Pomerantz, Prof. Dr. S. H. Gellman
Department of Chemistry, University of Wisconsin-Madison
1101 University Avenue, Madison, WI 53706 (USA)
Fax: (+1)608-265-4534
E-mail: gellman@chem.wisc.edu
M. M. Mꢀller, Prof. Dr. D. Hilvert
Laboratorium fꢀr Organische Chemie, ETH Zꢀrich
Hꢁnggerberg HCI F339, 8093 Zꢀrich (Switzerland)
Fax: (+41)44-632-1486
E-mail: hilvert@org.chem.ethz.ch
[**] This work was supported by the Schweizerischer Nationalfonds,
NIH grant GM056414 (to S.H.G.), and the Nanoscale Science and
Engineering Center at UW-Madison (NSF DMR-042588). We thank
Dr. Dennis Gillingham for help with substrate preparation.
Our design hypothesis was tested with b-peptides 2–4
(Scheme 2). b-Peptide 2 has three ACHC-ACHC-b3-hLys
triad repeats, a b3-hTyr residue to facilitate concentration
determination, and an N terminus that is capped with a
heptanoyl moiety to promote self-assembly.[23] Isomeric b-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200804996.
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Circular dichroism (CD) measurements provided prelimi-
nary insight into the folding and self-assembly of b-peptides
2–4; although CD is an intrinsically low-resolution method,
previous correlations with 2D NMR spectroscopy and
analytical ultracentrifugation (AU) data provide a basis for
interpretation.[22] CD data indicate that 2 is highly 14-helical
and self-assembled in aqueous buffer, that 3 is comparably
folded but not self-assembled,[22] and that 4 is largely unfolded
(Figure S1 in the Supporting Information). The dramatic
conformational difference between 2 and 3, which contain
ACHC residues, and 4, which lacks ACHC residues, highlights
the strong 14-helix stabilization provided by preorganization
at the residue level. Further characterization by NMR
spectroscopy (2–4) and AU (2) supports the conclusion that
2 forms large assemblies, while 3 and 4 undergo relatively
little self-association (Figure S2 in the Supporting Informa-
tion). The observation that 2 avidly self-associates, while 3
and 4 do not, indicates that the heptanoyl unit alone is
insufficient to induce self-association. Instead, there appears
to be a cooperative effect from pairing this terminal unit with
a b-peptide segment that forms a stable helical conformation
and displays a large, uninterrupted hydrophobic surface.
Comparable cooperative effects have been observed among
terminally modified a-peptides.[23]
The b-peptides were tested for their ability to promote the
retroaldol cleavage of 1, an anionic substrate that is expected
to bind well to the cationic catalysts. The reaction was
monitored by a standard coupled assay in which lactate
dehydrogenase (LDH) was used to catalyze the reduction of
pyruvate (a retroaldol product) by NADH (NADH =
reduced nicotinamide adenine dinucleotide). b-Peptide 2
promotes the retroaldol cleavage of 1 with multiple turnovers
(Figure S3 in the Supporting Information) and significant rate
accelerations over the uncatalyzed reaction. In contrast, b-
peptides 3 and 4 are relatively poor catalysts under identical
conditions (Figure 1a). The 20- to 50-fold lower activity for
these control b-peptides shows that the activity of 2 requires
both a well-folded helix and a properly arrayed set of b3-hLys
side chains.
Scheme 2. a) Sequences of b-peptides 2–6. b) Helical wheel diagram
of b-peptide 2 (left) and a cartoon showing b-peptide self-assembly
(right; the partial protonation state shown is purely hypothetical).
peptide 3 has a scrambled sequence that does not match the
three-residue repeat of the 14-helix; the three b3-hLys
residues of 3 should therefore be dispersed around the 14-
helix periphery rather than held in close alignment, as in 2.
Comparison of the activities of 2 and 3 allows us to determine
whether the spatial arrangement of the b3-hLys residues is
important for catalysis. b-Peptide 4 is an analogue of 2 in
which all of the preorganized ACHC residues are replaced by
flexible b3-hVal residues. Comparison of the activities of 4 and
2 allows us to assess the importance of helical folding for
catalysis.
The impact of N-terminal acyl group modification indi-
cates that b-peptide self-assembly is important for generation
Figure 1. Kinetic characterization of b-peptide aldolases. Retroaldol reactions were performed in quartz cuvettes with b-peptide (50 mm), substrate
1 (0.5 mm), NADH (0.1 mm), and LDH (1 UmLÀ1) in tris(hydroxymethyl)aminomethane hydrochloride (tris–HCl, 50 mm, pH 8) containing NaCl
(150 mm), at 308C. Product formation was coupled to the oxidation of NADH through the LDH-catalyzed reduction of pyruvate and monitored
spectroscopically at 340 nm (eNADH =6220mÀ1 cmÀ1). Rates were corrected for background retroaldol cleavage and elimination reactions.
a) Specific activities of b-peptides 2–6. b) pH rate profile for b-peptide 2. Additional LDH (10 UmLÀ1) was added to compensate for the reduced
a1ÀpH
dehydrogenase activity at high pH values. The rate profile was fitted to the equation kcat/Km =kHA/(1 + 10pK
+ 10pHÀpK ). c) Michaelis–Menten
a2
plot for 2. The dependence of rate on substrate concentration was determined both in continuous and discontinuous assay formats. Reactions
were performed in Tris–HCl (50 mm, pH 8) containing NaCl (150 mm), b-peptide 2 (50 mm), and substrate (0.2–12 mm) at 308C. NADH (0.5 mm)
and LDH (2 UmLÀ1) were added for continuous monitoring.
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Communications
of the catalytic species. Replacement of the heptanoyl group
10À3 minÀ1, Km = 0.5 mm, kcat/kuncat = 24000).[2a] The small
difference in rate enhancement between a 10-residue b-
peptide and a 200-residue protein underscores the catalytic
potential in b-peptide scaffolds. Furthermore, unlike helices
formed by a-peptides, the b-peptide 14-helix is very stable
when preorganized ACHC residues are incorporated into it.
In fact, b-peptide 2 retains high aldolase activity even at 808C
(kcat = (3.5 Æ 0.2) minÀ1, Km = (5.5 Æ 1.0) mm, kcat/kuncat = 1300;
Figure S6 and S7 in the Supporting Information), which firmly
illustrates the robust nature of this scaffold.
Although only 10 residues long and designed according to
very simple principles, b-peptide 2 displays excellent catalytic
properties. Our results suggest that b-peptidic structures may
be versatile scaffolds for engineering catalytic activities.
Nevertheless, the prototypic b-peptide catalyst presented
here is not optimal. The high Km value likely reflects the lack
of a defined substrate-binding pocket. The change from a
secondary to a tertiary structure should allow creation of true
active sites in which functional groups can be effectively
oriented for highly efficient catalysis. Recent approaches
toward higher-order foldamer structures,[8,11,24] and the diver-
sity of backbone chemistry available to folding oligomers in
general,[5,6] present exciting opportunities for the develop-
ment of more sophisticated catalysts.
with an acetyl group to generate b-peptide 5 reduces the
characteristic CD signature for self-assembly, but not the CD
signature for 14-helical secondary structure (Figure S1 in the
Supporting Information); the catalytic activity of 5 is an order
of magnitude less than that of 2. b-Peptide 6, which lacks the
acyl group altogether, is monomeric up to a concentration of
approximately 1 mm;[10a] and is more than 20-times less active
than 2. Some a-peptides designed for amine-mediated
catalysis benefit from self-assembly;[17] the interpeptide
interactions in these systems lead to a significant enhance-
ment of a-helical folding. In contrast, the benefit of self-
assembly for b-peptide catalysis is unlikely to be caused by an
increase in 14-helicity, because ACHC-rich sequences such as
those of 2, 3, 5, and 6 afford very high populations of the
helical conformation in aqueous solution even when the b-
peptides remain monomeric.[21,22] The favorable effects that
arise from the clustering of b-peptide molecules may reflect a
further reduction in the pKa value of the b3-hLys side-chain
ammonium group, which occurs as a result of high local
positive-charge density or an increase in the hydrophobicity
of the environment of catalytic amines. Alternatively, an
interface between helical b-peptides could act as a primitive
substrate-binding pocket. These postulated effects could
operate in tandem.
Received: October 13, 2008
Revised: November 11, 2008
Published online: December 18, 2008
As suggested by the mechanism proposed in Scheme 1,
the reaction catalyzed by 2 is pH-dependent. A bell-shaped
pH rate profile is observed, with a maximum rate at pH 9 and
pK1 = (8.8 Æ 0.2) and pK2 = (9.2 Æ 0.2) (Figure 1b). Although
the available data do not allow assignment of the inflections
to specific ionizing groups, the lower value is consistent with
the participation of a b3-hLys side-chain amino group that has
an unusually low pKa value. The decrease in activity at high
pH value could indicate bifunctional acid–base catalysis, or
reflect a change in the aggregation state of the b-peptide.
Additional evidence for the postulated mechanism was
obtained by trapping imine intermediates by reduction with
NaCNBH3. Signals that correspond to peptides modified with
pyruvate product, one or two aldol substrates, and one aldol
plus one pyruvate group were detected by LC–MS (Figure S4
in the Supporting Information).
Keywords: aldol reaction · enzyme models ·
.
homogeneous catalysis · peptides · preorganization
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