Positional screening and NMR structure determination of side-chain-to-side-chain cyclized β3-peptides

Article abstract of DOI:10.1039/c1ob06422c

Many β-peptides fold in a 14-helical secondary structure in organic solvents, but similar 14-helix formation in water requires additional stabilizing elements. Especially the 14-helix stabilization of short β-peptides in aqueous solution is critical, due to the limited freedom for incorporating stabilizing elements. Here we show how a single lactam bridge, connecting two β-amino acid side-chains, can lead to high 14-helix character in short β³-peptides in water. A comparative study, using CD and NMR spectroscopy and structure calculations, revealed the strong 14-helix inducing power of a side-chain-to-side-chain cyclization and its optimal position on the β³-peptide scaffold with respect to pH and ionic strength effects. The lactam bridge is ideally incorporated in the N-terminal region of the β³-peptide, where it limits the conformational flexibility of the peptide backbone. The lactam bridge induces a 14-helical conformation in methanol and water to a similar extent. Based on the presented first high resolution NMR 3D structure of a lactam bridged β³-peptide, the fold shows a large degree of high order, both in the backbone and in the side-chains, leading to a highly compact and stable folded structure.

Full text of DOI:10.1039/c1ob06422c

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PAPER
Positional screening and NMR structure determination of
side-chain-to-side-chain cyclized b3-peptides†
Esther Vaz,a Sonja A. Dames,b Matthias Geyer*a and Luc Brunsveld*a,c
Received 19th August 2011, Accepted 7th November 2011
DOI: 10.1039/c1ob06422c
Many b-peptides fold in a 14-helical secondary structure in organic solvents, but similar 14-helix
formation in water requires additional stabilizing elements. Especially the 14-helix stabilization of short
b-peptides in aqueous solution is critical, due to the limited freedom for incorporating stabilizing
elements. Here we show how a single lactam bridge, connecting two b-amino acid side-chains, can lead
3
to high 14-helix character in short b -peptides in water. A comparative study, using CD and NMR
spectroscopy and structure calculations, revealed the strong 14-helix inducing power of a side-chain-
3
to-side-chain cyclization and its optimal position on the b -peptide scaffold with respect to pH and ionic
3
strength effects. The lactam bridge is ideally incorporated in the N-terminal region of the b -peptide,
where it limits the conformational flexibility of the peptide backbone. The lactam bridge induces a
14-helical conformation in methanol and water to a similar extent. Based on the presented first high
3
resolution NMR 3D structure of a lactam bridged b -peptide, the fold shows a large degree of high
order, both in the backbone and in the side-chains, leading to a highly compact and stable folded
structure.
structural point of view and as well-defined scaffolds for the
Introduction
design of protein interaction modules5 and possible antibacterial
or antifungal agents.6
The design and synthesis of oligomeric structures that can fold
and adopt predictable and stable conformations in solution, called
foldamers, has received significant attention.1 The controlled
folding of synthetic molecules in aqueous solutions is an especially
important goal, since such predictable and stable secondary
structures are attractive for the generation of biologically active
molecules.2 b-Peptides, which are composed of homologated b-
amino acids, provide a good scaffold for the design of foldamers,
since a wide-variety of monomeric building blocks is accessible
and the resulting oligomers can form a plethora of secondary
structures.3 One of the most studied b-peptide secondary struc-
tures is the 14-helix.4 This oligoamide helix is defined by the
formation of i, i–2 C O ◊ ◊ ◊ H–N backbone hydrogen bonds, in
a 14-atom ring. As a result every third residue has the same
position along the outside of the helix. The folding of b-peptides
into 14-helices has attracted significant attention, both from a
For a number of biological applications, the stabilization of
short b-peptides with stable helical folds in aqueous buffers is of
particular interest. Several design strategies have been developed
to address the stabilization of 14-helix folding in water. The
vast majority of these strategies make use of the fact that the
amino acid side-chains in the i and i+3 positions are located
in close proximity on the outside of the helix and therefore
can be utilized for physical or chemical stabilization of the 14-
helical fold. The first reports of 14-helix stabilization in water
were shown with b-peptides incorporating six-membered ring
constrained residues, such as trans-2-aminocyclohexanecarboxylic
acid.7 These residues feature a preferred conformation, highly
beneficial for 14-helix formation. A number of groups have shown
3
that 14-helix formation in b-peptides exclusively built up of b -
amino acids could be achieved by oppositely charged side-chains
at the i and i+3 positions.8 It has further been reported that
additional stabilization of the helical fold can be achieved by
favourable interactions of the charged side-chains with the helix
macrodipole, i.e. basic residues near the N-terminus and acidic
residues near the C-terminus. Subsequent studies have shown that
residues with side-chain branching adjacent to the backbone, such
aMax Planck Institute of Molecular Physiology, Otto-Hahn Strasse
11, 44227, Dortmund, Germany. E-mail: matthias.geyer@mpi-dortmund.
mpg.de
bTU Mu¨nchen, Lichtenbergstrasse 4, 85747, Garching, Germany
cLaboratory of Chemical Biology, Department of Biomedical Engineering,
TU Eindhoven, Den Dolech 2, 5612AZ, Eindhoven, The Netherlands.
E-mail: l.brunsveld@tue.nl; Fax: (+31)40-2478367
3
3
as b -homovaline (b -hVal) promote 14-helicity and can replace
some of the charged side-chains, reducing the number of required
electrostatic side-chain interactions from two to only one face
of the helix.9 In analogy with a-helix stabilization strategies for
† Electronic supplementary information (ESI) available: experimental
details, additional CD data and 1D/2D NMR spectra. See DOI:
10.1039/c1ob06422c
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a-peptides,10 covalent linking of side-chains at the i and i+3
positions in b -peptides was also shown to be highly effective
Design and synthesis
3
3
A small family of 20 heptameric b -peptides (Scheme 1, 1–10)
has been previously designed to study if rigidification of the
backbone structure by electrostatic interactions or by side-chain-
to-side-chain lactam bridges promotes folding into 14-helical
conformations.12 In order to assess the helix stabilizing efficacy of
both strategies, the rigidification elements were varied in length,
for 14-helix stabilization. Disulfide bridging of two side-chains
represented the first example in this respect, leading to 14-
helix stabilization in methanol.11 Strong stabilization of 14-helical
structures in water was first achieved by covalent linkage of side-
chain residues by amide bonds.12 More recently, stable 14-helices
in aqueous solution were prepared using ring-closing metathesis
to connect the side-chains of non-natural homologated amino
acids.13,14 Based on this work, the first NMR solution-structure
analysis of a constrained peptide was presented.13
3
position, and orientation in the middle of the b -peptide sequences.
Circular dichroism (CD) and NMR spectroscopy measurements
showed that an appropriate covalent side-chain-to-side-chain
linkage provided better helical structure stabilization in water than
the corresponding salt-bridge interaction between side-chains. We
observed that the seven to eight atom-bridge length (between b -
Lys and b -Glu or b -Asp) and positioning of the covalent bridge
Each type of 14-helix stabilization strategy has its specific
advantages, for example with respect to synthetic access or helix
3
3
stability.12b 14-Helix stabilization of b -peptides by covalent side-
3
3
chain linkages appears to require only one bridge element for the
induction of helicity in a peptide of average length.12 Covalent
linkage of the side-chains lowers the pH and salt concentration
sensitivity of the helical fold. The more hydrophobic nature of the
14-helix, introduced by virtue of the covalent bridge, might further
favor cellular uptake.14b As a result, covalent side-chain bridging
appears to be a favorable approach, especially for the stabilization
3
nearer to the N-terminus of the b -peptide greatly favored folding
into a 14-helix (7b and 8b).12 These results led us to undertake and
report here a more detailed analysis of the optimal positioning
of the amide side-chain linkage with respect to 14-helix inducing
power. We therefore designed and prepared an enlarged library
3
of heptameric, amide side-chain cyclized b -peptides (Scheme 1,
1–16) with i to i+3 linkages at each of the possible positions
within the b -peptides (1,4; 2,5; 3,6; 4,7). For comparison, the
analogous salt-bridge stabilized b -peptides were also prepared.
The conformations of the resulting peptides were analyzed by
CD and NMR spectroscopy. Also we report here the first high
resolution NMR 3D structure of a lactam bridged b -peptide.
The b -peptide library (Scheme 1) was prepared by solid-
phase peptide synthesis on TentaGel R PHB resin, which has
high swelling capacity and low initial loading, suitable for on-
bead cyclizations. Allyloxycarbonyl carbamate (Alloc) and allyl
ester were chosen as orthogonal protecting groups for the amine
3
of short 14-helix motifs in b -peptides. Concomitantly, molecular
3
details on the exact placement and structural characteristics of
the side-chain stabilized short 14-helices are required to guide the
design of biologically active analogues. Here we describe the design
of a set of heptameric, linear and cyclized peptides composed of
3
3
3
b -amino acids and their structural characteristics concerning 14-
3
helix stability. Special focus is on the placement of the side-chain-
3
to-side-chain lactam bridge within the b -peptide scaffold. The
3
generated library of salt and lactam bridged b -peptides (Scheme
1) was structurally characterized by CD and NMR spectroscopy.
This approach resulted in an optimized stable 14-helical platform
3
3
and carboxylic acid functional groups of the b -amino acids,
for short b -peptides.
respectively, which subsequently would form either the salt- or the
3
covalent amide bridge.12 b -Peptides 11–16 were newly synthesized
using microwave irradiation and HBTU, HOBt and DIEA as
coupling reagents. Piperidine in DMF was used for Fmoc cleavage.
After complete assembly of the heptamer, the allyl and Alloc side-
chain protecting groups were selectively removed with [Pd(Ph3)4]
3
in dichloromethane and phenylsilane. For some of the b -peptides
this led to a concomitant partial loss of the terminal Fmoc
group, as detected via LC-MS. In these cases, in order to avoid
isomer formation during the subsequent cyclization step, a Boc-
3
protected b -amino acid was alternatively used as N-terminal
building block, thus avoiding undesired liberation of the peptide
3
N-terminus. The non-commercial Boc-protected b -amino acids
were prepared via the Arndt–Eistert homologation method (see
Supporting Information†).
After assembly and deprotection of the heptameric sequences,
3
the b -peptide resin was divided into two batches for either
3
salt-bridged stabilized b -peptide (via direct cleavage from the
3
resin) or covalently rigidified b -peptide (via on-bead cyclization
and subsequent cleavage from resin) formation. The on-bead
3
cyclization for b -peptides 1b–10b was previously reported by us to
proceed smoothly using HATU and HOBt as cyclization reagents
in NMP.12 This method was also effective for the cyclization of
peptides 15b and 16b. However, those conditions were found to
be less effective for cyclization of peptides 11b–14b, bearing the
lactam bridge as a 1,4-linkage at the complete peptide N-terminus.
3
Scheme 1 Library of heptameric b -peptides (1–16) containing either
electrostatic side-chain interactions (a series) or a covalent amide bridge
(b series) positioned at different linkage locations in the b -peptide from
3
the N- to the C-terminus.
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The cyclization in this case might be hindered due to a higher
degree of conformational freedom at the complete end of the
peptide sequence. Other coupling reagents such as PyBOP and
BTFFH or the use of collidine as base instead of DIEA were all
tested, but did not improve the cyclization efficiency. In order to
influence the conformation of the b-peptide during cyclization,
several solvents were screened (NMP, DMF, 1 : 1 THF/NMP, 1 : 1
DCM/NMP, CH3CN). The usage of HATU with DIEA in 1 : 1
THF/NMP finally proved to be the best condition for on-bead
cyclization at the N-terminus of the peptide sequence. Standard
cleavage from resin using 96% TFA, water and TIPS required 3 h
to reach a complete removal of the tBu and Boc protecting groups.
After preparative RP-HPLC purification the linear (30% average
yield) and cyclic peptides (10% average yield) were obtained in
moderate to good yields and high purity, as determined by LC-
MS (see Supporting Information†).
(Fig. 1A), all linear and cyclic peptides showed CD signatures
characteristic for 14-helix formation withMRE values at 215nm of
about -10 000 deg cm2 mol-1 (see also Supporting Information†).
3
This suggests that all b -peptides display significant and similar
extents of 14-helicity in this helix-stabilizing solvent. For most of
3
the b -peptides in methanol, the 14-helix inducing power of the
lactam bridge is slightly lower compared to that of the salt-bridge.
In organic solvent, both the bridge type and position thus have
3
only minor effects on the 14-helicity in the respective b -peptides.
In aqueous solution (pH 7.4) more significant and striking
differences were observed between the linear and cyclic peptides
as well as within the specific sub-libraries. In water the position
and type of bridge element significantly influenced the 14-helical
3
character of the b -peptides (Fig. 1B, Fig. 2).
Structure analysis by CD spectroscopy
3
Circular dichroism (CD) measurements were performed on all b -
peptides (1–16, a and b series) in both methanol and buffered water.
3
The 14-helix of b -peptides typically shows a very characteristic
CD signature composed of a maximum in the ellipticity around
195–198 nm and a minimum at 213–215 nm. From this conserved
3
shape the 14-helical content of b -peptides with analogous se-
quences can be evaluated according to the mean residue ellipticity
(MRE) at 215 nm.2,3 Fig. 1 shows representative CD signatures of
3
cyclic b -peptides from our library (~150 mM) in methanol and in
aqueous 10 mM sodium-phosphate buffer, pH 7.4. In methanol
Fig. 2 (A) Effect of the type (blue = salt-bridge; red = lactam-bridge)
and position of the bridge on the helical structure observed by CD
measurements at 215 nm in water (aqueous 10 mM sodium-phosphate
buffer at pH 7.4); and (B) visualization of the specific N-terminal
preference for the lactam bridge in the cyclic peptides and C-terminal
preference of the salt bridge element in linear peptides. All measurements
were performed at 20 ◦C and ~150 mM b -peptide concentration.
3
First, the orientation of the bridge has a radical effect on
3
14-helix stability. b -Peptides 5, 6, and 10 showed a complete
3
3
absence of 14-helical structure. In these b -peptides, the b -hLys
of the bridge motif is positioned towards the C-terminus and
3
3
the b -hAsp, or b -hGlu component towards the N-terminus.
For example, for Lin(3,6)-AspLys (6a) and Cy(3,6)-AspLys (6b),
respectively, the MRE values fall to -500 and -100 deg cm2 dmol-1.
Thus, the orientation of the bridging partners along the sequence
has a large impact on the stability of both, the salt- and the
3
Fig. 1 CD signatures of cyclic b -peptides 6b, 8b12 and 12b (~150 mM) in
methanol (A) and in aqueous 10 mM sodium-phosphate buffer at pH 7.4
(B). All measurements were performed at 20 ◦C.
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lactam-bridged peptides. The interactions of side-chains with the
helical macrodipole play an important role in the stabilization of
14-helices.9 The H-bonding pattern within the 14-helix generates a
macrodipole with a partial positive charge at the C-terminus and
concomitant negative charge at the N-terminus. For the salt-bridge
peptides 1–3) MRE values are comparable between the cyclic
and linear variants, although the lactam bridge variant of 3 (3b)
features a notably higher 14-helical character than its salt-bridge
counterpart. However in the (4,7) series, the salt-bridge stabilized
3
3
b -peptides outcompete the lactam-bridge stabilized b -peptides.
These results demonstrate that ionic side-chain interactions result
in increasing stabilities of the secondary structure when the bridge
3
stabilized b -peptides, the localization of a positively charged
3
residue close to the C-terminus, as in b -peptides 5a, 6a, and 10a,
3
thus interferes with the 14-helix macrodipole. Similarly and in-
line with results observed for a-peptides,15 the direction of the
dipole of the lactam bridge is also influencing 14-helix stability.
is located close to the C-terminus of the b -peptide. Clearly, these
observations are supposed to result from a number of molecular
3
interactions operating simultaneously within the b -peptide. For
3
3
For b -peptides 5b, 6b, and 10b an unfavorable dipole interaction
the salt-bridge series, positioning of the negatively charged b -
3
3
is expected, which analogously to their linear counterparts, acts
destabilizing on the 14-helix structure.
hGlu or b -hAsp close to the b -peptide C-terminus is probably
highly effective in stabilizing the macrodipole of the 14-helix. It has
been reported that when positioned more centrally on the linear
Second, the length of the side-chains in the bridging elements
has a significant influence on 14-helix formation in the cyclic
3
b -peptide, a basic side-chain is moderately stabilizing, whereas
3
lactam bridged b -peptides. Indicated by the very low MRE values
an acidic side-chain is slightly destabilizing.9c In line with this
3
3
for b -peptides 4 and 14, the shortest lactam bridge element
observation, the b -peptides with salt bridges between positions
3
3
between a b -hOrn and a b -hAsp turned out to be detrimental
for 14-helix formation. Apparently, a short lactam bridge does
not match the molecular requirements for H-bond formation in
the 14-helix. Most probably, the short linker induces significant
constraints in the folded peptide, counteracting the 14-helix
1,4 and 2,5 (11a–14a and 7a–10a) are less stable than the 4,7
3
analogues (15a–16a), which feature a b -hLys in the middle of the
3
sequence and b -hGlu at its C-terminus. For the lactam bridged
3
series, the intrinsic flexibility of the b -peptide is expected to be
much more important for the helix stability than charge effects. In
3
3
folding motif. In contrast, those cyclic b -peptides with longer
this respect it has been reported that in b -peptides, the N-terminus
3
3
lactam bridges, featuring either at least a b -hLys or a b -hGlu,
all showed high 14-helicity, in many cases surpassing the 14-
helicity induced by the equivalent salt bridge. When the lactam
bridge is provided with enough molecular flexibility, by virtue of
longer side-chains, the bridging element nicely complements the
14-helix folding motif (vide infra NMR results) leading to large
MRE values. Small variations in the MRE values between the
different longer linker types probably resulted from effects such
as differences in the polarity of the newly introduced amide bond
typically features a lower population of H-bonding than the C-
terminus.9c Stabilizing effects imposed by side-chain cyclization are
thus expected to be more pronounced when positioned near the N-
3
terminus. This is especially eminent in short b -peptides, as studied
here, since these will intrinsically be less structured than previously
3
studied longer b -peptides.8,9 Consistent with these observations,
3
optimal helix stabilization of the short b -peptides analyzed here
is achieved by using a covalent lactam bridge near the N-terminus.
3
and its interaction with the b -peptide surface. For the salt bridge
3
3
3
pH and salt stability
stabilized b -peptides the side-chain lengths (b -hLys vs. b -hOrn
3
3
and b -hGlu vs. b -hAsp) had overall only a minor effect on the
stability of the 14-helix, reflecting a higher side-chain flexibility.
Third, the relative positioning of the bridging element along
The effect of changes in the environmental conditions on 14-
helicity for either type of bridging element was evaluated from
CD spectroscopy measurements at different pH values and salt
3
the b -peptide scaffold influences its 14-helix stability. The results
3
concentrations. For the selected b -peptides 7, 9, 12, and 16, pH
shown in Fig. 2 demonstrate that a favorable proximity of the
dependent measurements were performed in the regime between
pH 1.7 and 9.6 (Fig. 3). Analysis of the characteristic intensities at
215 nm showed appreciable pH sensitivity of both the linear and
3
bridge to a specific b -peptide terminus exists, albeit with different
3
preferences for the two bridge types. Cyclic b -peptides featuring
an appropriate lactam bridge close to the N-terminus, i.e. between
positions 1,4 (11b–13b) and 2,5 (7b–9b), typically exhibit strong
MRE values at 215 nm, indicating significant 14-helix character.
3
3
cyclic b -peptides. All b -peptides showed a significant decrease of
3
Compared to the cyclic lactam series at positions 3,6 and 4,7 (b -
peptides 1b–6b and 15b–16b) and compared to their open chain
3
salt bridge analogues (7a–9a and 11a–13a), b -Peptides 7b–9b and
11b–13b showed the highest 14-helical content in water with MRE
3
values at 215 nm in the range of -10 000 deg cm2 dmol-1. Indeed, b -
peptides 7b and 8b even feature MREs of -11 000 deg cm2 dmol-1,
3
constituting the highest 14-helical content of all b -peptides in
water characterized in this study. Furthermore the MRE values
3
of the lactam-bridged b -peptides in water have not diminished
with respect to the MRE value in methanol. This illustrates the
high stability of the lactam-bridged 14-helix structures upon going
from a helix stabilizing solvent, such as methanol, to water.
3
In contrast to the lactam-bridged b -peptides, the salt bridged
3
versions showed an opposite behavior regarding the sequential
Fig. 3 MRE values at 215 nm of b -peptides 7, 9, 12, and 16 for the
pH-range from 1.7 to 9.6.
3
positioning of the bridging element. In the (3,6) series (b -
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3
3
14-helicity when increasing the pH from 7.4 to 9.6. Lowering the
pH to 3.6 or even 1.8 instead had a smaller effect on the 14-helix
population. The destabilizing effect of high and low pH on 14-helix
stability probably relates to changes in the protonation states of
b -peptides by NMR spectroscopy. Initially, two sets of b -
peptides, 6a,b and 8a,b, were selected because of their opposite
structural characteristics in aqueous solution as indicated by the
3
CD data. Based on the MRE values at 215 nm, b -peptides 6a,b
showed a complete absence of 14-helix structure, whereas b -
3
3
the amine and carboxylate functionalities within the b -peptides.
Especially the destabilizing interaction of the different protonated
peptides 8a,b, especially 8b, showed a high degree of 14-helicity.
An initial indication whether the b -heptapeptides under study
3
states of the terminal amine and carboxylic groups with the 14-
3
helix macrodipole, common to both the linear and cyclic b -
form an ordered secondary structure can be obtained from the
1
peptides, is playing a major role for the effect. Nevertheless,
almost all lactam-stabilized peptides were more stable than the
corresponding salt-bridge stabilized peptides over the whole pH
degree of the dispersion of NMR signals. H-NMR experiments
3
of b -peptides ◦6a, 6b, 8a and 8b were obtained in buffered water
solution at 10 C (Fig. 5). The cyclic peptides 6b and 8b showed
3
regime. The only exception in this respect is b -peptide 16b for
overall a greater dispersion of amide proton signals than their
3
which the bridging element is positioned between residues 4 and 7.
linear counterparts 6a and 8a, respectively. For b -peptides 6a and
3
The folding of the b -peptides of this study is stabilized in part
6b, some of the amide proton signals (i.e. at 8.10 and 8.19 ppm)
are exchange broadened. This observation could be explained by
conformational averaging between different secondary structures
or between a folded and unfolded state. These results provide
another hint toward the instability of potential 14-helical structure
and are in line with the absence of 14-helix signature in the CD
by electrostatic interactions. The importance of electrostatic inter-
actions on 14-helix stability can be screened by increasing the salt
3
concentrations (e.g. NaCl). If the fold of a particular b -peptide is
significantly stabilized by electrostatic interactions, it is expected to
be destabilized in the presence of high salt concentrations.5,12 Fig. 4
shows the influence of increasing the NaCl concentration from 0 to
1.6 M on the MRE at 215 nm for a selected set of salt- and lactam-
3
3
spectra of the respective b -peptides in water. In contrast, for b -
peptides 8a and 8b, all amide protons, including the amide proton
of the lactam bridge in 8b, can be detected as about equally intense
3
bridged b -peptides that all have stabilizing element located near
3
3
the N-terminus. For all studied b -peptides the MRE decreased
signals. This indicates a much more stable fold for these b -peptides
with increasing concentration of the electrolyte. As expected, the
salt effect is significantly more pronounced for the b -peptides
and most likely a 14-helix in agreement with the observed CD data.
3
Consistent with the larger absolute value of the MRE at 215 nm for
3
with an intrahelical salt bridge as 14-helix stabilizing element.
the cyclic b -peptide 8b, which indicates higher 14-helix stability,
3
Accordingly, b -peptides 8a and 12a, showed a rapid decrease of
it also shows a greater dispersion of the NMR signals compared
the helical content upon increasing the electrolyte concentration.
In the presence of 1.6 M sodium chloride the negative MRE values
at 215 nm have diminished by 70%, indicating that reduction of
the ionic interaction by high salt greatly destabilizes the 14-helical
to the linear peptide 8a.
3
fold. b -Peptides 8b and 12b, bearing a side-chain-to-side-chain
covalent bond, showed only a decrease of 39% at the highest
electrolyte concentration, suggesting that the 14-helical structure
is still significantly populated. The observed destabilization of the
3
lactam-bridged b -peptides at high salt results probably from a
charge-macrodipole interaction (see above, pH screening).
3
Fig. 5 NH region of b -peptides 6a, 6b, 8a and 8b in H2O/D2O 9 : 1 at
pH 7.4 and 10 ◦C.
The H/D exchange rates of amide protons with solvent protons
are sensitive to the secondary structure content of the peptide in
solution. A low exchange rate indicates that protons are shielded
from interaction with the bulk water by an ordered structure, e.g.
by a hydrogen bonded helix. In contrast, a fast H/D exchange
indicates the amide to be readily accessible to the solvent, either
because of an unfolded nature or because of rapid interchange
between different conformations. Because CD measurements for
3
Fig. 4 MRE values at 215 nm for b -peptides 8 and 12 in aqueous buffer
at NaCl concentrations from 0 to 1.6 M.
3
b -peptide 8b indicated a high degree of 14-helix secondary
structure in methanol and water, its H/D exchange rates were
determined.16 A time course series of H-NMR spectra of b -
peptide 8b dissolved in CD3OD at 2 ◦C was obtained (Fig. 6).
Initial rapid recordings of the 1H-NMR spectra showed all seven
amide proton signals of 8b in the regime between 7.6 and 8.8 ppm.
1
3
Structure analysis by NMR spectroscopy
3
The high 14-helix stability of some of the lactam-bridged b -
peptides argued for a more detailed structural analysis of selected
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3
Fig. 6 H/D exchange series of b -peptides Cy(2,5)-LysAsp (8b) in 100%
CD3OD at 2 ◦C. The peak at 8.52 ppm (labeled *) corresponds to formic
acid.
In time, the amide protons exchange for deuterium, leading to
a decrease in amide proton signal intensity. The observed H/D
exchange half-times range from about 15 min to over 20 h. A
clear difference in H/D exchange rates is observed for the different
3
Fig. 7 Sequential NOE path of the b -heptapeptide 8b extracted from
ROESY experiments in MeOH. Each quadrant highlights specific 14-helix
characteristic, intraresidue and medium range correlations.
3
positions within the b -peptide. Whereas, the flanking amide bonds
and also the lactam bridge exchange more rapidly, the residues
3
3
embedded between the macrocyclic lactam system, b -hLys2, b -
3
hOrn3, and b -hVal4, showed very long H/D exchange half-times
terminus, [Ha(i)–Hb(i+3)]. Fig. 7 shows selected regions from the
ROESY spectrum of b -peptide 8b in methanol. In methanol as
well as in water all proton signals could be observed and assigned.
Furthermore, in both solvents the two protons attached to the
Ca carbons could be assigned stereo-specifically for the axial and
equatorial position, supporting formation of secondary structure
and a high degree of order in both solvents.
3
indicative of a very stable conformation in this region.
3
To further assess the structure of the b -peptides in solution
and to analyze their helical character, homonuclear 2D NMR
3
spectra were recorded for a number of b -peptides (8a, 8b, 12b),
both in methanol and in aqueous solvent. Total correlation
spectroscopy (TOCSY) experiments were acquired to identify
3
3
the characteristic residue spin systems of the b amino acids
The conformational stability of b -heptapeptide 12b in
and to assign the proton resonances (Table 1 and Supporting
Information†). The obtained assignments were confirmed based
on intraresidue and sequential NOE-connectivities from ROESY
experiments recorded with mixing times of 150 and 300 ms (Fig.
7 and Supporting Information†). The ROESY spectra showed
further medium-range NOEs characteristic for 14-helix structure,
e.g. between amide protons and protons at b-positions two or
three residues apart ([HN(i)–Hb(i+2)] and [HN(i)–Hb(i+3)]), or
between Ca-protons and Cb-protons three residues toward the C-
methanol was further assessed from temperature shift experiments.
Going from 271 K to 283 K relative amide proton shift were as
listed (in ppm): Lys1 Hh -0.07; Val2 -0.03; Orn3 -0.05; Asp4
-0.03; Val5 0.00; Ser6 -0.08; Tyr7 -0.09 (see also Supporting
Information†). The observation of negative temperature coeffi-
cients for the analyzed range of 12 K is in full agreement with those
observed for the amide resonances of a-peptides and proteins.
Assuming a linear dependence of HN shift versus temperature
change, as has been observed previously,17 the values of these
3
Table 1 1H NMR chemical shifts of the cyclic b -heptapeptide 8b in aqueous solution or MeOH at 283 K
Solvent
HN
Ha
Ha
Hb
Hg
Others
ax
eq
3
b -Val1
H2O
MeOH
H2O
MeOH
H2O
MeOH
H2O
MeOH
H2O
MeOH
H2O
MeOH
H2O
MeOH
—
—
2.58
2.80
2.50
2.76
2.15
2.66
2.20
2.57
2.31
2.59
2.22
2.27
2.13
2.12
2.65
2.60
2.29
2.34
2.29
2.39
2.37
2.44
2.45
2.78
2.27
2.50
2.32
2.41
3.32
3.42
4.09
4.48
4.05
4.28
3.81
4.12
4.42
4.59
4.02
4.32
4.23
4.48
1.84
2.00
1.49, 1.51
1.53, 1.66
1.31, 1.43
1.61, 1.63
1.58
dCH3: 0.87
dCH3: 1.05
3
b -Lys2
8.02
8.17
7.73
8.35
7.69
8.18
8.01
8.62
7.97
7.85
7.76
7.64
dCH2: 1.18, 1.22; eCH2: 1.37 zCH2: 3.02, 3.07; hNH: 7.94
dCH2: 1.38, 1.45; eCH2: 1.52 zCH2: 3.09, 3.38; hNH: 7.99
dCH2: 1.48; eCH2: 2.83
dCH2: 1.49; eCH2: 2.84, 2.90
dCH3: 0.77
dCH3: 0.93
3
b -Orn3
3
b -Val4
1.72
3
b -Asp5
2.13, 2.22
2.30, 2.30
3.29
3.35, 3.45
2.40, 2.67
2.60
3
b -Ser6
3
b -Tyr7
eH: 6.95; zH: 6.64
eH: 6.95; zH: 6.63
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gradients correspond to: Lys1 Hh -5.8 ppb K-1; Val2 -2.5 ppb
K-1; Orn3 -4.2 ppb K-1; Asp4 -2.5 ppb K-1; Val5 0.00 ppb
K-1; Ser6 -6.7 ppb K-1; Tyr7 -7.5 ppb K-1. It is commonly
assumed that coefficients more positive than -6 ppb K-1 indicate
intramolecular H-bonded HN protons in aqueous solution. The N-
terminal five amino acids of the (1,4) lactam-bridged peptide 12b
showed all coefficients higher than -6 ppb K-1, thereby suggesting
stabilization of the b-peptide backbone by the cyclization.
All structure calculations were performed with XPLOR-NIH18
using torsion angle and Cartesian coordinate space molecular
dynamics. The parameter and topology files “parallhdg.pro” and
“topallhdg.pro” were modified to incorporate structural parame-
ters (bonds, angles, dihedral angles, improper angles, charge etc.)
3
for the b-amino acids appearing in the sequence of the cyclic b -
heptapeptide 8b and included in the Supporting Information.†
3
3
Cyclization between the side-chains of b -hLys2 and b -hAsp5
was enabled by defining a new patch, which is similar to the one
used for the definition of disulfide bonds. In addition, the distance
Structure calculation of a cyclic b3-peptide
3
between the side-chain amide nitrogen (Nh of b -hLys2) and the
d
3
˚
side-chain C of b -hAsp5 was restrained to 1.5–5.3 A. For one set
of calculations, the ROEs were calibrated exactly according to the
ROE cross peak intensity, which is referred to a ‘calibrated’. For
a second set of calculations the cross peak intensities were used to
simply group the ROEs into 4 bins with upper bounds set to 2.3
Based on a comparison of the CD data (Fig. 1) and the
ROESY peak pattern (Supporting Information†), the 14-helix
3
fold of the (2,5) lactam bridged b -peptide 8b is highly similar
in both solvents. The presence of certain 14-helix typical NOE-
correlations for the same peptide in water, were already reported
earlier.12b Based on the ROESY data recorded in methanol, it
was possible to calculate a high-resolution NMR 3D structure of
˚
˚
˚
˚
A, 2.8 A, 3.6 A, and 5.8 A, respectively. This is referred to as ‘bin’.
The lower bound corresponded approximately to the sum of the
van der Waals radii. For all distance restraints r-6 sum averaging
was used. In addition, stereo-specific assignments were obtained
for all a-methylene proton pairs.
3
the (2,5) lactam bridged b -peptide 8b (Fig. 8). The ROESY data
in methanol was used, because the NMR signals were overall
better resolved (Supporting Information†). Presumably due to
higher conformational flexibility and/or increased exchange of the
backbone amide with solvent protons, the ROESY data for the
peptide in water showed overall weaker and thereby also about
20% fewer cross peaks. Moreover, NOE-restraints involving the
A total of 121 distance restraints were derived from unambigu-
ous ROE assignments (Table 2), which corresponds to about 17
restraints per residue. Hydrogen bonds restraints were defined
based on hydrogen-deuterium exchange experiments and the
observed ROE correlations. The HN-O distance bounds for residue
3
backbone Cb-proton around 4–5 ppm of the b -amino acid could
˚
pairs 2–4, 3–5, 4–6 and 5–7 were 1.8–2.3 A. We also performed a
be more reliably derived since the respective cross peaks were
not disturbed by a strong water signal at about 4.7 ppm. The
influence of water compared to methanol on the 14-helix fold was
estimated from a refinement of the structures calculated based on
the methanol restraints in water. This appeared valid due to the
above mentioned similarity of the fold in both solvents.
run without hydrogen bonds restraints as a reference, leading to
the same overall fold (see Supporting Information†). The 20 lowest
Table 2 Statistics of the 20 lowest energy structures of 8b, calculated
based on data recorded in methanol
Type of distance restraint
Distance restraints
Total
Intraresidue
Sequential
Medium range
Hydrogen bond restraints
Calibrated
Bin definition
125
86
13
22
4
125
86
13
22
4
For the structure calculation in vacuoa
Rmsd’s from experimental restraints
Distance (A)
˚
0.0477 0.0021 0.0323 0.0031
Rmsd’s from idealized geometry
Bonds
Angles
0.0044 0.0002 0.0034 0.0004
0.5383 0.0147 0.480 0.019
Improper
0.257 0.022
-13.5 4.1
0.210 0.018
-12.5 2.9
Lennard-Jones energy (kcal mol-1)
˚
Average rmsd to mean structure (A)
Residues 1–7 (bb/heavy)
0.42/0.84
0.61/1.20
Following the refinement in a water shella
Rmsd’s from experimental restraints
Distance (A)
Rmsd’s from idealized geometry
Bonds
Fig. 8 Superimposition of the 20 lowest energy structures of the
(2,5) lactam bridged b -heptapeptide 8b calculated based on the data
˚
3
0.0349 0.0020 0.0270 0.0020
recorded in methanol. The structures calculated in vacuo (A) represent
the conformation in methanol. Subsequent refinement in a water-shell was
done to obtain a structural model for the conformation in aqueous solvent
(B). This approach appeared valid due to the high similarity of the folds in
both solvents that is indicated by the presented CD and NMR data. Each
bundle is shown in three different perspectives (bottom view, front view,
top view). The grey tube in each bundle represents the backbone of the
14-helix. The side-chains on the three different regions on the 14-helix are
given in different color-coding with the lactam bridge shown in green.
0.0094 0.0007 0.0097 0.0006
1.309 0.0605 1.097 0.063
Angles
Improper
0.450 0.067
-98.8 10.2
0.421 0.065
-91.5 14.8
Total energy (kcal mol-1)
Average rmsd to mean structure (A)
˚
Residues 1–7 (bb/heavy)
0.34/0.74
0.49/1.04
˚
a
None of the structures had distance restraints violations > 0.5 A, rms(d) =
root mean square (deviation), bb = backbone (CA, CB, C, O, N).
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energy structures out of 200 calculated ones were additionally
refined in a water-shell (Fig. 8b).19 Structural statistics are given
in Table 2. As described above, refinement of the structure in a
water shell was performed to achieve a model of the structure that
allows to estimate the behaviour in aqueous solution (Fig. 8b).
folding of b-peptides in water remains a challenge. This is
especially true for short b-peptides, featuring only a limited
number of b-amino acids for fold stabilization. In this manuscript
we addressed this issue for the folding of b-peptide heptamers in
a 14-helical secondary structure in water. The incorporation of a
single lactam bridge, which connects two b-amino acid side-chains,
in the b-heptapeptide leads to high 14-helix character in water. By
executing a comparative study on a library of b-heptapeptides,
with varying lactam bridge positions, orientations and lengths,
3
The water-refined NMR structures of b -peptide 8b showed all
a well-defined compact 14-helix structure with only little fraying
of the backbone at the N- and C-termini. A full helix turn is
achieved every three residues and side-chains are superimposed in
three different segments around the helical axis. Only the side-
3
the optimal placement of the stabilizing motif on the b -peptide
3
3
chains of the N-terminal b -hVal and the C-terminal b -hTyr
show somewhat more structural heterogeneity, in line with the
specific position of the lactam bridge. The lactam bridge occupies
one of the three flanking sites and is thus fully consistent with
the hydrogen bonding pattern in the 14-helix. As seen from the
surface display, the peptide appears very compact due to the
cyclization and the small number of seven residues (Fig. 9). As
such, despite the 14-helix formation, a highly compact folded
structure is formed. The lactam bridge significantly limits the
scaffold could be deduced. CD and NMR spectroscopy, together
with structure calculations, revealed that the optimal position for
inducing 14-helicity is located at the N- terminus of the b-peptide,
and therewith markedly contrasts with the effects of a more
classical side-chain-to-side-chain salt-bridge. The lactam bridge
3
is ideally incorporated in the N-terminal region of the b -peptide,
because the conformational flexibility of the peptide backbone
is intrinsically the highest at this position. Stabilization of this
specific segment with a lactam bridge leads to the highest overall
14-helicity. The lactam bridge induces a 14-helical conformation
in methanol and water to a similar extent and with high order,
both in the backbone and in the side-chains, leading to a highly
compact and stable folded structure.
3
number of possible b -peptide conformations, which is reflected
in low backbone rmsd values (Table 2), thereby stabilizing the
14-helix backbone structure. This suggests that the respective
structure could serve as a scaffold for the positioning of side-chains
in a highly ordered 3D format. Such high order for the side-chains
has already previously been noted in a comparative study with b-
peptides incorporating six-membered ring constrained residues.12b
The presented first high-resolution NMR structure of the (2,5)
3
lactam bridged b -heptapeptide 8b is very compact. A lactam
bridge of appropriate length and position significantly limits the
3
number of b -peptide conformations, thereby stabilizing the 14-
helix backbone structure even in a very short peptide of only seven
3
residues. Even though the reported b -heptapeptides are model
3
peptides, the results imply that this small and stable 14-helical b -
peptide fold could serve as a stable scaffold for the positioning of
functionally important side-chains on a rigid backbone structure
3
for biologically active b -peptide sequences, e.g. for applications in
the area or protein recognition or membrane targeting. Moreover,
stabilization by covalent cyclization might be particularly suited
to minimize proteolytic digest in cells or blood and to enhance
membrane transport.
Acknowledgements
We thank Bernhard Griewel for expert technical assistance with
NMR measurements and Ingrid Vetter for helpful discussions
on template building. This work was supported by a Sofja Ko-
valevskaja Award from the Alexander von Humboldt Foundation
and the BMBF to L.B. and the Spanish Ministerio de Educacio´n
y Ciencia (postdoctoral fellowship E.V.).
Fig. 9 Structure of one lowest energy conformation of the cyclic 14-helix
8b peptide as calculated from NMR data recorded in MeOH. Shown
are two different perspectives as stick (top) and as surface representation
(bottom). Despite 14-helix formation, the molecule appears compact but
not elongated due to the (2,5) lactam bridge and the short size of seven
residues. Note that the NH ◊ ◊ ◊ OC hydrogen bond pairing in the backbone
Notes and references
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