Oligo(4-aminopiperidine-4-carboxylic acid): An unusual basic oligopeptide with an acid-induced helical conformation

Article abstract of DOI:10.1021/ja106118w

In sharp contrast with helical polypeptides carrying basic side chains, Api₈, a basic oligopeptide containing the non-natural achiral amino acid 4-aminopiperidine-4-carboxylic acid (Api), adopts a helical conformation only in acidic media. Alkaline titration of a protonated Api₈ oligomer appended with a leucine derivative at its N-terminus showed that disruption of its helical conformation occurs in a pH range of 7-10. NMR studies indicated that the piperidine groups in Api₈, when nonprotonated, possibly interact with the proximal amide protons in the peptide backbone and hamper the formation of the H-bonding network responsible for the helical conformation. The helical structure is induced not only by protonation but also by acylation of the piperidine groups.

Full text of DOI:10.1021/ja106118w

Published on Web 09/02/2010
Oligo(4-aminopiperidine-4-carboxylic acid): An Unusual Basic Oligopeptide
with an Acid-Induced Helical Conformation
Joon-il Cho,† Masahiro Tanaka,† Sota Sato,‡ Kazushi Kinbara,† and Takuzo Aida†,*
Department of Chemistry and Biotechnology and Department of Applied Chemistry, School of Engineering,
The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
Received July 10, 2010; E-mail: aida@macro.t.u-tokyo.ac.jp
Abstract: In sharp contrast with helical polypeptides carrying
basic side chains, Api8, a basic oligopeptide containing the non-
natural achiral amino acid 4-aminopiperidine-4-carboxylic acid
(Api), adopts a helical conformation only in acidic media. Alkaline
titration of a protonated Api8 oligomer appended with a leucine
derivative at its N-terminus showed that disruption of its helical
conformation occurs in a pH range of 7-10. NMR studies
indicated that the piperidine groups in Api8, when nonprotonated,
possibly interact with the proximal amide protons in the peptide
backbone and hamper the formation of the H-bonding network
Figure 1. Schematic representations of Api octapeptides and their transition
to a helical conformation induced by H+.
responsible for the helical conformation. The helical structure is
induced not only by protonation but also by acylation of the
piperidine groups.
Natural polypeptides containing basic amino acids such as lysine
and arginine play crucial roles in biological events.1 Conformational
studies of their synthetic analogues have indicated that protonation
of the basic side chains of these amino acid residues results in a
helix-to-coil transition as a result of disruption of the H-bonding
network along the peptide backbone by an electrostatic repulsion.2
Such a protonation-induced conformational change has inspired
synthetic chemists to design a variety of pH-responsive nonpeptidic
analogues appended with basic functional groups.3 However, with
only a few exceptions,3c-e all previously reported pH-responsive
helical motifs having basic functional groups, including foldamers,3a
adopt a helical conformation only in nonacidic media.3 Here we
report that oligo(4-aminopiperidine-4-carboxylic acid) (Apin) is the
first basic oligopeptide that adopts a stable helical conformation
only in acidic media.
Api is a non-natural amino acid bearing a piperidine group and
has no chiral center. By analogy to oligopeptides containing other
R,R-disubstituted achiral amino acids,4 Apin is presumed to adopt
a helical conformation as a result of not only intramolecular
H-bonding interactions but also steric repulsion among the R-sub-
stituents. However, homotropic Apin has never been reported; only
Api-containing copolypeptides5 are known. Thus, we first estab-
lished an iterative synthetic method for Apin starting from Api2.6
For a conformational study of Apin using circular dichroism (CD)
spectroscopy, Api8 carrying N-acetylleucine (L- or D-LeuAc) at its
N-terminus [LeuAc(Api8)OBn; Figure 1] was synthesized6 with an
expectation that Leu as a chiral auxiliary could allow Api8 to adopt
a prevailing one-handed helical conformation. However, to our
surprise, LeuAc(Api8)OBn did not show any CD sign typical of
helical structures. We later found that protonation of the piperidine
groups is quite essential for LeuAc(Api8)OBn to adopt a helical
conformation (Figure 1).
Figure 2. (a) CD spectra at 20 °C for LeuAc(Api8)OBn (0.03 mM) at pH
4 (solid curves) and pH 10 (broken curves). (b) Plots of ∆ε at 226 nm for
LeuAc(Api8)OBn (0.03 mM) vs pH at 20 °C. (c) CD spectra at 20-80 °C
for LeuAc(Api8)OBn (0.03 mM) at pH 4. (d) CD spectra at 20 °C for
LeuFmoc(BocApi8)OBn (0.15 mM) in MeCN (solid curves) and MeOH
(broken curves).
As shown in Figure 2a, an acidic aqueous solution (pH 4) of
L-LeuAc(Api8)OBn (0.03 mM) at 20 °C displayed negative-signed
Cotton effects at 210 (π-π*) and 226 nm (n-π*). As expected,
the CD spectrum of D-LeuAc(Api8)OBn under conditions identical
to the above was the mirror image of that observed for the L-form.
Since the ratio of the ∆ε values at 210 and 226 nm (∆ε226/∆ε210
)
was almost unity, the octameric Api moiety (Api8) in
LeuAc(Api8)OBn most likely adopts an R-helical conformation.7 The
observed CD spectral profile remained virtually unchanged upon a
decrease in the pH (e.g., to 2). In sharp contrast, under basic
conditions with pH g 10, LeuAc(Api8)OBn showed a CD spectral
feature (Figure 2a) with a shape similar to that of monomeric
LeuAcOBn,6 indicating that Api8 without protonation adopts a
† Department of Chemistry and Biotechnology.
‡ Department of Applied Chemistry.
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C O M M U N I C A T I O N S
Figure 4. 1H NMR spectra of tertBu(Api)(Aib)OMe (5 mM) in (a) CH3NO2
and (b) CD3NO2 at 20 °C.6
broad peaks (Figure 3a), both of which became very weak upon
selective irradiation of the water signal.6 Hence, we conclude that
Api8 under nonacidic conditions is devoid of H-bonds along the
peptide backbone.
By using tertBu(Api)(Aib)OMe (Aib ) 2-aminoisobutyric acid)
(Figure 4) as a model compound for the repeating Api units, we
found that the nonprotonated piperidine groups in Api8 possibly
hamper the H-bonding interactions of the amide units in the peptide
backbone. tertBu(Api)(Aib)OMe bears two amide NH groups that
are located in similar steric environments but have different
geometries with respect to the piperidine nitrogen atom. Interest-
ingly, when CD3NO2 (pKa ) 10.2)10 was used as the solvent, the
1H NMR spectrum of tertBu(Api)(Aib)OMe hardly showed the amide
NH signal due to Hb as a result of H-D exchange, while the signal
due to Ha remained intact (Figure 4).11 In contrast, when the
piperidine group of tertBu(Api)(Aib)OMe was protonated, both Ha
and Hb were detected.6 The same was true for tertBu(BocApi)(Ai-
b)OMe, a Boc-protected version of tertBu(Api)(Aib)OMe, in
CD3NO2.6 Since in either case the intrinsic acidities of Ha and Hb
are likely comparable to one another, the above results clearly
indicate that the piperidine nitrogen atom in tertBu(Api)(Aib)OMe,
when nonprotonated, interacts with amide N-Hb regioselectively
and activates it for proton exchange. The observed regioselectivity
is likely due to a restricted conformation of the piperidine group.
Such an interaction could also occur in Api8, causing its H-bonding
network that is responsible for the helical conformation to be
disrupted in nonacidic media.
Figure 3. 1H NMR spectra at 20 °C for Ac(Api8)NHMe (5 mM) in water
at (a) pH 9 and (b) pH 6. The sample tube included an inner sealed tube
containing a D2O solution of 4,4-dimethyl-4-silapentane-1-sulfonic acid
sodium salt (DSS, 15 mM) as an internal standard. Arrows indicate
H-bonded pairs.
nonhelical conformation. Alkaline titration of an acidic (pH 2)
aqueous solution of LeuAc(Api8)OBn resulted in an abrupt CD
intensity change at pH 7-10 (Figure 2b). Although the pH value
at the inflection point of this transition is less than the pKa value of
piperidine (11.1),8 we conclude that protonation of the piperidine
units allows LeuAc(Api8)OBn to adopt an R-helical conformation.
The helical structure is thermally stable, as the CD spectral profile
of LeuAc(Api8)OBn at pH 4 still maintained the characteristics of
the R-helix even upon heating to 80 °C and recovered the original
intensity (64 f 100%) completely upon cooling to 20 °C (Figure
2c). We also found the presence of a critical chain length for
protonated Apin to adopt a stable helical conformation. A shorter-
chain homologue such as LeuAc(Api4)OBn is likely a critical
oligomer, which at pH 2 displayed only a small CD spectral feature
of the helical conformation.6 The pH dependence of the confor-
mational stability of R-helical Api8 observed here is contrary to
those reported for ordinary polypeptides bearing basic functional
groups.2 In relation to this interesting contrast, we found that Api8
adopts a helical conformation when an electron-withdrawing group
such as tert-butoxycarbonyl (Boc) is attached to the piperidine
nitrogen atoms to decrease their basicity. For example,
LeuFmoc(BocApi8)OBn (Figure 1) in MeCN and MeOH at 20 °C
displayed a CD spectral feature typical of helical conformations
(Figure 2d).9
To elucidate a possible role of the piperidine groups in the
conformational characteristics of Api8, we measured 1H NMR
spectra of Ac(Api8)NHMe (Figure 1) in water at 20 °C (Figure 3).
Under acidic conditions (pH 6), the amide NH region (Figure 3b)
displayed nine well-resolved signals (H1-H9). In an 1H NMR
saturation-transfer experiment upon selective irradiation of the water
signal (4.6 ppm), amide NH signals H4-H9 decreased in intensity
by 40-50%, whereas signals H1-H3 displayed more significant
intensity decreases (70-90%).6 The observed intensity changes are
caused by the amide-water proton exchange. On the basis of the
exchange rates, as evaluated by the saturation-transfer method,6
the amide NHs in the former set (H4-H9) are likely involved in
the H-bonding network, but the remaining three (H1-H3) are free.
Together with 1H-1H correlations evaluated by 2D rotational
Overhauser effect spectroscopy (ROESY),6 all of the amide NH
signals in Figure 3b were reasonably assigned to the R-helical
conformation of protonated Ac(Api8)NHMe. In sharp contrast, under
basic conditions (pH 9), the amide NH region exhibited only two
In conclusion, we have demonstrated that oligomeric 4-aminopi-
peridine-4-carboxylic acid (Apin) is the first basic peptide that adopts
a helical conformation only in acidic media. When the piperidine
groups of Apin are nonprotonated, they possibly interact with the
proximal amide NH protons in the peptide backbone and hamper
the formation of the H-bonding network responsible for the helical
conformation. Utilization of Apin as a building block certainly
provides many new possibilities for designing pH-responsive
functional peptides and related chemistry.
Acknowledgment. We thank Professor Makoto Fujita for his
generous support in NMR measurements.
Supporting Information Available: Details of syntheses, charac-
terization data, and CD and NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
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