Helical Oligopeptides of a Quaternized Amino Acid with Tunable Chiral-Induction Ability and an Anomalous pH Response

Article abstract of DOI:10.1002/chem.201605460

A series of octamer (8-mer) and hexadecamer (16-mer) oligopeptides of 4-aminopiperidine-4-carboxylic acid (Api) with l-leucine as a chiral auxiliary at their N or C termini were synthesized. By using circular dichroism spectroscopy, the conformational profiles of the peptides were systematically studied, which revealed that the α-helix-formation ability of the peptides is determined by the combination of parameters, which includes peptide length, state of the piperidine groups in the Api units, and position of the chiral auxiliary. When the piperidines were in the free-base state, the peptides showed a low propensity to form helical structures. However, the protonation and acylation of the piperidines enhanced the formation of helical structures, such that the order for helix-formation ability was protonated>acylated>free base. In terms of peptide length, the 16-mers generally showed higher helix-formation ability than the corresponding 8-mers, and one of the 16-mers showed helicity at the highest level reported thus far for oligopeptides of a similar length. It was also found that the sensitivity of the helical structure towards the state of the piperidine groups changed drastically depending on the chiral auxiliary position; the N-terminal chiral peptides were more sensitive than the C-terminal chiral analogues.

Full text of DOI:10.1002/chem.201605460

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Accepted Article
Title: Helical Oligopeptides of a Quaternized Amino Acid with Tunable
Chiral-Induction Ability and an Anomalous pH Response
Authors: Joonil Cho, Yasuhiro Ishida, and Takuzo Aida
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Helical Oligopeptides of a Quaternized Amino Acid with Tunable
Chiral-Induction Ability and an Anomalous pH Response
Joonil Cho,[a],[b] Yasuhiro Ishida,*[b] and Takuzo Aida*[a]
Abstract: A series of octamer (8mer) and hexadecamer (16mer)
oligopeptides of 4-aminopiperidine-4-carboxylic acid (Api) with L-
leucine as a chiral auxiliary at their N- or C-termini were synthesized.
Using circular dichroism spectroscopy, the conformational profiles of
the peptides were systematically studied, which revealed that the α-
helix formation ability of the peptides is determined by the
combination of parameters including peptide length, state of the
piperidine groups in the Api units, and position of the chiral auxiliary.
When the piperidines are at the free-base state, the peptides show
low propensity to form helical structures. However, the protonation
and acylation of the piperidines enhance the formation of helical
structures, where the order in helix formation ability is: protonated >
acylated > free-base. In terms of peptide length, the 16mers
generally show higher helix-formation ability than the corresponding
8mers, and one of the 16mers shows helicity at a highest level for
the oligopeptides with similar length. It was also found that the
sensitivity of the helical structure toward the state of the piperidines
changes drastically, depending on the chiral auxiliary position; the N-
terminal chiral peptides are more sensitive than the C-terminal chiral
ones.
Introduction
The handedness of most helical biopolymers and synthetic
polymers is determined by the absolute configuration of their
constituent monomer units.[1,2] In sharp contrast to such static
helices, some synthetic polymers are known to adopt dynamic
helical conformations.
Pioneering examples[3] include
polyisocyanides, polymethacrylates, and polyisocyanates,
whereas more-recent examples[4] feature polyacetylene
derivatives. These dynamic helical polymers have attracted
particular attention as chiroptical sensors, because their helical
sense can switch in response to chiral stimuli or to the
environment of the polymer.[1b,5]
Figure 1. General tendency of conformational profiles of the Api peptides with
three different states of the secondary amine groups in the piperidine groups:
free-base (center), protonated (top), and acylated (bottom) states.
In the 1980s, Toniolo and co-workers reported that a
sufficiently long synthetic oligopeptide of 2-aminoisobutyric acid
(Aib) shows a dynamic helical inversion in solution.[6,7] Although
Aib is achiral, its oligomers adopt a helical conformation
because of steric repulsion between its side chains.[8] More
recently, it has been reported that, by incorporating chiral amino
acids, the helical structure of oligomeric Aib can be biased to
either
a
right- or left-handed conformation.[9–11]
Although
structural diversification has been achieved by replacing Aib with
other α,α-dialkylamino acids,[12–14] most of these monomers are
not amenable to side-chain functionalization, limiting their range
of potential applications.
[a]
[b]
Dr. J. Cho, Prof. Dr. T. Aida
Department of Chemistry and Biotechnology, School of Engineering,
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)
E-mail: aida@macro.t.u-tokyo.ac.jp
Dr. J. Cho, Dr. Y. Ishida
With these aspects in mind, we focused our attention on 4-
aminopiperidine-4-carboxylic acid (Api),[15] which bears
a
reactive secondary amino group (Figure 1, center). Although
some oligopeptides containing Api have been reported,[16] the
conformational properties of Api oligopeptides had not been
explored until our previous work on Api octamers (8mers).[17]
We showed that Api 8mers with desired terminal functionalities
can be synthesized by a solution-phase iterative method.
Emergent Bioinspired Soft Matter Research Team
Center for Emergent Matter Science, RIKEN
2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
E-mail: y-ishida@riken.jp
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spectroscopy, enantiopure Leu (LLeu was mainly used) was
incorporated at either of the N- or C-terminus of the Api peptides
to bias the equilibrium of the two enantiomeric conformations
toward one state. These systematic studies fully addressed the
open questions mentioned above and they clearly demonstrated
the potential utility of Api peptides as new motifs for the study of
polymers and peptides with dynamic helical structures.
Results and Discussion
Synthesis of Api Peptides
The
synthesized by a solution-phase method, as previously reported
(Scheme S1).[17]
The target 8mers with N-terminal chiral
auxiliary [Ac(LLeu)(Api)8OBn (= Leu-Api8), Ac(LLeu)(HApi)8OBn
Fmoc-terminated
peptide
Fmoc(BocApi)8OBn
was
L
(= Leu-HApi8), and Ac(LLeu)(BocApi)8OBn (= Leu-BocApi8)] and
with C-terminal chiral auxiliary [Ac(Api)8(LLeu)OBn (= Api8-LLeu),
Ac(HApi)8(LLeu)OBn (= HApi8-LLeu), and Ac(BocApi)8(LLeu)OBn
(= BocApi8-LLeu)] were prepared from Fmoc(BocApi)8OBn by the
introduction of a chiral auxiliary at the N- and C-termini,
conversion of the N-terminal end-capping group, and conversion
of the piperidine groups in Api units, as shown in Scheme S2.
L
L
Figure 2. Molecular structures of Api 8mers and 16mers carrying an LLeu
moiety at the N- or C-terminus with various states of the secondary amine
groups in the piperidine moieties: free-base (blue), protonated (red), and
acylated (green) (Ac = acetyl; Boc = tert-butoxycarbonyl; Bn = benzyl).
The target 16mers were synthesized by a similar route to the
8mers, but with some modifications because the corresponding
precursor Fmoc(BocApi)16OBn was not available. Condensation
of Fmoc(BocApi)8OH with H(BocApi)8OBn was sluggish and was
accompanied by the formation of unknown side-products due to
the labile nature of the Fmoc group (Scheme S1) under the
condensation conditions. In an alternative approach (Scheme
S3), we condensed Ac(LLeu)(BocApi)8OH with H(BocApi)8OBn to
give Ac(LLeu)(BocApi)16OBn (= LLeu-BocApi16), which was then
converted into Ac(LLeu)(HApi)16OBn (= LLeu-HApi16) and
Ac(LLeu)(Api)16OBn (= LLeu-Api16). In a similar manner, the
Furthermore, we demonstrated that Api 8mers substituted with
enantiopure leucine (L- or D-form; LLeu or DLeu) at their N-
termini display an interesting change in conformation in
response to changes in pH, in which protonation of the
piperidine groups triggers a nonhelical-to-helical conformational
transition (Figure 1, center to top). In sharp contrast to
conventional basic peptides,[18] the Api 8mer is the first basic
oligopeptide that adopts a helical conformation in acidic media
target
16mers
with
C-terminal
chiral
auxiliary
only.
As confirmed by 1H NMR spectroscopy,[17] the
[Ac(Api)16(LLeu)OBn (= Api16-LLeu), Ac(HApi)16(LLeu)OBn (=
HApi16-LLeu), and Ac(BocApi)16(LLeu)OBn (= BocApi16-LLeu)] were
prepared.
conformational locking induced by the formation of a hydrogen-
bonding network around the peptide backbone can be
manipulated through involvement of the basic piperidine groups
(Figure 1, center). In addition, it was preliminarily confirmed that
the acylation of the piperidine groups also induce Api peptides to
adopt an α-helical structure, because the acylation diminishes
the basicity of the secondary amine lope pairs, thereby releasing
the conformational locking (Figure 1, center to bottom).[19]
Circular Dichroism (CD) Spectroscopy of Api Peptides
Conformational features of the Api peptides were investigated by
CD spectroscopy. For comparing the CD profiles of the Api
peptides between the three states of the piperidine units (free-
base, protonated, and acylated), MeOH was chosen as a
solvent, because of its ability to dissolve all of these Api peptides
(Figures 3, 4, and S1–S3). For comparing the helicity of the
protonated Api peptides with other reported oligopeptides,
aqueous HCl (pH 4–5) was used, because previous examples
were usually measured in not MeOH but aqueous media (Table
1 and Figures S4 and S5). For the pH titration experiment of the
protonated/free-base Api peptides, aqueous media were used,
in order to control the pH values accurately (Figure 5). In all
cases, CD intensity was converted into mean-residue molar
ellipticity ([θ]MRE = [θ]/number of amino acid residues).
Despite these interesting observations,
a number of
questions about Api oligopeptides remain unanswered: (i) Which
structural elements ensure the helical conformation?, (ii) How
large is the helical content?, and (iii) How can the pH
responsiveness be modulated? Another open question is (iv)
Which of the protonation and acylation of the piperidine groups
induce the helical structures more efficiently?
To address these issues, we have synthesized a series of
Api octamers (8mers) and hexadecamers (16mers) with varying
the state of the piperidine groups (Figure 2) and have compared
their conformational profiles. To monitor the conformational
properties of the Api peptides by circular dichroism (CD)
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Table 1. Helicities of typical canonical oligopeptides with high α-helical
tendencies compared with those of the Api oligopeptides
[a]
Number of
residues
[θ]MRE_n-π*
Helicity[b]
(%)
Peptide
(deg cm2 dmol–1
)
DPAEA2KA3GR[c]
S2DVSTAQA3YKLHED[d]
LLeu-HApi8
12
17
9
–7900
–9900
25
29
17
32
–5000[e]
–11000[e]
LLeu-HApi16
17
[a] Mean residue molar ellipticity values at the absorption band of amide n–π*
transition. [b] Estimated according to the equation reported in ref. 21.
Percentage helicity = 100 × [θ]MRE_n-π*
/
max[θ]MRE_n-π*, where max[θ]MRE_n-π* = –
40000 × [1 – (2.5/m)] and m = number of amino acid residues. [c] Ref. 21.
[d] Ref. 22. [e] Calculated from the CD spectrum measured at [peptide] = 30
µM in aqueous HCl (pH 4.0) at 0 ºC.
LLeu-Api16 probably interact with neighboring amide NH groups
and hinder their hydrogen bonding with carbonyl groups along
the peptide backbone (Figure 1, center). In sharp contrast, the
protonated oligopeptides LLeu-HApi8 and LLeu-HApi16 showed
CD patterns typical of
a right-handed α-helical structure
(Figures3a and 3c, red); the two characteristic Cotton effects at
211 (π–π*) and 225 nm (n–π*) were explicit, and the ratio of
their intensities was almost unity.[20] As shown in our previous
study, the formation of an α-helical structure was unambiguously
1
confirmed for the analogous Api 8mer [Ac(HApi)8NHMe] by a H
NMR saturation-transfer experiment and by 1H–1H correlations
evaluated by 2D rotational Overhauser effect spectroscopy
Figure 3. CD spectra in MeOH at 20 °C of the Api peptides with the free-base
(Apin, blue), protonated (HApin, red), and acylated (BocApin, green) states (n =
8 or 16). a) N-terminal chiral 8mers. b) C-terminal chiral 8mers. c) N-terminal
chiral 16mers. d) C-terminal chiral 16mers. For the concentrations of the Api
peptides, see Experimental Section.
(ROESY).[17]
In the protonated state, the hydrogen-bond
accepting sites in the piperidine groups are occupied by protons,
so that the amide NH groups in the peptide backbone can form
the hydrogen-bonding networks characteristic of α-helical
structures (Figure 1, top). As described in the introduction, the
response of Api peptides toward acidic and basic conditions is
opposite to that of conventional basic peptides.[18]
Artifacts such as the contamination of linear dichroism
signals are likely to be excluded, because the D-forms of the Api
peptides, synthesized from DLeu by the same routes as the L-
forms, showed CD spectra that were essentially the mirror
images of those of the L-forms (Figure S1). In addition, for the
CD spectra of all the Api peptides, essentially no concentration
dependence was observed, indicating that the aggregation of
Api peptides is negligible in the present CD measurement
conditions (Figures S2 and S3).
Although the CD spectra of the protonated peptides LLeu-
HApi8 and LLeu-HApi16 were quite similar in pattern, they were
notably different in intensity. The [θ]MRE of LLeu-HApi16 was
L
about twice that of Leu-HApi8 (Figures 3a and 3c, red). When
the medium was changed from MeOH to aqueous HCl (pH 4.0),
the difference in [θ]MRE was further enhanced; the [θ]MRE of LLeu-
HApi16 was 2.3 times greater than that of LLeu-HApi8 (Figure S4).
These observations show that chain elongation significantly
stabilizes the α-helical conformation of Api oligopeptides. Note
Chiroptical Profiles of Api Peptides (i): Effects of the State
of the Piperidine Groups
L
that the average fractional helicity of Leu-HApi16, as estimated
from the CD intensity at 225 nm (n–π*), is comparable to or
even greater than that of a typical α-helical oligopeptide (17
residues) specifically designed to form a stable α-helix (Table
We at first focused on the effects of the peptide length and the
state of the piperidine groups, with fixing the chiral auxiliary
position at the N-terminus (Figures 3a and 3c). CD spectra of
the free-base peptides Leu-Api8 and Leu-Api16 show a single
positive peak at around 210 nm, which are obviously different
from the CD profiles of α-helices (Figures 3a and 3c, blue). As
we clarified in our previous report by using H–D exchange as a
probe,[17] the nonprotonated piperidine groups in LLeu-Api8 and
L
1).[21–23] Considering that Leu-HApi16 has only one stereogenic
L
L
center at its N-terminus, whereas the reference oligopeptides
consist exclusively of enantiopure amino acids, we can conclude
that the present Api oligopeptide realizes a highly efficient
transfer of chiral information.
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As described in the introduction, acylation of the piperidine
groups is also anticipated to induce helical structures (Figure 1,
center to bottom). Indeed, the acylated 16mer LLeu-BocApi16,
bearing tert-butoxycarbonyl units on the secondary amines of
the piperidine groups, displayed CD bands at 210 and 225 nm,
characteristic of an α-helical conformation (Figure 3c, green).
Contrary to this, the acylated 8mer LLeu-BocApi8 displayed CD
profiles quite different from α-helices (Figure 3a, green).[19]
Therefore, the effect of acylation on helix induction (Figure 1,
center to bottom) is more dependent on the length of the
oligopeptide chain than on protonation (Figure 1, center to top).
In addition, the helicity induced by acylation is lower than that
induced by protonation (Table 1).
In the following sections, we focus on the 16mers, because
the helix-formation ability of the 16mers superior to the 8mers
would make the discussion simpler.
Figure 4. CD spectra in MeOH of Api 16mers at various temperatures from 20
to 60 °C: a) LLeu-HApi16 (red) and LLeu-BocApi16 (green) and b) HApi16-LLeu
(red), BocApi16-LLeu (green), and Api16-LLeu (blue). For the concentrations of
the Api peptides, see Experimental Section.
Chiroptical Profiles of Api Peptides (ii): Effects of the
Position of the Chiral Auxiliary
The conformational features of the C-terminal chiral 16mers
were also studied in the similar manner to the N-terminal chiral
ones (Figure 3d). At the protonated state, the C-terminal chiral
16mer (HApi16-LLeu) adopted a left-handed α-helical structure
characterized with two positive Cotton effects (Figure 3d, red),
whose handedness was opposite to the N-terminal chiral 16mer
(LLeu-HApi16).[24] Considering that these 16mers possess chiral
auxiliaries based on the same amino acid (LLeu), the
inconsistency of their helical handedness apparently seems
strange, but similar helicity inversion was reported for a pair of
Aib oligopeptides, having an L-amino acid at either of the N- or
chiral and the C-terminal chiral 16mers, one or two piperidine
groups located near to the chiral auxiliary can interact with the
adjacent amide NH groups in the peptide backbone (Figures S6,
blue), so that the chiral information transfer from the chiral
auxiliary to the achiral Api oligomers (Figures S6, red) is
‘disrupted’. In the N-terminal chiral 16mer, the piperidine groups
in charge of the ‘disrupting’ are on the 3rd and 4th residue from
the N-terminus (Figure S6a, blue). Meanwhile, the ‘disrupting’
residue from the C-terminus (Figure S6b, blue), whose
secondary amine, located very near to the terminus, is exposed
to exterior solvent and therefore prone to be solvated.
Accordingly, the ‘disrupting’ of the C-terminal chiral 16mer is
partially decelerated even at the free-base state.
C-terminus.[25]
It should also be noted that the helical
handedness is determined not only by the position of chiral
auxiliary but by the balance of several parameters; even a slight
modification of the C-terminal chiral auxiliary, replacing the
endcapping group from the ester to the corresponding amide,
caused the helicity inversion, giving the same handedness to the
N-terminal chiral peptide (Figure S5).[25]
Chiroptical Profiles of Api Peptides (iii): Response to
Changes in Temperature
With five α-helical 16mers (LLeu-HApi16, LLeu-BocApi16, HApi16-
LLeu, BocApi16-LLeu, and Api16-LLeu) in hand (Figures 3c and
3d), we investigated the stability of their conformations to
changes in temperature (Figure 4).
With varying the state of the piperidine groups, the helicity of
the C-terminal chiral 16mers changed only slightly (Figure 3d),
unlike the change of the N-terminal chiral 16mers (Figure 3c).
Indeed, the [θ]MRE of C-terminal chiral 16mer in the acylated
state (BocApi16-LLeu; Figure 3d, green) is slightly lower than that
in the protonated state (HApi16-LLeu; Figure 3d, red).
Noteworthy, the C-terminal chiral 16mer retained certain helicity
even at the free-base state (Api16-LLeu; Figure 3d, blue),
although the helicity is the lowest among the three states.
Overall, these observations imply that the sensitivity of the
present helical structures toward the state of the piperidine unit
can be modulated by the position of the chiral auxiliary; the C-
terminal chiral 16mer is less sensitive than the N-terminal chiral
16mer. As a further confirmation, the 8mers also showed the
similar tendency (Figures 3a and 3b).
To clarify the effects of piperidine state on the heat tolerance
of the helical structures, we at first focus on the data of the C-
terminal chiral 16mers, which can adopt helical structures at any
state of the piperidine groups (Figure 4b). Upon heating from 20
to 60 °C, the protonated 16mer (HApi16-LLeu) showed relatively
small change in its CD profiles (Figure 4b, red), where [θ]MRE
slightly decreased (9%) with respect to that of the original state
at 20 °C. Meanwhile, the acylated (BocApi16-LLeu) and free-base
16mers (Api16-LLeu) were more prone to heat-induced
denaturation (Figure 4b, green); on heating from 20 to 60 °C, the
[θ]MRE of the acylated and free-base 16mers decreased by 19%
and 28%, respectively. Thus, the heat tolerance of the helical
structures of the Api peptides is in the following order:
protonated > acylated > free-base. As a confirmation for the
generality of the above rule, the N-terminal chiral 16mers (LLeu-
Although the studies on detailed mechanism are now
ongoing in our group, these observations can be elucidated by
the following hypothesis. In the free-base form of the N-terminal
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The pH-titration plot, i.e. the plot of the relative changes in
the CD intensity at 226 nm as a function of the pH (Figure 5c),
gives
a more profound insight on the difference in pH
responsiveness between the N-terminal chiral and the C-
terminal chiral 16mers. On increasing the pH from 2.0 to 11.2,
the N-terminal chiral 16mer underwent the structural transition at
an earlier stage (closed circles) than the C-terminal chiral 16mer
(open circles). Indeed, the pH midpoint for the N-terminal chiral
16mer is 8.1, while that of the C-terminal chiral 16mer is 9.4.
Such a notable difference in pH responsiveness between the N-
terminal chiral and the C-terminal chiral 16mers might be
elucidated by difference in the environment of the ‘disrupting’
piperidines, as described above (Figure S6).[27]
Conclusions
A series of Api 8mers and 16mers were prepared as a new motif
for the study of polymers and peptides with dynamic helical
structures, and their conformational profiles were investigated
with varying the peptide length, the state of the piperidine groups
in the Api units, and the position of chiral auxiliary. These
systematic studies revealed the following facts.
(i) Effect of peptide length: Chain elongation of the Api
peptides enhances the tendency to form an α-helix (Figure
3). The 16mers generally show higher helix formation
ability than the corresponding 8mers, and one of the
16mers shows a helicity of 32%, which is at a highest level
for the oligopeptides with similar length (Table 1).
Figure 5. CD spectra ([peptide] = 30 µM) in aqueous media at 20 °C of a)
LLeu-HApi16 and b) HApi16-LLeu upon titration from pH 2 to 11. (c) Plots of the
relative changes in [θ]MRE (= {[θ]MRE – [θ]MRE_pH2} / {[θ]MRE_pH11 – [θ]MRE_pH2}) at
226 nm as a function of the pH value. The broken lines indicate the pH
midpoints.
(ii) Effect of the state of the piperidine groups: Depending on
the state of the piperidine groups, helix formation ability of
the Api peptides changes as follows: protonated > acylated
> free-base (Figure 3). Heat tolerance of the helical
structures also changes in the same manner (Figure 4).
(iii) Effect of the position of the chiral auxiliary [1]: The position
of chiral auxiliary affects the helical handedness (Figure 3).
In the protonated state of the piperidines, the N-terminal
chiral and the C-terminal chiral peptides adopt right- and
left-handed helical structures, respectively. It should also
be noted that the helical handedness is determined not
only by the position of chiral auxiliary but by the balance of
several parameters, including the structure of the
endcapping groups (Figure S5).[25]
(iv) Effect of the position of the chiral auxiliary [2]: The position
of the chiral auxiliary affects the sensitivity of the helical
structures toward the state of the piperidine groups. The
N-terminal chiral peptides are more sensitive than the C-
terminal chiral ones (Figure 3).
(v) Effect of the position of the chiral auxiliary [3]: The position
of the chiral auxiliary affects pH responsiveness of the
helical structures (Figure 5). For the structural transition
induced by the increase of pH, the C-terminal chiral
peptide shows higher pH midpoint (pH 9.4) than the N-
terminal chiral one (pH 8.1).
HApi16 and LLeu-BocApi16) also showed the same tendency in
heat tolerance (protonated > acylated; Figure 4a), although the
data of the free-base state (LLeu-Api16) are missing because of
its poor helix-formation ability. Relatively low tolerance of the
helical structure acylated peptides (LLeu-BocApi16 and BocApi16-
LLeu) might be attributed to the steric hindrance of the Boc
group.
Chiroptical Profiles of Api Peptides (iv): Response to
Changes in pH
We next investigated the pH responsiveness of the Api 16mers
(HApi16 ⇌ Api16) by a titration method. Thus, the protonated
16mers (LLeu-HApi16 or HApi16-LLeu) were dissolved in aqueous
HCl (pH 2.0), and the resultant solutions were titrated with
aqueous NaOH while the resulting conformational change was
monitored by CD spectroscopy. At the initial and final states of
the titration, the CD profiles of the N-terminal chiral and the C-
terminal chiral 16mers were in consistent with the analogous
measurement in MeOH (Figures 3c and 3d).[26] On increasing
pH from 2.0 to 11.2, the N-terminal chiral 16mer underwent a
drastic helical-to-nonhelical structural transition (Figure 5a),
while the C-terminal chiral 16mer just decreased its helicity
(Figure 5b).
As described in the introduction, the Api peptides are the first
basic oligopeptide that adopt a helical conformation in acidic
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3H), 0.93 (d, J = 6.3 Hz, 3H) ppm; 13C NMR (125.77 MHz; CD3CN;
50 °C): δ 176.33, 176.30, 176.08, 175.76, 175.54, 175.21, 155.66,
155.63, 155.55, 155.44, 155.39, 80.35, 80.29, 80.23, 80.18, 80.11, 80.05,
50.43, 59.24, 59.22, 59.00, 58.84, 58.60, 58.57, 58.49, 41.2–40.2, 28.90,
28.87, 28.84, 25.73, 23.30, 22.94 ppm; HR-MALDI-TOF-MS calcd. for
C96H159N17O27Na ([M + Na]+) 2005.1484, found 2005.1486.
media exclusively. The conformational features of the 16mers
clarified here, which are notably improved from those of the
8mers, should expand the utility of Api peptides. Furthermore,
pH responsiveness of the Api peptides can be modulated by the
position of the chiral auxiliary unit (N- or C-terminus).
Considering these features, as well as the potential of the
secondary amines in the piperidine groups for modification, such
as acylation, alkylation, quaternization, metal coordination, etc.,
the Api peptides developed here should serve as versatile
precursors for various functionalized helical peptides.
Ac(LLeu)(BocApi)16OBn (= LLeu-BocApi16
)
To a stirred CH2Cl2 solution (1 mL) of Ac(LLeu)(BocApi)8OH (198 mg, 0.10
mmol) at 0 °C, were added HOAt (14 mg , 0.10 mmol), HATU (38 mg,
0.10 mmol), and TMP (40 µL, 0.30 mmol) at 0 °C, and the mixture was
stirred for 3 h at 0 °C. Then, H(BocApi)8OBn (192 mg, 0.10 mmol) was
added at 0 °C to the reaction mixture. After being stirred at 0 °C for 30
min, the reaction mixture was allowed to warm to rt and stirred for 4 days.
Then, the solution was diluted with CH2Cl2 (10 mL), washed with
aqueous HCl (1 M, 3 × 10 mL) and brine (10 mL), dried over MgSO4, and
evaporated to dryness under a reduced pressure at rt. The residue was
subjected to silica gel column chromatography eluted with AcOEt/CHCl3
(1:1–2:1, v/v) to afford Ac(LLeu)(BocApi)16OBn as a white solid (136 mg,
35 µmol, 35%). 1H NMR (500.16 MHz; CD3CN; 50 °C): δ 8.05 (brs, 1H),
7.80 (s, 2H), 7.76 (s, 1H), 7.73 (s, 1H), 7.69 (s, 1H), 7.64 (s, 1H), 7.62 (s,
1H), 7.57 (s, 1H), 7.46 (s, 1H), 7.42 (s, 1H), 7.38–7.29 (m, 5H), 7.28 (s,
1H), 7.12 (s, 2H), 6.76 (brs, 1H), 5.05 (s, 2H), 4.21 (m, 1H), 3.97–2.71 (m,
64H), 2.24–1.70 (m, 64H), 1.97 (s, 3H), 1.70–1.52 (m, 3H), 1.45–1.37 (m,
144H), 0.96 (d, 6H) ppm; 13C NMR (125.77 MHz; CD3CN; 50 °C): δ
176.39, 176.31, 176.19, 176.01, 175.95, 175.89, 174.87, 174.71, 174.47,
174.41, 155.75, 155.68, 155.49, 137.58, 129.51, 129.19, 80.41, 80.36,
80.32, 80.24, 80.21, 80.18, 80.15, 80.10, 80.06, 80.02, 79.97, 79.91,
67.55, 59.64, 59.55, 59.03, 58.97, 58.70, 58.68, 58.57, 58.54, 58.45,
58.42, 57.42, 41.75–39.07, 35.93–34.65, 30.59–29.88, 25.79, 23.51,
23.39, 22.04 ppm; HR-MALDI-TOF-MS calcd. for C191H309N33O51Na ([M +
Na]+) 3904.2492, found 3904.2426.
Experimental Section
Materials and Methods
All reagents were purchased from Kanto Kagaku, Aldrich, Tokyo Kasei,
and Wako Pure Chemical. Unless otherwise noted, they were used as
received. For column chromatography, Wakogel silica C-300HG (particle
size 40–60 µm) or C-400HG (particle size 20–40 µm) was used.
Analytical TLC was performed with 0.25-mm thick silica gel 60F plates
with a fluorescent indicator (254 nm).
1H and 13C NMR spectra were recorded on a JEOL model GSX270
spectrometer, operating at 270.05 and 67.80 MHz for 1H and 13C NMR,
respectively, and a JEOL model JNM-ECA500 spectrometer operating at
500.16 MHz and 125.77 MHz for 1H and 13C NMR, respectively.
Chemical shifts were determined with respect to partially or non-
deuterated solvents [(CH3)4Si] as internal references. Electronic
absorption (UV-vis) and circular dichroism (CD) spectra were recorded
on a JASCO model U-best V-560 spectrometer and a JASCO model J-
820 spectropolarimeter, respectively. High-resolution matrix-assisted
laser desorption/ionization TOF-MS (HR-MALDI-TOF-MS) spectra were
recorded on a Bruker model AutoFlex Speed mass spectrometer on a
positive mode by using poly(ethylene glycol) (Mn = 1000, 2000, and
4000) as an internal reference, sodium trifluoroacetate as a protonation
reagent, and a mixture of α-cyano-4-hydroxycinnamic acid (CHCA) and
2,5-dihydroxybenzoic acid (DHB) as a matrix. Ac(L/DLeu)(BocApi)8OBn (=
Ac(LLeu)(HApi)16OBn (= LLeu-HApi16
)
To a stirred AcOEt solution (5 mL) of Ac(LLeu)(BocApi)16OBn (154 mg, 40
µmol) was introduced HCl gas over 15 min at rt, and the reaction mixture
was stirred at rt for 30 min. The precipitate in the resultant mixture was
collected by filtration, washed with hexane, and dried overnight at 60 °C
under a reduced pressure to afford Ac(LLeu)(HApi)16OBn as a white
hygroscopic solid (114 mg, 40 µmol, quant). 1H NMR (500.16 MHz;
CD3OD; 25 °C): δ 8.91(s, 1H), 8.88 (s, 1H), 8.53 (s, 1H), 8.41 (s, 1H),
8.39 (s, 1H), 8.22 (s, 1H), 8.18 (s, 1H), 8.12 (brs, 1H), 8.06 (s, 3H), 8.00
(s, 1H), 7.94 (s, 1H), 7.90–7.80 (m, 3H), 7.61 (s, 1H), 7.45–7.35 (m, 5H),
5.19 (ABq, 2H, ΔνAB = 15.3 Hz, JAB = 12.0 Hz), 3.95 (brs, 1H), 3.75–2.92
(m, 64H), 2.22 (s, 3H), 2.87–2.12 (m, 64H), 1.85–1.62 (m, 3H), 1.07 (d, J
= 6.3 Hz, 3H), 1.01 (d, J = 6.3 Hz, 3H) ppm; HR-MALDI-TOF-MS calcd.
for C111H181N33O19Na ([M – 16HCl + Na]+) 2303.4104, found 2303.4090.
L/DLeu-BocApi8),
Ac(L/DLeu)(Api)8OBn
(=
L/DLeu-Api8),
Ac(L/DLeu)(HApi)8OBn (= L/DLeu-HApi8), and H(BocApi)8OBn were
synthesized according to the reported methods[17] with minor modification
in the deprotection step of benzyl ester of Fmoc(BocApi)nOBn (n = 2, 4,
8).[28]
Peptide Synthesis
Ac(LLeu)(Api)16OBn (= LLeu-Api16
)
The synthesis and characterization of the L-form peptides are described
here. The D-form peptides were synthesized in the same methods to
their antipodes, by using the starting materials with the corresponding
stereochemistry.
To a stirred aqueous solution (0.1 mL) of Ac(LLeu)(HApi)16OBn (57 mg,
20 µmol) was added an aqueous solution of methanolic KOH solution
(0.1 M, 3.3 mL) at rt. After being stirred at rt for 5 min, the reaction
mixture was concentrated to dryness under a reduced pressure at rt.
The resultant residue was suspended in MeOH (1 mL) and filtrated
through a hydrophobic porous membrane (pore diameter = 0.20 µm,
DISMIC–31jp). The filtrate was concentrated to dryness under a reduced
pressure at rt to afford Ac(LLeu)(Api)16OBn as a white solid (46 mg, 20
µmol, quant). 1H NMR (500.16 MHz; CD3OD; 25 °C): δ 7.39–7.21 (m,
5H), 5.11 (ABq, 2H, ΔνAB = 13.3 Hz, JAB = 12.6 Hz), 3.98 (brs, 1H), 3.25
(m, 64H), 2.03 (s, 3H), 2.45–1.77 (m, 64H), 1.85–1.62 (m, 3H), 1.06 (d, J
= 6.9 Hz, 3H), 0.99 (d, J = 6.3 Hz, 3H) ppm; HR-MALDI-TOF-MS calcd.
for C111H181N33O19Na ([M + Na]+) 2303.4104, found 2303.4144.
Ac(LLeu)(BocApi)8OH
To a solution of Ac(LLeu)(BocApi)8OBn (414 mg, 0.20 mmol) in MeOH (6
mL) was added 10 wt% Pd/C (414 mg) at rt, and the resultant mixture
was degassed and purged with hydrogen. After being stirred at rt for 12
h, the catalyst was filtered off through a Celite pad, and the solid was
washed with a mixture of CH2Cl2/MeOH (10:1, v/v). The filtrate and
washing were combined and concentrated under reduced pressure to
dryness. The resultant solid was suspended in hexane, and then filtered
and washed with hexane. The solid residue was dried under reduced
pressure to afford Ac(LLeu)(BocApi)8OH as a white solid (368 mg, 186
µmol, 93%). 1H NMR (500.16 MHz; CD3CN; 50 °C): δ 7.66 (brs, 1H),
7.55 (brs, 1H), 7.52 (s, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.41 (brs, 1H),
7.27 (s, 1H), 7.12 (s, 1H), 3.92 (brs, 1H), 3.85–2.55 (m, 32H), 2.40–1.85
(m, 32H), 1.70–1.52 (m, 3H), 1.43–1.38 (m, 72H), 0.95 (d, J = 6.3 Hz,
Fmoc(BocApi)8(LLeu)OBn
By following the same procedure used for the preparation of
Ac(BocApi)8(LLeu)OBn, Fmoc(BocApi)8OH (377 mg, 184 µmol) and
TsOH•H(LLeu)OBn (73 mg, 184 µmol) were converted to
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10.1002/chem.201605460
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FULL PAPER
Fmoc(BocApi)8(LLeu)OBn as a white solid (352 mg, 156 µmol, 85%). 1H
NMR (500.16 MHz; CDCl3; 50 °C): δ 7.77 (d, J = 8.0 Hz, 2H), 7.52 (d, J =
7.5 Hz, 2H), 7.43 (m, 2H), 7.36–7.26 (m, 7H), 7.22 (s, 1H), 7.16 (s, 1H),
7.13 (s, 1H), 7.07 (brs, 2H), 6.77 (brs, 1H), 5.84 (brs, 1H), 5.09 (m, 2H),
4.65 (brs, 1H), 4.57 (m, 2H), 4.21 (t, J = 6.0 Hz, 1H), 4.03–2.50 (m, 32H),
2.37–1.56 (m, 35H), 1.52–1.38 (m, 72H), 0.90 (m, 6H) ppm; 13C NMR
(125.77 MHz; CDCl3; 50 °C): δ 174.83, 174.76, 174.62, 174.58, 174.35,
173.31, 172.94, 165.74, 156.67, 154.69, 154.58, 154.53, 154.47, 154.33,
154.28, 154.26, 143.01, 142.97, 141.52, 141.46, 135.89, 128.38, 128.14,
128.12, 128.04, 127.90, 127.21, 124.56, 120.35, 80.41, 80.27, 80.15,
80.05, 79.93, 79.67, 79.45, 79.28, 67.13, 66.58, 58.86, 58.72, 58.06,
57.95, 57.63, 57.59, 57.54, 52.06, 47.24, 40.17–38.30, 34.74–33.37,
28.55–28.31, 24.51, 22.77, 21.67 ppm; HR-MALDI-TOF-MS calcd. for
C116H173N17O28Na ([M + Na]+) 2275.2528, found 2275.2499.
5.11 (m, 2H), 4.46 (m, 1H), 3.34–2.43 (m, 32H), 2.39–1.53 (m, 38H),
0.90 (m, 6H) ppm; HR-MALDI-TOF-MS calcd. for C63H101N17O11Na ([M +
Na]+) 1294.7759, found 1294.7719.
Ac(BocApi)16(LLeu)OBn (= BocApi16-LLeu)
To a stirred CH2Cl2 solution (1 mL) of Ac(BocApi)8OH (187 mg, 100 µmol)
at 0 °C, were added HOAt (14 mg , 100 µmol), HATU (38 mg, 100 µmol),
and TMP (40 µL, 300 µmol), and the mixture was stirred for 3 h at 0 °C.
Then, H(BocApi)8(LLeu)OBn (203 mg, 100 µmol) was added at 0 °C to the
reaction mixture. After being stirred at 0 °C for 30 min, the reaction
mixture was allowed to warm to rt and stirred for 4 days. Then, the
solution was diluted with CH2Cl2 (10 mL), washed with aqueous HCl (1 M,
3 × 10 mL) and brine (10 mL), dried over MgSO4, and concentrated to
dryness under a reduced pressure at rt. The residue was subjected to
silica gel column chromatography eluted with AcOEt/CHCl3 (1:1–2:1, v/v)
to afford Ac(LLeu)(BocApi)16OBn as a white solid (136 mg, 35 µmol, 35%).
1H NMR (500.16 MHz; CD3OD; 50 °C): δ 7.39–7.27 (m, 5H), 5.11 (ABq,
2H, ΔνAB = 12.3 Hz, JAB = 12.6 Hz), 4.50 (m, 1H), 3.98–2.68 (m, 64H),
2.55–1.54 (m, 70H), 1.50–1.39 (m, 144H), 0.93 (d, J = 6.3 Hz, 3H), 0.90
(d, J = 6.3 Hz, 3H) ppm; 13C NMR (125.77 MHz; CD3OD/CDCl3 = 2:1
(v/v); 50 °C): δ 177.98, 176.37, 176.29, 176.20, 176.16–175.84, 175.60,
175.10, 173.62, 173.56, 155.93, 155.88, 155.79–155.56, 136.67, 129.15,
129.03, 128.96, 81.42–81.23, 81.21, 81.16, 81.09, 80.95, 80.83, 80.68,
80.10, 80.06, 80.02, 67.49, 59.75, 59.60, 58.45, 58.35, 58.27, 58.20,
58.10, 57.98, 41.59–39.24, 35.47–34.26, 28.96–28.58, 25.14, 23.28,
21.75 ppm; HR-MALDI-TOF-MS calcd. for C191H309N33O51Na ([M + Na]+)
3904.2492, found 3904.2445.
H(BocApi)8(LLeu)OBn
To a stirred THF solution (3 mL) of Fmoc(BocApi)8(LLeu)OBn (338 mg,
150 µmol) was added diethylamine (0.3 mL, 3 mmol) at rt, and the
reaction mixture was stirred at rt for 3 h. The reaction mixture was then
evaporated to dryness under a reduced pressure at rt. The residue was
chromatographed with CH2Cl2/MeOH (100:0–100:8, v/v) as an eluent, to
allow the isolation of H(BocApi)8(LLeu)OBn (293 mg, 144 µmol, 96%). 1H
NMR (500.16 MHz; CDCl3; 50 °C): δ 8.91 (brs, 1H), 7.44 (s, 1H), 7.42 (s,
1H), 7.40 (s, 1H), 7.33–7.26 (m, 5H), 7.21 (s, 1H), 7.05 (s, 1H), 6.55 (s,
1H), 5.11 (ABq, 2H, ΔνAB = 12.6 Hz, JAB = 12.6 Hz), 4.59 (m, 1H), 4.04–
2.65 (m, 32H), 2.39–1.53 (m, 35H), 1.50–1.41 (m, 72H), 0.91 (d, J = 4.1
Hz, 3H), 0.90 (d, J = 4.1 Hz, 3H) ppm; 13C NMR (125.77 MHz; CDCl3;
50 °C): δ 178.66, 174.64, 174.45, 174.38, 173.90, 173.81, 173.63,
173.20, 154.69, 154.62, 154.59, 154.42, 136.23, 128.36, 128.04, 127.93,
80.77, 80.69, 80.52, 80.20, 79.79, 79.53, 79.23, 66.42, 59.04, 58.87,
58.09, 58.02, 57.80, 57.44, 57.25, 56.39, 51.39, 40.49–38.26, 34.68–
33.75, 29.43, 28.57–28.35, 24.52, 23.02, 21.53 ppm; HR-MALDI-TOF-
MS calcd. for C101H163N17O26Na ([M + Na]+) 2053.1847, found 2053.1816.
Ac(HApi)16(LLeu)OBn (= HApi16-LLeu)
By following the same procedure used for the preparation of
Ac(LLeu)(HApi)16OBn, Ac(BocApi)16(LLeu)OBn (105 mg, 27 µmol) was
converted into Ac(HApi)16(LLeu)OBn as a white hygroscopic solid (77 mg,
27 µmol, quant). 1H NMR (500.16 MHz; CD3OD; 25 °C): δ 8.81(s, 1H),
8.68 (s, 1H), 8.52 (brs, 1H), 8.50 (s, 1H), 8.35 (s, 1H), 8.30 (s, 1H), 8.21
(brs, 1H), 8.12 (s, 1H), 8.02 (brs, 2H), 7.99 (s, 1H), 7.93 (s, 1H), 7.91 (s,
1H), 7.80–7.73 (m, 3H), 7.68 (s, 1H), 7.52–7.42 (m, 5H), 5.14 (ABq, 2H,
ΔνAB = 14.2 Hz, JAB = 11.6 Hz), 3.90 (brs, 1H), 3.72–2.91 (m, 64H), 2.18
(s, 3H), 2.94–2.10 (m, 64H), 1.82–1.67 (m, 3H), 1.02 (d, J = 6.1 Hz, 3H),
Ac(BocApi)8(LLeu)OBn (= BocApi8-LLeu)
To a stirred CH2Cl2 solution (1 mL) of H(BocApi)8(LLeu)OBn (145 mg, 71
µmol) and DIPEA (14 µL, 78 µmol) was added acetic anhydride (8 µL, 78
µmol) at 0 °C, and the mixture was allowed to warm to rt and stirred for
12h. Then, the solution was diluted with CH2Cl2 (10 mL), washed
successively with hydrochloric acid (1 M, 3 x 10 mL) and brine (10 mL),
dried over MgSO4, and concentrated to dryness under a reduced
0.99 (d,
J = 6.1 Hz, 3H) ppm; HR-MALDI-TOF-MS calcd. for
C111H181N33O19Na ([M – 16HCl + Na]+) 2303.4104, found 2303.4090.
Ac(Api)16(LLeu)OBn (= Api16-LLeu)
pressure at rt.
The residue was subjected to silica gel column
By following the same procedure used for the preparation of
Ac(LLeu)(Api)16OBn, Ac(HApi)16(LLeu)OBn (46 mg, 16 µmol) was
converted into Ac(Api)16(LLeu)OBn as a white hygroscopic solid (36 mg,
16 µmol, quant). 1H NMR (500.16 MHz; CD3OD; 25 °C): δ 7.45–7.21 (m,
5H), 5.11 (m, 2H), 4.47 (m, 1H), 3.37–2.48 (m, 64H), 2.45–1.75 (m, 64H),
1.75–1.51 (m, 3H), 0.91 (m, 6H) ppm; HR-MALDI-TOF-MS calcd. for
C111H181N33O19Na ([M + Na]+) 2303.4104, found 2303.4078.
chromatography using CH2Cl2/MeOH (100/5–100/8 in v/v) as an eluent,
to afford Ac(BocApi)8(LLeu)OBn as a white solid (146 mg, 70 µmol, 99%).
1H NMR (500.16 MHz; CD3OD; 50 °C): δ 7.39–7.26 (m, 5H), 5.11 (ABq,
2H, ΔνAB = 12.0 Hz, JAB = 12.1 Hz), 4.50 (m, 1H), 4.04–2.63 (m, 32H),
2.52–1.69 (m, 35H), 1.61–1.56 (m, 3H), 1.51–1.39 (m, 72H), 0.93 (d, J =
6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H) ppm; HR-MALDI-TOF-MS calcd. for
C103H165N17O27Na ([M + Na]+) 2095.1953, found 2095.1930.
Ac(HApi)8(LLeu)OBn (= HApi8-LLeu)
Circular Dichroism (CD) Spectroscopy
By following the same procedure used for the preparation of
Ac(LLeu)(HApi)16OBn, Ac(BocApi)8(LLeu)OBn (112 mg, 54 µmol) was
converted into Ac(HApi)8(LLeu)OBn as a white hygroscopic solid (84 mg,
54 µmol, quant.). 1H NMR (500.16 MHz; CD3OD; 25 °C): δ 8.94 (brs,
1H), 8.76 (s, 1H), 8.28 (s, 1H), 8.03 (brs, 1H), 7.90 (s, 1H), 7.79 (s, 1H),
CD measurements in MeOH were performed by using a quartz cuvette
with 1 mm optical path length (Figures 3, 4, and S1–S3). The sample
solutions with the following peptide concentrations were used. Also note
that essentially no concentration dependence was observed for the CD
spectra of all peptides (Figures S2 and S3).
7.56 (brs, 1H), 7.47–7.28 (m, 5H), 5.15 (ABq, 2H, ΔνAB = 12.3 Hz, JAB
=
12.1 Hz), 4.57 (m, 1H), 3.82–2.91 (m, 32H), 2.86–1.94 (m, 35H), 1.84 (m,
2H), 1.63 (m, 1H), 0.94 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H) ppm;
HR-MALDI-TOF-MS calcd. for C63H101N17O11Na ([M – 8HCl + Na]+)
1294.7759, found 1294.7726.
Protonated 8mers
Free-base 8mers
Acylated 8mers
(LLeu-HApi8 and HApi8-LLeu):
(LLeu-Api8 and Api8-LLeu):
(LLeu-BocApi8 and BocApi8-LLeu):
300 µM
300 µM
200 µM
200 µM
200 µM
100 µM
Protonated 16mers (LLeu-HApi16 and HApi16-LLeu):
Free-base 16mers (LLeu-Api8 and Api8-LLeu):
Ac(Api)8(LLeu)OBn (= Api8-LLeu)
Acylated 16mers
(LLeu-BocApi16 and BocApi16-LLeu):
By following the same procedure used for the preparation of
Ac(LLeu)(Api)16OBn, Ac(HApi)8(LLeu)OBn (62 mg, 40 µmol) was
converted into Ac(Api)8(LLeu)OBn as a white solid (50 mg, 40 µmol,
quant). 1H NMR (500.16 MHz; CD3OD; 25 °C): δ 7.40–7.20 (m, 5H),
CD measurements in aqueous media were performed by using a quartz
cuvette with 10 mm optical path length (Table 1 and Figures 5, S4, and
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FULL PAPER
S5). The sample solutions with the peptide concentration of 30 µm were
used.
[7]
[8]
For recent study on kinetics of Aib oligopeptides, see: M. Kubasik, J.
Kotz, C. Szabo, T. Furlong, J. Stace, Biopolymers 2005, 78, 87–95.
a) C. Toniolo, E. Benedetti, Macromolecules 1991, 24, 4004–4009; b) A.
Bavoso, E. Benedetti, B. Di Blasio, V. Pavone, C. Pedone, C. Toniolo,
G. M. Bonora Proc. Natl. Acad. Sci. USA 1986, 83, 1988–1992; c) C.
Toniolo, G. M. Bonora, V. Barone, A. Bavoso, E. Benedetti, B. Di
Blasio, P. Grimaldi, F. Lelj, V. Pavone, C. Pedone Macromolecules
1985, 18, 895–902.
Mean residue molar ellipticity ([θ]MRE) was calculated from ellipticity (θ)
using the following equations:
[θ]MRE = (100 × θ)/(C × L × m)
θ : ellipticity (degree)
C : peptide concentration (mol L–1
)
[9]
a) E. Benedetti, M. Saviano, R. Iacovino, C. Pedone, A. Santini, M.
Crisma, F. Formaggio, C. Toniolo, Q. B. Broxterman, J. Kamphuis,
Biopolymers 1998, 46, 433–443; b) B. Pengo, F. Formaggio, M. Crisma,
C. Toniolo, G. M. Bonora, Q. B. Broxterman, J. Kamphuis, M. Saviano,
R. Iacovino, F. Rossi, E. Benedetti, J. Chem. Soc. Perkin Trans. 2 1998,
1651–1658.
L : light path length of cuvette (cm)
m: number of amino acid residues
pH Titration Experiments
Typically, LLeu-HApi16 (4.3 mg, 1.5 µmol) was dissolved in water (50 mL)
to afford an aqueous solution with the peptide concentration of 30 µM.
The pH value of the aqueous solution was adjusted to 2.0 by adding an
aqueous solution of HCl (6.0 M). To the aqueous solution was added an
aqueous solution of NaOH (5.4 × 10–n M, n = 1–3) in a stepwise manner
with magnetic stirring. After each step of the NaOH addition, pH of the
aqueous solution was measured. Then, a part of the aqueous solution
[10] a) Y. Inai, Y. Kurokawa, T. Hirabayashi, Biopolymers 1999, 49, 551–
564; b) Y. Inai, Y. Kurokawa, A. Ida, T. Hirabayashi, Bull. Chem. Soc.
Jpn. 1999, 72, 55–61.
[11] a) B. A. F. Le Bailly, L. Byrne, J. Clayden, Angew. Chem. 2016, 128,
2172–2176; Angew. Chem. Int. Ed. 2016, 55, 2132–2136; b) J. Solà, M.
Helliwell, J. Clayden, J. Am. Chem. Soc. 2010, 132, 4548–4549; c) J.
Clayden, A. Castellanos, J. Solà, G. A. Morris, Angew. Chem. 2009,
121, 6076–6079; Angew. Chem. Int. Ed. 2009, 48, 5962–5965.
[12] a) B. A. F. Le Bailly, J. Clayden, Chem. Commun. 2016, in press, DOI:
10.1039/c6cc00788k; b) C. Toniolo, M. Crisma, F. Formaggio, C.
Peggion, Biopolymers 2001, 60, 396–419.
(~3 mL) was taken into
a quartz cuvette and subjected to CD
measurement. After the CD measurement, the portion taken in the
quartz cuvette was combined with the mother liquor and subjected to the
next step of the NaOH addition. The pH titration experiment of HApi16
LLeu was conducted in the same manner.
-
[13] a) N. Ousaka, Y. Takeyama, H. Iida, E. Yashima, Nat. Chem. 2011, 3,
856–861; b) N. Ousaka, Y. Inai, J. Org. Chem. 2009, 74, 1429–1439; c)
Y. Inai, N. Ousaka, T. Okabe, J. Am. Chem. Soc. 2003, 125, 8151–
8162; d) Y. Inai, Y. Ishida, K. Tagawa, A. Takasu, T. Hirabayashi, J. Am.
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Acknowledgements
This work was partly supported by Incentive Research Fund (No.
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10.1002/chem.201605460
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FULL PAPER
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2 and 1,
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(1 atm) in THF/MeOH (3:1, v/v).
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Table of Contents
FULL PAPER
Dynamic Helices: As a new motif
for the study of dynamic helices,
hexadecamers of 4-aminopiperidine-
4-carboxylic acid (Api), bearing L-
leucine as a chiral auxiliary at either
N- or C-terminus, was developed.
Although the Api hexadecamer
possesses only one stereogenic
center, it showed notably high
helicity. The helicity of Api peptides
was readily modulated by changing
the peptide length, state of the
piperidine groups in the Api units,
and position of the chiral auxiliary.
J. Cho, Y. Ishida*, and T. Aida*
Page 1 – Page 8
Helical Oligopeptides of a
Quaternized Amino Acid with
Enhanced Chiral-Induction Ability
and an Anomalous pH Response
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