Modifying oligoalanine conformation by replacement of amide to ester linkage

Article abstract of DOI:10.1002/chir.22823

Oligo(lactic acid) is an ester-analogue of short oligoalanine sequence and adopts a rigid left-handed helical structure. In this study, oligo(lactic acid) was incorporated into oligoalanine sequences and their conformations were studied by vibrational circular dichroism and electronic circular dichroism spectroscopy. The results suggested that oligo(lactic acid) moiety in these sequences maintains a left-handed helix and increases the conformational propensity of the oligoalanine moiety to form a left-handed polyproline type II-like helix. The importance of the chirality of oligo(lactic acid) moiety for the oligoalanine conformation was also studied. The results obtained in this study should be useful in developing ester-containing oligopeptides that function better than normal peptides.

Full text of DOI:10.1002/chir.22823

Received: 6 December 2017
DOI: 10.1002/chir.22823
Revised: 28 December 2017
Accepted: 29 December 2017
S P E C I A L I S S U E A R T I C L E
Modifying oligoalanine conformation by replacement of
amide to ester linkage
Takahiro Hongen | Tohru Taniguchi
| Kenji Monde
Frontier Research Center for Advanced
Abstract
Material and Life Science, Faculty of
Advanced Life Science, Hokkaido
University, Sapporo, Japan
Oligo(lactic acid) is an ester‐analogue of short oligoalanine sequence and
adopts a rigid left‐handed helical structure. In this study, oligo(lactic acid)
was incorporated into oligoalanine sequences and their conformations were
studied by vibrational circular dichroism and electronic circular dichroism
spectroscopy. The results suggested that oligo(lactic acid) moiety in these
sequences maintains a left‐handed helix and increases the conformational pro-
pensity of the oligoalanine moiety to form a left‐handed polyproline type II‐like
helix. The importance of the chirality of oligo(lactic acid) moiety for the
oligoalanine conformation was also studied. The results obtained in this study
should be useful in developing ester‐containing oligopeptides that function bet-
ter than normal peptides.
Correspondence
Tohru Taniguchi, Kenji Monde, Frontier
Research Center for Advanced Material
and Life Science, Faculty of Advanced Life
Science, Hokkaido University, Kita 21
Nishi 11, Sapporo 001‐0021, Japan.
Email: ttaniguchi@sci.hokudai.ac.jp;
kmonde@sci.hokudai.ac.jp
Funding information
Suhara Memorial Foundation; Akiyama
Life Science Foundation; Japan Society for
the Promotion of Science (JSPS), Grant/
Award Numbers: 15H03111 and 26702034
KEYWORDS
electronic circular dichroism, lactic acid, polyproline type II helix, vibrational circular dichroism,
β‐strand
1 | INTRODUCTION
oligoalanines may adopt multiconformational states
including not only PPII7-9 but also α‐helix,10,11 β‐strand,
Polyproline type II (PPII) helix is a left‐handed 31‐helix
found in various proteins such as collagen, elastin, and
casein.1-3 Polyproline type II helix does not form intramo-
lecular hydrogen bonds, unlike right‐handed α‐helix, which
forms hydrogen bonds between the C═O group of the ith
residue and the NH group of the (i + 4)th residue. As a
result, PPII adopts a rather extended structure and its
carbonyl and amino groups are readily accessible by other
molecules for intermolecular interactions. These structural
properties render PPII numerous possibilities as functional
motifs for protein‐protein interactions, molecular scaffold,
peptide‐based drugs, antifreeze agent, and so on.4-6 How-
ever, designing a peptide sequence forming a PPII helix
without proline residues is sometimes difficult because of
the lack of stabilizing hydrogen bonds. For example,
and the resultant self‐assembled fibrils.12,13 As another
example, we have witnessed that antifreeze glycopeptide
H‐[Ala‐Thr‐Ala]n‐OH exists as PPII when its Thr residues
are glycosylated with N‐acetylgalactosamine, while it shows
an electronic circular dichroism (ECD) spectrum typical for
disordered structures when it is unglycosylated.14,15
A
method to facilitate the formation of PPII helix should
benefit medicinal chemistry and biochemistry by using
peptides and their analogues.
Modification of amide linkages in the peptide main
chain to different ones (eg, urea, carbamate, and aminoxy)
has been effective in regulating their secondary structures
and hence their functions.16,17 Considering future applica-
tions to biological systems, ester linkage is promising
because of (1) low toxicity, (2) easy availability (eg, solid‐
phase synthesis using α‐hydroxy acids and genetic code
expansion),18-20 and (3) ability to alter polypeptide
conformation due to the impaired hydrogen bonding
[This article is part of the Special Issue: Proceedings 16th International
Conference on Chiroptical Spectroscopy, Rennes France 2017.]
396
© 2018 Wiley Periodicals, Inc.
wileyonlinelibrary.com/journal/chir
Chirality. 2018;30:396–401.

HONGEN ET AL.
397
property.18,21 Among many α‐hydroxy acids, lactic acid
(Lac) is often incorporated into oligopeptides: For exam-
ple, a recent study found that substitution of an Ala
residue to Lac changed the conformations of pentapep-
tides and heptapeptides from the 11‐helix to the 14/15‐
helix.21 Meanwhile, to the best of our knowledge, there
has been no study on the conformations of oligopeptides
containing di‐ and longer oligo(Lac)s. Recently, the
solution conformation of poly(L‐Lac), a biodegradable
thermoplastic polyester, was studied by Ho′s and our
groups by means of vibrational circular dichroism (VCD)
spectroscopy.22,23 A negative‐positive VCD couplet at
around 1760 cm−1 observed under several conditions sug-
gested that poly(L‐Lac) adopts a fairly stable left‐handed
103‐like helix in solution,23-25 unlike multiconformational
oligoalanines. Moreover, conformational studies on sev-
eral oligo(L‐Lac)s indicated that 4 ester linkages are suffi-
cient to fix their helicity.23 These results inspired us to
use oligo(L‐Lac) sequences as a rigid structural unit for
ester‐containing peptides.
TABLE 1 The structures of the hexamers
Structurea
1 (R1 = Ac)
1a (R1 = Me)
R1‐LLac6‐OMe
2a
H‐LAla6‐OBn
3 (R1 = Ac, R2 = OMe) R1‐LAla3‐LLac3‐R2
3a (R1 = H, R2 = OH)
4
5
6
Ac‐LLac3‐LAla3‐OMe
Ac‐LAla3‐DLac3‐OMe
Ac‐DLac3‐LAla3‐OMe
aAla = ―NHCH(CH3)CO―; Lac = ―OCH(CH3)CO―; Bn = ―CH2Ph.
In this current work, we prepared several hexamers
composed of oligo(Lac) and oligoalanine and studied
their conformations by using VCD and ECD spectros-
copies. The results indicated that the left‐handed helical
conformations of oligo(Lac)s in these hexamers are
indeed rigid and also induce PPII helix formation of adja-
cent oligoalanine moieties. The chiroptical spectra were
analyzed in an empirical manner, because our prelimi-
nary density functional theory geometry optimization
calculations of the oligoalanine moiety of these hexamers
resulted in β‐strand, not PPII, conformations.
2 | MATERIALS AND METHODS
All the samples were prepared by means of solution‐
phase synthesis by using HATU and HOBt (see
Supporting Information). The purity of each molecule
1
was confirmed by H‐NMR and 13C‐NMR spectroscopies.
Vibrational circular dichroism and infrared (IR) spectra
were measured for 16 and 6000 scans, respectively, using
a BioTools Chiralir‐2X. All spectra were recorded by
using a 100‐μm CaF2 cell at a resolution of ~8 cm−1 at
ambient temperature and were presented after
subtracting solvent spectra obtained under the identical
experimental conditions. Prior to the VCD measurements
using CD3OD, each sample was once dissolved in an
excess amount of CD3OD, heated for 10 minutes, and
then dried by vacuum pump to perform amide H‐D
exchange. Electronic circular dichroism and UV spectra
were measured on a Jasco J‐820 spectrometer by using
a 0.1‐cm quartz cell.
FIGURE 1 (top) Vibrational circular dichroism and (bottom)
infrared (bottom) spectra of (A) 1, (B) 3, and (C) 4 in CD3CN
(black), CDCl3 (gray), and CD3OD (red)

398
HONGEN ET AL.
properties. To obtain more insights into the properties
of ester‐containing peptides, the conformations of the
hexamers were further studied.
3 | RESULTS AND DISCUSSION
3.1 | Synthesis and general properties of
hexamers
3.2 | Conformations of the oligoester
moiety of hexamers
Several hexamers were synthesized by combining a Lac
trimer and Ala trimer as listed in Table 1. LAla trimer
was placed before or after Lac trimer (3 and 4) to study
L
The VCD spectra of 1, 3, and 4 were measured first in
CDCl3 and CD3CN to analyze the conformation of the
oligoester moiety through comparison with previously
studied 1a in CDCl3.23 Especially, we were interested to
see if there were any differences in the helicity of 3 and
4, as an ester C═O is capable of acting as a weak hydrogen
bond acceptor with an NH group.
the difference in the influences in the peptide conforma-
tion. Using DLac and LAla, diastereomeric Ac‐LAla3‐
DLac3‐OMe (5) and Ac‐DLac3‐LAla3‐OMe (6) were also
synthesized to examine the influences of the helicity of
Lac trimer on the conformation of LAla trimer. H‐
LAla6‐OBn (2a) was used as a standard hexaalanine
because the other LAla hexamers synthesized in this
study were not soluble enough for spectral measure-
ments in water (pH 1‐13), CH3OH, trifluoroethanol,
DMF, CHCl3, and so on. Some oligoalanines have been
known difficult to dissolve because of aggregate
formations.13
Aiming at future applications of ester‐substituted
oligopeptides in various biological fields, we first carried
out a cell viability assay of H‐LAla3‐LLac3‐OH (3a)
against mouse embryonic fibroblast cells and confirmed
that it was indeed low in toxicity (Figure S1). Initially,
we also planned to test the antifreeze activity (an activity
to inhibit the growth of ice crystal at low temperature) of
the hexamers synthesized in this work because of their
expected conformational similarity with an antifreeze
glycopeptide hexapeptide that forms a PPII helix.14 How-
ever, this plan was abandoned due to the insolubility of
3a in pure water. Despite this observation, 3a showed
much higher solubility than 2a and H‐LAla6‐OH in
various solvents such as CH3OH, 50% CH3OH‐H2O,
and acetonitrile, which exemplified the usefulness of
ester substitution for modifications of oligopeptide
L
As shown in Figure 1A, Lac hexamer 1 measured in
CDCl3 (gray line) and CD3CN (black line) exhibited a
strong IR and VCD signals in the ester C═O stretching
region at around 1750 cm−1. In accordance with our
previous observation on 1a, the negative‐positive VCD
couplet (from lower to higher frequencies) suggested a
left‐handed helical structure.22-25 Meanwhile, heteroge-
neous hexamers 3 and 4 in CD3CN presented 3 character-
istic IR absorption bands originating from ester C═O
stretching (~1750 cm−1), amide I (~1675 cm−1), and
amide II vibrational modes (~1525 cm−1) (black lines in
Figure 1B and C and Table 2). The Δε values of the ester
carbonyl VCD couplet of 3 and 4 were smaller than those
of 1, as expected from the smaller numbers of ester link-
ages (7, 4, and 3 consecutive ester linkages for 1, 3, and
4, respectively). Nonetheless, both 3 and 4 exhibited a
negative‐positive couplet, which is indicative of the left‐
L
handed helical properties of their Lac3 moiety. No shift
was recognized for the ester C═O IR peak positions
between 1 and 3 (1755 cm−1), which should reflect the
absence of hydrogen bonds involving ester C═O groups
(Table 2). A minor red‐shift observed for 4 (1747 cm−1
)
TABLE 2 Comparison of the peak positions of the exciton couplets of hexamers measured at 0.025 M
1
3
4
5
6
Solvent
CD3CN
CD3OD
CD3CN
CD3OD
CD3CN
CD3OD
CD3CN
CD3CN
Ester C═O stretching
ν[cm−1] at IRmax
1755
1751
1767
1759
1751
1767
1755
1744
1763
1759
1751
1771
1747
1744
1767
1751
1747
1767
1755
1767
1751
1747
1767
1744
a
ν[cm−1] at Δε1
ν[cm−1] at Δε2
a
Amide I or I′
ν[cm−1] at IRmax
—
—
—
—
—
—
1674
1678
—
1659
1643
1682
1678
1862
—
1667
1659b
1678b
1674
1682
ν[cm−1] at Δε1
(1678)c
(1682)c
a
ν[cm−1] at Δε2
a
aWavenumber at the extrema of the first or second Cotton effect (Δε1 or Δε2, respectively).
bDetermined by the measurement at 0.017 M.
cThe first Cotton effect appeared at the same or higher frequencies compared with the infrared peak maximum.

HONGEN ET AL.
399
seemed within a range of red shifts intrinsic to shorter
oligo(Lac)s.23 Possibilities of significant hydrogen bonds
involving the ester groups of 4 were also excluded by the
similar VCD patterns between 3 and 4 in the amide I
and II regions. These observations led us to speculate that
the LLac3 moieties of 3 and 4 were hardly involved in
hydrogen bonding with their LAla3 moieties and main-
tained left‐handed helical properties.
relevant, protic solvents. The hexamers listed in Table 1
could not be dissolved in pure water, but most of these
were soluble enough for VCD measurement in methanol
and ECD measurement in 50% CH3OH‐H2O. LAla hexamer
2a was studied only by ECD spectroscopy due to its poor
solubility. When measured in 50% CH3OH‐H2O, 2a exhib-
ited an ECD spectral shape that resembled to β‐strand
(Figure 2a).26 The ECD spectral shape of 2 is similar to
that of H‐LAla6‐OMe,27 indicating little contribution from
the C‐terminal benzyl group to its ECD spectral shape.
The conformations of 1, 3, and 4 in CD3OD were then
studied by VCD spectroscopy (Figure 1, red line). The VCD
signals of their ester carbonyl groups were slightly broad-
ened possibly because of interactions with solvent but, as
3.3 | Conformations of the oligopeptide
moiety of hexamers
We then investigated the influence of LLac3 moiety to the
conformation of LAla3 moiety in more physiologically
FIGURE 2 (top) Electronic circular
dichroism and (bottom) ultraviolet spectra
of (A) 1 and 2a, (B) 3, (C) 4, (D) 5, and (E)
6 in 50% CH3OH‐H2O measured at various
temperatures

400
HONGEN ET AL.
moiety was confirmed by VCD measurement (Table 2 and
Figure 3). The ECD measurements of 5 and 6 were carried
out in 50% CH3OH‐H2O, resulting in a spectral pattern
indicative of a β‐strand structure (Figure 2D and E).27
Although right‐handed helical DLac3 could not induce a
right‐handed helical LAla3 conformation, this study
showed the importance of the combination of the chirality
of oligoester and oligopeptide moieties.
4 | CONCLUSION
In this work, we incorporated oligo(Lac)s, a rigid
L
structural motif we previously found, into Ala hexamer
sequences and studied their conformations. These model
compounds themselves may be difficult to use for
biological systems due to the poor solubility to pure water,
albeit low toxicity; however, this study provided useful
insights into future applications of ester‐substituted
oligoesters. First, oligo(L‐Lac) sequences maintained a
left‐handed helical structure even when incorporated into
L
FIGURE 3 Comparison of vibrational circular dichroism spectra of
(A) 5 (0.025 M) and (B) 6 (0.025 M). All spectra were measured in CD3CN
oligopeptides. Second, left‐handed helical Lac3 induced
the formation of a left‐handed PPII‐like helix of adjacent
LAla3 on both the N‐ and C‐terminal sides. Third, the
chirality of oligoester sequences is important for the
conformational outcome of neighboring oligopeptides.
Last, introduction of ester linkage to oligoalanine
increased its solubility to various solvents possibly due
to suppression of aggregation. These insights should be
useful in developing peptide analogues containing ester
linkage with various amino acids and α‐hydroxy acids
that function better than normal oligopeptides.
expected, maintained negative‐positive patterns. The amide
C═O stretching (amide I') of 3 and 4 also presented a nega-
tive first Cotton effect and a weak positive second Cotton
effect. These results suggested the left‐handed helical ten-
dencies of both the triester and tripeptide moieties in 3 and 4.
The tripeptide conformations were further studied by
ECD spectroscopy. As shown in Figure 2B,C, 3 and 4 in
50% CH3OH‐H2O at 25°C exhibited a positive band at
~217 nm and a negative band at ~193 nm, which are charac-
teristic to PPII helix.* Therefore, substitution of amide link-
age in LAla hexamers to ester linkage increased the
conformational propensity of the oligopeptide moiety to form
a PPII‐like structure. In a similar manner to other reported
LAla oligomers,7,8 3 and 4 underwent helix‐strand transition
upon increasing temperature with the helix of 3 being slightly
more stable than that of 4, as evidenced by ECD spectroscopy.
ACKNOWLEDGMENTS
This work was partially supported by the Japan Society for
the Promotion of Science (JSPS) (nos. 26702034 and
15H03111), the Shimadzu Science Foundation, the
Akiyama Life Science Foundation, and the Suhara
Memorial Foundation. This work was inspired by JSPS
Asian CORE Program “Asian Chemical Biology
Initiative” and JSPS A3 Foresight Program.
3.4 | Influence of the helicity of oligoester
moiety to oligopeptide moiety
Left‐handed helical LLac3 stabilized a left‐handed PPII‐
like conformation of an adjacent LAla3. This finding inter-
ested us to study which LAla3 conformation (eg, left‐
handed PPII, right‐handed α‐helix, and β‐strand) is
induced by inversion of the helicity of the oligoester moi-
ety. To this end, we studied the conformations of hexamers
5 and 6, of which the right‐handed helicity of the DLac3
ORCID
Tohru Taniguchi http://orcid.org/0000-0002-4965-7383
Kenji Monde http://orcid.org/0000-0002-1424-1054
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How to cite this article: Hongen T, Taniguchi T,
Monde K. Modifying oligoalanine conformation by
replacement of amide to ester linkage. Chirality.
2018;30:396–401. https://doi.org/10.1002/chir.22823
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