Simultaneous synthesis and self-assembly of cyclic diphenylalanine at hydrothermal condition

Article abstract of DOI:10.1246/cl.2006.636

Hydrophobic aniino acid, L-phenylalanine, formed insoluble aggregates with unique morphologies at hydrothermal condition. The analyses by infrared spectrum and gel permeation chromatography indicate the highly efficient synthesis of cyclic diphenylalanine

Full text of DOI:10.1246/cl.2006.636

6
36
Chemistry Letters Vol.35, No.6 (2006)
Simultaneous Synthesis and Self-assembly of Cyclic Diphenylalanine
at Hydrothermal Condition
ꢀ
Takanari Togashi, Mitsuo Umetsu, Hiroyuki Tsuchizaki, Satoshi Ohara, Takashi Naka, and Tadafumi Adschiri
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University,
2-1-1 Katahira, Aoba-ku, Sendai 980-8577
(Received March 17, 2006; CL-060332; E-mail: ajiri@tagen.tohoku.ac.jp)
Hydrophobic amino acid, L-phenylalanine, formed insoluble
(1)
(2)
aggregates with unique morphologies at hydrothermal condition.
The analyses by infrared spectrum and gel permeation chroma-
tography indicate the highly efficient synthesis of cyclic di-
phenylalanine probably due to solid–liquid phase separation.
The formed cyclic diphenylalanine simultaneously self-assem-
bled to form nanorod structure in the hydrothermal condition.
(
a)
(
(
a)
b)
(b)
(
(
c)
d)
(
c)
d)
700 1650 1600 1550 1500
(
1
3200 2800 2400 2000
-1
-1
Recent approach of the hydrothermal organic synthesis for
macromolecules and polymers is attracting an interest in the field
of green chemistry. Amino acid is one of the low molecules
which have been synthesized from carbon dioxide by hydrother-
Wavenumber/ cm
Wavenumber/ cm
Figure 1. FT-IR spectra of L-Phe (a) and the insoluble aggre-
ꢁ
gates formed at 220 C in a 170 mM L-Phe solution in 30 min
(
b) and 120 min (c), in a 710 mM L-Phe solution in 30 min (d).
1
mal reaction, and the polymerization of amino acid through
dehydration between amino acids has been also reported in
reaction time and L-Phe concentration leads to the formation
6
2
sub- or supercritical water. Cycled hydrothermal treatment of
3
of amide bond from carboxyl and amine groups. The IR bands
ꢂ1
ꢂ
glycine results in the elongation of oligopeptides, and rapid
quench of temperature increases the yield of oligopeptide,
at 1590 cm is considered to be some COO vibration, which
ꢂ
might indicate residual COO groups in the insoluble aggre-
gates.
4
although it is still much less than 10%.
In this study, we report a highly efficient dehydration be-
tween hydrophobic amino acids in subcritical water. Amino acid
is the monomer composing peptide and protein in vivo, but the
self-assembly of the peptide is recently utilized in the field of
nanobiotechnology.5 We, further, show that the synthesized
product forms an unique assembled structure.
The aggregates formed by the hydrothermal reaction were
insoluble in water, but it should be noted that the water-insoluble
aggregates could be dissolved in THF; consequently, the dis-
solved samples were analyzed by gel permeation chromatogra-
phy (GPC) to identify the molecules composing the aggregates
(Figure 2). The chromatogram from the aggregate formed at
the low L-Phe concentration (170 mM) in the reaction time of
30 min, had only a single peak (Figure 2a) which was considered
to be derived from acetyl phenylalanine by mass analysis.
Whereas, the aggregates formed in 120 min showed another
component observed at 29 mL of the elution volume where
the purchased normal or cyclic diphenylalanines are eluted
(Figure 2b). Further, the aggregate formed at the higher concen-
tration (710 mM) mainly showed the dimeric components, inde-
pendently of the reaction time (Figures 2c and 2d). In order to
identify the molecular structure, we measured the molecular
weight of the compound at the elution volume of 29 mL by
L-Phenylalanine (L-Phe) was saturated in distilled water at
ꢁ
2
5 and 100 C (170 and 710 mM, respectively), and then the
.28 mL of L-Phe solutions were loaded into a pressure-resistant
4
vessels (SUS316) with 5 mL of inner volume. The reactor was
ꢁ
heated at 220 C under the pressure of 22 MPa in an electric
furnace for various reaction times.
For all the cases, white insoluble aggregates were observed
in the reacted solutions, even after they were washed by excess
distilled water for the removal of unreacted L-Phe. This implies
that the L-Phe was changed to more hydrophobic substances. In
order to analyze the chemical structure of the aggregates, attenu-
ated total reflectance Fourier-transform infrared (ATR FT-IR)
spectra were measured for these precipitates (Figure 1). For
3
100x10
8
0
0
0
0
0
ꢂ1
ꢂ
L-Phe, an intense band at 1560 cm corresponding to COO
(a)
6
4
2
stretching vibration (Figure 1-(1a)) were observed. For the
aggregate formed from a 170 mM L-Phe solution, the IR band
disappeared (Figure 1-(1b)), but instead, two weak bands
(b)
(c)
(
d)
ꢂ1
ꢂ1
appeared at 1590 and 1660 cm , and the band at 1660 cm
became strong with both reaction time and L-Phe concentration
27
28
29
30
31
32
Elution volume /mL
3þ
increased (Figures 1-(1c) and 1-(1d)). The IR band from NH
ꢂ1
Figure 2. Results of GPC analysis for the synthesized compo-
nents in insoluble aggregates at concentration of 170 mM at
group around 2400 cm also decreased by the hydrothermal
reaction, and the aggregate formed from the highly-concentrated
L-Phe solution showed no absorbance for this band (Figure
(
a) 30 min, (b) 120 min and 710 mM at (c) 30 min, (d) 120 min.
A 100 mL of sample (0.2 mg/mL) was applied to a combined
GPC column line (Shodex KF801 and 803L) equilibrated with
THF. The eluant was monitored by refractive index.
ꢂ1
1
-(2)). Considering that the IR band at 1660 cm can be as-
signed to ꢀC=O vibration from amide bond, the increase of
Copyright ꢀ 2006 The Chemical Society of Japan

Chemistry Letters Vol.35, No.6 (2006)
637
ꢁ
Table 1. The yield of the synthesized precipitates at 220 C
from the 170 and 710 mM L-Phe solutions in the reaction time
of 30 and 120 min
(a)
(c)
170 mM
Yield/%
710 mM
Yield/%
Reaction time
30 min
20 min
0.7
6.8
22.2
34.0
(b)
(d)
1
MALDI-TOF mass spectrum. The spectrum for the dimeric form
showed the same molecular mass as the cyclic diphenylalanine
þ
(
c-Phe-Phe, ½M þ Hꢃ ¼ 295:064). The reverse-phase HPLC
analysis also supported that most of the components are
c-Phe-Phe. These results indicate that the hydrothermal reaction
formed c-Phe-Phe rather than normal Phe-Phe. The complete
disappearance of the COO and NH bands in the IR spectrum
for the aggregates formed in the highly-concentrated L-Phe
solution supports the GPC and mass results (Figure 1d).
Figure 3. SEM images of the insoluble aggregates after hydro-
thermal reaction for 30 min (a) and 120 min (b) at the L-Phe con-
centration of 170 mM, and for 30 min (c) and 120 min (d) at the
L-Phe concentration of 710 mM.
ꢂ
3þ
rod-like structure (Figure 3). The GPC results clearly demon-
strate that the rod structure contains c-Phe-Phe while the dimeric
component was not observed for the microsphere. This suggests
that the c-Phe-Phe promotes the formation of the rod-like struc-
ture. Recently, the formation of nanotube by normal Phe-Phe has
been reported. Song et al. revealed that the peptide concentration
is critical for the formation of nanotube or spherical vesicles.9
Our study is the first report for the formation of microsphere
and rod-like structure by c-Phe-Phe. Our results suggest that c-
Phe-Phe also behaves like normal Phe-Phe. Reches et al. propose
Table 1 lists the yield of synthesized c-Phe-Phe by the hy-
drothermal reaction. The yield was estimated from the results
of chromatography. Although longer reaction time led to in-
crease the c-Phe-Phe yield, the L-Phe concentration also promot-
ed the synthesis of c-Phe-Phe. At the high concentration of
L-Phe, even at the reaction time of 30 min, the cyclic peptide
was obtained with high yield, while the yield of the cyclic
peptide formed at the low concentration was as low as 6.8% at
1
20 min of reaction time. We also changed the reaction tempar-
ꢁ
10
ture and pressure, in the region from 180 to 250 C, and from 10
to 30 MPa, respectively; however, the change was not critical for
the yield of c-Phe-Phe. In the case of glycine, the hydrothermal
reaction results in the formation of cyclic diglycine (diketopiper-
the pathway for the aggregation of Phe-Phe; Phe-Phe, firstly,
forms an extended sheet by hydrogen bonds and aromatic stack-
ing interactions, and then the extended sheet is transformed to
the spherical or rod structure. The Phe-Phe might form the ani-
sotropic extended sheet to form the formation of rod-structure.
In conclusion, we attained highly efficient synthesis of dehy-
drated L-Phe, cyclic Phe-Phe, by hydrothermal reaction, proba-
bly due to solid–liquid phase separation during the reaction,
and also showed that the synthesized cyclic Phe-Phe self-assem-
bles to form rod structure.
7
azine). However, the yields are much less than 10%. This is
probably due to the reverse hydrolysis and the subsequent reac-
tion of diketopiperazien with the bi-products. We think that the
phase separation for the formation of c-Phe-Phe might be critical
for suppressing the further reaction to reduce its yield. Tsukahara
et al. reported that the oligomerization of glycine is promoted by
7
the addition of lipid at hydrothermal condition. This might
imply that hydrophobic region by lipid vesicles stabilizes the
dehydrated polypeptide at hydrothermal condition. A similar
effect can be expected for the aggregates formation in the reac-
tion atmosphere, namely, anti-promoting reaction might proceed
by the phase separation of the c-Phe-Phe formed.
This work was supported by Industrial Technology Re-
search Grant Program in ‘05 from New Energy and Industrial
Technology Development Organization (NEDO) of Japan.
References
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1
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5
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Published on the web (Advance View) May 20, 2006; doi:10.1246/cl.2006.636