Pressure-induced oligomerization of alanine at 25 °C

Article abstract of DOI:10.1039/c5cc03665h

Pressure-induced oligomerization was found from high-pressure experiments at 25 °C on alanine powder soaked in its saturated aqueous solution. The oligomerization to alanylalanine occurred at 5 GPa. The maximum yields of alanylalanine and trialanine were, respectively, 1.1 × 10^-3 and 1.3 × 10^-4 at 11 GPa.

Full text of DOI:10.1039/c5cc03665h

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Pressure-induced oligomerization of alanine at
25 8C
Cite this: Chem. Commun., 2015,
51, 13358
Chikako Fujimoto,a Ayako Shinozaki,ab Koichi Mimura,b Tamihito Nishida,c
Hirotada Gotou,d Kazuki Komatsua and Hiroyuki Kagi*a
Received 2nd May 2015,
Accepted 8th July 2015
DOI: 10.1039/c5cc03665h
www.rsc.org/chemcomm
Pressure-induced oligomerization was found from high-pressure which was suggested by notably large pressure-dependence of
experiments at 25 8C on alanine powder soaked in its saturated the OH stretching frequency (À107 cmÀ1 GPaÀ1), the condensa-
aqueous solution. The oligomerization to alanylalanine occurred at tion reaction occurred.9
5 GPa. The maximum yields of alanylalanine and trialanine were,
respectively, 1.1 Â 10À3 and 1.3 Â 10À4 at 11 GPa.
In amino acids, hydrogen bonding and Coulombic interactions
strongly affect their crystal structure. With increasing pressure,
these interactions change drastically. Some of the amino acids
Intermolecular interaction changes drastically with increasing undergo phase transitions.10 Pressure-dependence in the crystal
pressure, inducing chemical reactions of organic materials structure of L-alanine up to 13.6 GPa was investigated precisely
without a catalyst. Pressure-induced chemical reactions have using neutron powder diffraction and single-crystal X-ray diffrac-
been reported for several types of organic materials. For example, tion.11 Increased pressure decreases intermolecular distances,
+
formation of naphthalene, biphenyl, terphenyl and so on has particularly those between NH3 and COOÀ, and might induce
been reported for high-pressure experiments on benzene, a chemical reactions of amino acids in addition to the continuous
simplest aromatic hydrocarbon, up to 16 GPa at room temperature.1 changes in structure and phase transformation.
Recently, crystalline one-dimensional sp3-bonded carbon nano-
Actually, high pressure can be a crucially important environ-
threads were recovered to ambient pressure from compression of ment for peptide formation from amino acids. Reportedly,
benzene at 20 GPa at room temperature.2 Above that pressure, an shock compression up to 26.3 GPa and 1170 K caused the
irreversible chemical reaction occurred to form an amorphous formation of glycylglycine and triglycine from glycine.12 Static
phase.3 The overlap of p bonds and the decrease in the inter- compression up to 100 MPa and 150 1C for 1–32 days results in
molecular distance of benzene molecules with increasing pres- oligomerization of glycine molecules up to 10-mer.13 Experiments
sure induced oligomerization and amorphization,1,4 which under much higher pressure (1.0–5.5 GPa) and higher tempera-
are enhanced by increasing temperature and selective laser ture (180–400 1C) conditions were conducted for durations from
irradiation.4,5 Theoretical and experimental studies reported 2 h to 24 h and the formation of pentamers of glycine and alanine
that pressure-induced oligomerization and polymerization also was reported.14 Both high temperature and high pressure were
occurred in chain unsaturated hydrocarbons such as propene,6 simultaneously applied to samples free from water in the pre-
butadiene,7 and acetylene.8 Furthermore, pressure-induced vious experiments. However, it remains unclear whether heating
condensation of trimethylsilanol was observed from Raman and or compression is the more important factor for oligomerization
IR absorption spectroscopies at pressures up to 3.3 GPa at room of amino acids.
temperature. Along with increased hydrogen bonding interaction
To investigate pressure-induced peptide formation from amino
between trimethylsilanol molecules with increasing pressure, acids at room temperature, we conducted high-pressure experi-
ments on L-alanine by the static compression method, without
heating. We analyzed the recovered samples using GC-MS and
found the formation of alanylalanine and trialanine.
a Geochemical Research Center, Graduate School of Science, The University of
Tokyo, Hongo, Tokyo 113-0033, Japan. E-mail: kagi@eqchem.s.u-tokyo.ac.jp;
Fax: +81 3 5841 4119; Tel: +81 3 5841 7625
L-Alanine powder with saturated L-alanine aqueous solution
was used as a sample, which was compressed by a large-volume
opposed-anvil assembly as presented in Fig. 1.1 Alanine (99.0%
purity, Wako Pure Chemical Inds. Ltd) powder of 7 mg to 10 mg
was loaded into a gasket and soaked in approximately 5 mg of
alanine-saturated aqueous solution which was prepared using
b Department of Earth and Planetary Sciences, Graduate School of Environmental
Studies, Nagoya University, Nagoya 464-8601, Japan
c Division of Earth and Environmental Sciences, Graduate School of Environmental
Studies, Nagoya University, Nagoya 464-8601, Japan
d Institute for Solid State Physics, The University of Tokyo, Kashiwa,
Chiba 277-8581, Japan
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Fig. 1 Double-toroidal anvil assembly. The center part was made of sintered
diamond surrounded by tungsten carbide and a binding ring made of
stainless steel.
pure water (UV-sterilized Milli-Q water). The alanine-saturated
solution was added as a pressure-transmitting medium. An
encapsulating gasket of stainless steel (SUS304) was used to
prevent the leakage of the liquid sample. Gaskets were cleaned in
acetone ultrasonically for 5 min and were wiped using Kimwipes
with pure water for 10 min while wearing laboratory gloves. The
sample chamber volume was about 9.4 mm3.1 High-pressure
experiments were conducted at room temperature (25 1C) on a
CAPRICORN 500 ton press at the Institute for Solid State Physics,
The University of Tokyo. The relationship between the load and
generated pressure was obtained from the cell volume of NaCl,
a standard pressure calibration material (Brown 1999), using a
synchrotron X-ray facility (KEK, Photon Factory, AR-NE7).
Four experimental runs were conducted with maximum
pressures of approximately 5 GPa (25 ton), 7 GPa (40 ton),
9 GPa (60 ton), and 11 GPa (70 ton) with preservation times at
maximum pressure for 1 h. Pressure was increased to maximum
pressure for 15–30 min depending on the target pressure and
was decreased to ambient pressure for the same duration after
1 hour preservation. Details of experimental parameters are pre-
sented in Table 1. After recovery, a gasket encapsulating alanine
solution was stored immediately in 5 mL of pure water contain-
ing 4.19 mg of norvaline as standard for quantification.
Fig. 2 Mass chromatograms of m/z = 168 + 211 + 282 recovered from
5 GPa, 7 GPa, 9 GPa, and 11 GPa (run numbers 1, 2, 3, and 4, respectively).
Numbered peaks 1, 2, and 3 respectively correspond to the derivatives of
norvaline, alanylalanine, and trialanine. The solid star (m/z = 168) derived
from a background compound that accompanied the derivatization pro-
cess respectively corresponds to the derivatives of norvaline, alanylalanine,
and trialanine. The solid star (m/z = 168) derived from a background
compound that accompanied the derivatization process.
and derivatized by 1.25 M HCl-isopropanol (Fluka Chemical), and
then by trifluoroacetic anhydride (TFAA) (Tokyo Chemical Indus-
try Co. Ltd) to produce N-TFA acylated-(Ala)n-O-isopropyl ester.
The derivatized samples were identified and quantified using
GC-MS (JMS-K9; JEOL Co.) equipped with a capillary column
(0.25 mm i.d. Â 30 m, 0.25 mm film thickness, HP-5; Agilent
Technology Co.). The GC column temperature was operated as
follows: maintained at 50 1C for 3 min, then increased to 225 1C
Run products in the encapsulated gasket were extracted by
breaking the gasket in the norvaline-containing aqueous solution
used for the storage. Samples were analyzed using a previously
reported procedure.12 The extracted sample solutions were dried
at 3.5 1C minÀ1, followed by an increase to 325 1C at 5 1C minÀ1
.
It was finally held at 325 1C for 10 min, with He carrier gas (flux;
1 mL minÀ1). The ionization voltage of the mass spectrometer
was set to 70 eV. The scan range of m/z was 40–550. Concen-
tration of alanylalanine (alanine dimer) contained in the starting
alanine reagent (99.0% purity, Wako Pure Chemical Inds. Ltd)
was 0.01 mmol molÀ1. That of trialanine (alanine trimer) was
below the detection limit. Moreover, alanylalanine and tri-
alanine were not detected from the procedure blank obtained
from pure water loaded in the same gasket assembly with
application of pressure of 5 GPa. Alanine peptides longer than
tetramer, such as cycloalanylalanine, amines, and carboxylic acids,
Table 1 Summary of run conditions and the abundances of the oligomers
Run no.
Load (ton)
Pressure (GPa)
Sat Ala aq. (mL)
Alanine (mg)
Standard
—
—
—
1
25
5
2
45
7
3
60
9
4
70
11
5
5
5
5
6.43
9.82 6.99 7.85 10.16
Alanylalanine (mg)
0.11
0.01
ND
0.89 1.19 4.62 20.22
0.05 0.09 0.33 1.11
Alanylalanine (mmol molÀ1
Trialanine (mg)
)
ND
ND
ND
ND
0.18 2.28
0.01 0.13
Trialanine (mmol molÀ1
ND: not detected.
)
ND
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Fig. 3 Representative mass fragmental patterns of derivatives of alanylalanine and trialanine recovered from 11 GPa, compared with those of standard
materials.
cannot be detected by the present experimental procedure trialanine obtained from the run product of 11 GPa (run no. 4)
and conditions.
with those of purchased standard materials.
Fig. 2 shows mass chromatograms of m/z = 168 + 211 + 282
A solution containing known quantities of norvaline, alanyl-
of samples recovered from 5 GPa, 7 GPa, 9 GPa, and 11 GPa. alanine, and trialanine was derivatized in the same way as the
Each chromatogram is displayed by taking the most intense samples recovered from high pressure. The product of detection
peak to be 100%. Numbered peaks 1, 2, and 3 correspond efficiency and yield of derivatization was calibrated from the
respectively to norvaline, alanylalanine, and trialanine. The peak areas corresponding to the derivatives of norvaline, alanyl-
oligomers (peak 2 and peak 3) were identified by comparison alanine, and trialanine in the mass chromatograms. Using these
of their retention times and mass fragmental pattern with those calibration data, quantities (weights) of alanylalanine and tri-
of purchased standard materials. Fig. 3 presents a comparison of alanine in the recovered samples were obtained. Concentrations
mass fragmental patterns of derivatives of alanylalanine and of alanylalanine in all the run products were notably higher than
the background concentration (0.01 mmol molÀ1) in the starting
material (see Table 1). In contrast, trialanine was detected in
the two samples recovered from 9 GPa (run no. 3) and 11 GPa
(run no. 4). Fig. 4 depicts molar ratios of alanylalanine and
trialanine to starting alanine vs. pressure. With increasing pressure,
yields of alanylalanine and trialanine increased. The maxi-
mum yield (0.11%) of alanylalanine was comparable to the
oligomer yields described in reports of previous studies in
terms of the order of magnitude.14
Reports of previous studies described that the formation of
oligomers was promoted by energetically activating amino acid
with vacuum ultraviolet irradiation, ion irradiation, catalysis,
increasing temperature, and so on.15 In contrast, this study
clarified that oligomerization of amino acids occurs at high
pressure even at low temperatures without catalyst. It is note-
worthy that the oligomerization of alanine occurs without applica-
tion of additional activation at high pressure.
Solid alanine is in its zwitterion form. The N–HÁ Á ÁO groups
of the neighbouring molecules selectively approach each other
with increasing pressure.11 The oligomerization with dehydra-
Fig. 4 Molar ratios of alanylalanine and trialanine to starting alanine vs.
pressure.
tion is regarded as occurring when the intermolecular distance
13360 | Chem. Commun., 2015, 51, 13358--13361
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of alanine molecules shortens with increasing pressure as well as Notes and references
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pressure as portrayed in Fig. 4.
Our results demonstrated that oligomerization of alanine
occurred at pressures higher than 5 GPa even at 25 1C. The
maximum yields of alanylalanine and trialanine were 1.1 Â
10À3 and 1.3 Â 10À4, respectively, at 11 GPa. This finding will
open new windows for understanding the pressure-induced
peptide formation from amino acids.
This work was supported by JSPS KAKENHI Grant Number
25287147 and 26246039. Part of this work was conducted under
the Visiting Researcher’s Program of the Institute for Solid State
Physics, The University of Tokyo. The synchrotron radiation facility
was used at NE7 of KEK, PF-AR with the approval of the Photon
Factory Program Advisory Committee (Proposal No. 2013G609).
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This journal is ©The Royal Society of Chemistry 2015
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