Glutathione nanosphere: Self-assembly of conformation-regulated trigonal-glutathiones in water

Article abstract of DOI:10.1246/bcsj.20100048

A novel trigonal conjugate of glutathiones with a 1,3,5-tris(aminomethyl)- 2,4,6-triethylbenzene core was synthesized and its self-assembling behavior was investigated in water. Three glutathione units were regulated to orient on the same side of the benz

Full text of DOI:10.1246/bcsj.20100048

880
Bull. Chem. Soc. Jpn. Vol. 83, No. 8, 880–886 (2010)
© 2010 The Chemical Society of Japan
Glutathione Nanosphere: Self-Assembly of Conformation-Regulated
Trigonal-Glutathiones in Water
Kazunori Matsuura,*1,2 Keisuke Fujino,1 Takeshi Teramoto,1
Kazuya Murasato,1 and Nobuo Kimizuka1
1Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University,
744 Moto-oka, Nishi-ku, Fukuoka 819-0395
2PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi 332-0012
Received February 22, 2010; E-mail: ma2ra-k@mail.cstm.kyushu-u.ac.jp
A novel trigonal conjugate of glutathiones with a 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene core was
synthesized and its self-assembling behavior was investigated in water. Three glutathione units were regulated to orient
on the same side of the benzene ring, through steric repulsions between ethyl groups attached on the benzene core.
1
Concentration dependence of H NMR chemical shifts in D2O revealed formation of molecular assemblies with two
affinity constants (Ka = 4.75 © 102 and 6.76 © 104 M¹1), which reflect stepwise assembly directed by electrostatic
interactions, hydrophobic interactions, and hydrogen bonding. In scanning electron microscopy, hard spherical assemblies
with the size of 310 « 50 nm were observed at high concentration (10 mM), whereas slightly disordered spherical
assemblies were obtained at lower concentrations. The spherical assemblies self-assembled from the conformation-
regulated trigonal glutathiones showed regular morphology and enhanced rigidity compared to those formed from
conformationally non-regulated trigonal glutathiones.
The self-assembly of biomolecules, such as DNAs and
and the internal skeleton of tomato bushy stunt virus2,22 are
self-assembled from C3-symmetric protein subunits. By mim-
icking these C3-symmetric protein subunits, we have developed
artificial C3-symmetric self-assembly units for the construction
of unique bio-nanoassemblies.23,24 C3-symmetric DNA bearing
self-complementary sticky-ends spontaneously formed spher-
ical assemblies in water.23 Trigonal conjugates of ¢-sheet
forming peptides were self-assembled into nanospheres24a and
nanofibers,24b depending on molecular structures. Similarly,
C3-symmetric dipeptide WW conjugate was shown to self-
assemble into nanocapsules in methanol-water mixtures, as
reported by Gazit et al.25
Recently, ubiquitous biological tripeptide, gultathione (£-
Glu-Cys-Gly), has attracted much interest as a useful self-
assembly unit. Atkins et al. reported formation of organogel26a
and hydrogel26b from oxidized glutathione and glutathione-
pyrene conjugate, respectively. We have developed a C3-
symmetric glutathione conjugate (Trigonal-glutathione, TG)
which spontaneously self-assembled into nanospheres with the
size of 100-250 nm in water,27 although its symmetric mo-
lecular design was totally different from those of conventional
amphiphiles. In this paper, we describe synthesis and self-
assembly of a novel C3-symmetric glutathione conjugate in
water (Figure 1, CRTG). 1,3,5-Tris(aminomethyl)-2,4,6-tri-
ethylbenzene scaffold was employed, since it has been utilized
as C3-symmetric scaffold to design tripodal receptors.28 The
substituents on the 1,3,5-positions were reported to orient the
same side of benzene ring, and this feature was utilized to
assemble three glutathione arms on one side of the scaffold.
peptides, have attracted much attention because of their
potential for the design of elaborated nanostructures.1 Supra-
molecular protein assemblies in biological systems, such as
viral capsids, fragella, microtubles, amyloid fibrils, and
clathrin,2 provide excellent models for the de novo design of
artificial peptide nanoassemblies. Inspired by these biological
supermolecules, various kinds of peptide nanostructures have
been developed. Amphiphilic peptides have been designed to
form vesicle-like assemblies in water3,4 or monolayers at the
oil/water interfaces.5 ¡-Helix coiled-coil6-10 and ¢-sheet11-19
structures have been also employed as self-assembling motifs
to form peptide nanofibers and gels. For example, Woolfson
et al. developed orthogonal coiled-coil peptide units that
self-assembled into unique nanofibers, such as zig-zag and
branched ribbons.8 Zhang et al. reported an ionic comple-
mentary ¢-sheet-forming peptide FKFEFKFE which effi-
ciently self-assembled into left-handed helical ribbons.12a
Chmielewski et al. reported self-assembly of collagen peptide
into microstructures via metal coordination.20
The self-assembly of conventional peptides depends on their
amphiphilicity and capability to form secondary structures.
In order to innovate the molecular design of peptide-based
molecular assemblies, pre-organization of peptide chains
should be introduced since it might reduce the entropy loss
during self-assembling and afford unique assemblies even with
short peptides.
Such a pre-organization approach has been successfully
adopted by biological supermolecules. For example, clathrin21
Published on the web July 24, 2010; doi:10.1246/bcsj.20100048

K. Matsuura et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 8 (2010)
881
degassed water (10 mL). To the solution was added a solution
of 2 (0.151 g, 0.200 mmol) in degassed DMF (30 mL) at 0-5 °C
under nitrogen atmosphere. After adding diisopropylethyl-
amine (0.40 mL, 2.3 mmol), the mixture was stirred for 3 h
under the same conditions. After the solvent was evaporated
under vacuum, the residue was dissolved in water and then
lyophilized. The resulting colorless powder was purified
by reversed-phase HPLC eluting with a linear gradient of
acetonitrile/water (15/85 to 30/70 over 60 min) containing
0.1% TFA. The elution fraction containing CRTG was
lyophilized to a colorless powder (0.183 g, 60%). MALDI-
TOF-MS (matrix: ¡-CHCA): m/z calcd for C51H78N12O21S3:
1290.46 [M]+, found: 1290.29 [M]+. 1H NMR (300 MHz,
[CRTG] = 1 mM in D2O at 20 °C, TSP): ¤ 4.42 (dd, J = 5.1,
8.7 Hz, 3H), 4.33 (s, 6H), 3.78 (s, 6H), 3.71 (t, J = 6.6 Hz, 3H),
3.16 (s, 6H), 2.91 (dd, J = 5.1, 14.1 Hz, 3H), 2.77 (dd, J = 8.7,
14.1 Hz, 3H), 2.51 (q, J = 7.5 Hz, 6H), 2.39 (dt, J = 2.4,
7.5 Hz, 6H), 2.02 (dt, J = 6.6, 7.5 Hz, 6H), 1.00 (t, J = 7.5
Hz, 6H). Elemental Analysis calcd for C51H78N12O21S3¢
2CF3CO2H: C, 43.47; H, 5.31; N, 11.06%. Found: C, 43.53;
H, 5.30; N, 11.07%.
Molecular Mechanics Calculations. Molecular mechanics
calculations were carried out by using OPLS2005 force field in
MacroModel 9.1 program (SCHRÖDINGER). Total energy of
various conformations of trigonal-glutathiones in water was
calculated with the GB/SA solvation model29 of the program.
All geometric parameters (bond lengths, bond angles, and
dihedral angles) were optimized without any assumption by
using low mode/torsional sampling (MCMM) (step number:
10000) to search the global minimum conformation.
Figure 1. Structures of conventional trigonal-glutathione
(TG) and conformation-regulated trigonal-glutathione
(CRTG) at pH 3.
Experimental
General. Reagents were obtained from commercial sources
and used without further purification. Deionized water of high
resistivity (>18 M³ cm) purified with a Millipore Purification
System (Milli-Q water) was used as a solvent for trigonal-
glutathiones. 1H NMR spectra were recorded on a Bruker
AV300M spectrometer. Reversed-phase HPLC was performed
at ambient temperature with a Shimadzu LC-6AD liquid
chromatograph equipped with a UV-vis detector (220 nm,
Shimadzu SPD-10AVvp) using GL Science Inertsil ODS-3
columns (4.6 © 250 mm and 20 © 250 mm). MALDI-TOF
mass spectra were obtained on a PE Applied Biosystems
Voyager System 1180 MALDI-TOF mass spectrometer with
dithranol and ¡-cyano-4-hydroxycinnamic acid (¡-CHCA) as
matrix.
Estimation of Association Constants by 1H NMR.
1H NMR spectra of CRTG at different concentration in D2O
were recorded on a Bruker AV300M spectrometer at 18 °C with
sodium 3-(trimethylsilyl)-2,2,3,3-d4-propanoate (TSP) as ex-
ternal standard. The chemical shifts of each proton of CRTG
were plotted to the concentration. The obtained curves were
fitted by nonlinear regression analysis to the isodesmic model
(eq 1).30
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2Kac þ 1 ꢀ 4Kac þ 1
¤
max ꢀ ¤ ¼
ð¤max ꢀ ¤fÞ
ð1Þ
2Ka c2
2
Synthesis. 1,3,5-Tris(aminomethyl)-2,4,6-triethylbenzene
(1) was synthesized from 1,3,5-triethylbenzene according to a
literature method.28e
¤ denotes the chemical shift obtained from the spectra, ¤f and
max are the chemical shifts for the free and assembled species,
¤
1,3,5-Tris(iodoacetamidomethyl)-2,4,6-triethylbenzene (2).
A solution of iodoacetic acid N-hydroxysuccinimide ester
(1.00 g, 3.53 mmol) in acetone (10 mL) was added to a solution
of 1 (0.249 g, 1.00 mmol) in acetone (5 mL) at ¹5 °C. The
mixture was stirred in the dark for 8 h at the same temperature
and the resulting colorless precipitate was filtered and dried
under vacuum to provide compound 2 (0.225 g, 30%). MALDI-
TOF-MS (matrix: dithranol): m/z calcd for C21H30I3N3O3 +
respectively, Ka is the association constant, and c is the total
concentration of CRTG in the sample.
FT-IR Measurements in D2O.
Transmission FT-IR
spectrum of CRTG at 10 mM in D2O were recorded with a
Shimadzu FT-IR 8400S spectrophotometer using a liquid cell at
room temperature under nitrogen atmosphere.
Scanning Electron Microscopy (SEM). Aqueous solu-
tions of trigonal-glutathiones (0.1, 1.0, and 10 mM) were
prepared simply by dissolving in water without sonication or
heating. The samples were aged at 3 °C for 24 h. The pH was
adjusted by adding aqueous NaOH. 10 ¯L of the solutions were
applied to a carbon-coated Cu-grid (Oken Co., Ltd.), left for
60 s, and then removed. The grid was dried in vacuo and coated
with platinum (ca. 3 nm, Hitachi E-1030 ion sputter). The grids
were observed by SEM (Hitachi S-5000) with an acceleration
voltage of 15 kV at tilt angle of 0 and 30°.
1
Na+: 775.93 [M + Na]+, found: 775.79 [M + Na]+. H NMR
(300 MHz, DMSO-d6): ¤ 8.24 (t, J = 4.2 Hz, 3H), 4.29 (d, J =
4.2 Hz, 6H), 3.66 (s, 6H), 2.63 (q, J = 7.2 Hz, 6H), 1.09 (t, J =
7.2 Hz, 9H).
1,3,5-Tris{[2-(carboxymethylcarbamoyl)-2-(£-glutamyl-
amino)ethylthio]acetamidomethyl}-2,4,6-triethylbenzene
(Conformation-Regulated Trigonal-Glutathione, CRTG).
Reduced glutathione (0.187 g, 0.608 mmol) was dissolved in

882
Bull. Chem. Soc. Jpn. Vol. 83, No. 8 (2010)
Glutathione Nanosphere
Scheme 1. Synthesis of conformation-regulated trigonal-glutathione (CRTG).
Results and Discussion
Synthesis of Conformation-Regulated Trigonal-Gluta-
thione. Scheme 1 shows the synthesis of a conformation-
regulated trigonal-glutathione (CRTG) from 1,3,5-tris(amino-
1
methyl)-2,4,6-triethylbenzene (1). H NMR of compound 1 in
CDCl3 at ambient temperature showed that 1 exclusively takes
alternate substituent conformation.28 Compound 1 was con-
verted to the iodoacetamido derivative 2 by condensation
with activated ester. CRTG was prepared by substituting thiol
groups of reduced glutathione for the iodo groups of core
molecule 2. The final product was purified by reverse-phase
HPLC and confirmed by MALDI-TOF-MS (m/z = 1290) and
1H NMR. Aqueous solution of the purified CRTG was pH 3.
CRTG was soluble in water at the whole range of pH even at
high concentration (10 mM). 1H NMR of 1 mM CRTG in D2O
showed one set of relatively sharp signals which are assigned to
a kind of glutathione, linker arm, and ethyl group at the core,
respectively (Figures 3A and 3B). This suggests that substitu-
ents on CRTG in water preferentially adopted alternative
conformation, in which three glutathione arms were oriented to
the same side of the benzene ring, because it is known that
substituents on 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene
scaffold prevent free rotation.28
Figure 2. (A, B) Most stable conformation of CRTG (A)
and conventional TG (B) expected by molecular mechan-
ics caluculation (MacroModel). (C, D) Metastable con-
formation of CRTG (C) and conventional TG (D) at a
local minimum in which one arm of glutathione flipped.
Molecular Mechanics Calculation of Trigonal-Gluta-
thiones. In order to estimate conformations and potential
energies of CRTG and conventional TG in water, molecu-
lar mechanics calculations with GB/SA solvation model29
(MacroModel, OPLS2005 force field, MCMM algorism) were
carried out. Figure 2 shows the most stable and metastable
conformations of CRTG and conventional TG, respectively.
The “metastable conformation” means conformation at a local
minimum potential in which one arm of glutathione units
flipped (turned to the opposite face). In the most stable

K. Matsuura et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 8 (2010)
883
conformations of CRTG and TG, it was shown that the three
glutathione arms were oriented to the same side of the benzene
ring by forming intramolecular hydrogen bonds. The tripod
structure was more stabilized in CRTG, since energy gap
¦E between the most stable and metastable conformations
of CRTG (+43.95 kJ mol¹1) was much larger than that
of TG (+6.96 kJ mol¹1) as summarized in Table 1. As the
Analysis of Self-Assembling Behavior of CRTG Using
1
1H NMR. Figure 3A shows H NMR spectra of CRTG at
various concentrations in D2O. As the concentration increases,
signals assigned to ¡-protons of glycine (Hh) and glutamate
(Hg) shifted to the lower magnetic field, while the other signals
were independent of concentration. These results indicate that
CRTG is self-assembled by hydrogen bonds and/or electro-
static interactions between glutathione units upon increasing
rotation energy barrier on the C-C bond of ethane is about
¹1 31
12 kJ mol
,
the calculated energy gaps indicate that gluta-
concentration. FT-IR transmission spectrum of 10 mM CRTG
¹1
thione arms on CRTG do not flip at ambient temperature,
whereas those on TG can flexibly change their orientation. The
unique orientation of glutathione units on CRTG in Figure 2A
in D2O showed peaks at 1729, 1672, 1630, and 1462 cm ,
which are assigned to C=O stretching vibration of COOH, free
and hydrogen-bonded amide I bands, and C-N stretching
vibration, respectively (Figure 4).32 It is probable that amide
groups on the arms are not dominantly participating in the self-
assembly, because the Hi and Hf protons did not show shifts in
1H NMR spectra even at higher concentrations.
1
is supported by H NMR data as described below.
Table 1. Total Potential Energies of Trigonal-Glutathiones
Calculated by Molecular Mechanics
Figure 3C shows concentration dependence of chemical
shifts of Hh and Hg for CRTG. The association constants (Ka)
and chemical shifts of free species (¤f) were calculated by
nonlinear regression analysis according to the isodesmic model
(eq 1, see Materials and Methods).30 The analyses of the
chemical shifts of Hh and Hg afforded different affinity con-
Emost stable
/kJ mol
Emetastable
/kJ mol
¦E
/kJ mol
¹1 a)
¹1 b)
¹1 c)
CRTG
TG
¹4220.95
¹4243.26
¹4177.00
¹4236.29
+43.95
+6.97
¹1
¹1
stants (Ka = 6.76 © 104 M for Hh and Ka = 4.75 © 102 M
a) Total potential energy of most stable conformation shown
as Figures 2A and 2B. b) Total potential energy of meta-
stable conformation shown as Figures 2C and 2D. c) ¦E =
for Hg), respectively, as summarized in Table 2. These results
suggest that two different modes of association are involved in
the self-assembly of CRTG. The intermolecular interactions
Emetastable ¹ Emost stable
.
1
Figure 3. (A) H NMR spectra of CRTG at various concentrations (0.1-10 mM) in D2O (pD 3) at 18 °C. (B) Assignment of each
proton signal. (C) Dependence of the chemical shifts of Hg and Hh of CRTG on the concentration. The solid curves are theoretical
according to an isodesmic model (eq 1) using the parameters in Table 2.

884
Bull. Chem. Soc. Jpn. Vol. 83, No. 8 (2010)
Glutathione Nanosphere
Figure 6. The effect of pH on the nanostructure self-
assembled from CRTG (1 mM) in water. (A) pH 3, (B)
pH 5, (C) pH 7, and (D) pH 10. SEM samples were coated
with ca. 3 nm Pt.
Figure 4. FT-IR spectrum of CRTG at 10 mM in D2O.
Figure 5. SEM images of aqueous solution (pH 3) of CRTG at 0.1 (A), 1.0 (B), and 10 mM (C). SEM sample was coated with ca.
3 nm Pt. Scale bar indicates 500 nm.
concentration (10 mM), whereas irregular spherical assemblies
Table 2. Apparent Association Constants (Ka) in the Self-
were observed at lower concentrations (0.1 and 1.0 mM). The
observed difference in morphology might arise from differ-
ences in the assembly mode. It seems that interactions
involving glutathione C-termini (hydrogen bonds of carboxylic
acid and amide groups), which are dominant at lower
concentration (0.1 and 1.0 mM), afford irregular spherical
assemblies. At the higher concentration (10 mM), it is probable
that CRTG molecules in a spherical assembly are rearranged
by electrostatic interactions of N-termini (ammonium and
carboxylate ions), which afford regular spherical assemblies
with smooth surface. Time-dependent morphological changes
of the assemblies in the aqueous solutions were hardly
observed at room temperature at least for 1 month.
Figure 6 shows the effect of pH on the morphology of
CRTG (concentration, 1 mM). Upon increasing pH, the
spherical assemblies gradually collapsed. It is known that
reduced glutathione is ionized with pKa1 = 2.12 (carboxylic
acid of glutaminic acid), pKa2 = 3.59 (carboxylic acid of
glycine), pKa3 = 8.75 (thiol of cysteine), and pKa4 = 9.65
(ammonium of glutaminic acid).33 By using these pKa values,
the isoelectric point (pI) of CRTG is estimated to be ca. 2.9. It
indicates that CRTG possess nearly neutral net charge at pH 3,
and anionic charge at higher pH. Therefore, the disruption of
Assembly of CRTG at 18 °C
¹1
a)
b)
Ka/M
¤
f
¤
max
Hg of CRTG
Hh of CRTG
(4.75 « 0.35) © 102 3.76 « 0.01
(6.76 « 1.29) © 104 2.29 « 0.26
3.996
3.955
a) The chemical shift for the free species. b) The chemical shift
for the assembled species.
occurring even at lower concentration involve carboxylic acid
and amide groups near the Hh proton (1/Ka = 0.0015 mM),
which might form intermolecular hydrogen bonds. Contribu-
tion of glutathione N-terminal groups (ammonium and car-
boxylate ions) is indicated by the changes in Hg proton signals
(1/Ka = 2.1 mM), which becomes eminent at higher concen-
trations. Similar behavior was observed in 1H NMR of conven-
tional TG, but the association constant (Ka = 6.36 © 103 M
)
¹1
was lower than that of CRTG.27
Nanostructures of Trigonal-Glutathiones.
Scanning
electron microscopy (SEM) of CRTG showed the presence
of spherical assemblies with the size of 200-500 nm at the
range of 0.1-10 mM CRTG concentration (Figure 5). It is
notable that non-collapsed spherical assemblies (diameter,
310 « 50 nm) with smooth surface were observed at higher

K. Matsuura et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 8 (2010)
885
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The spherical assemblies formed from 10 mM CRTG
(310 « 50 nm by SEM, Figure 5C) were larger than those
formed from conventional TG (112 « 75 nm by SEM,
Figure 7B).27 In addition, the size distribution observed for
CRTG is apparently smaller compared to those of TG. The
nanoassemblies formed from 1 mM CRTG (Figures 5B and
6A) are all hard (non-collapsed) spheres, whereas the conven-
tional TG (concentration at 1 mM) afforded wrinkly collapsed
assemblies (Figure 7A). The difference in hardness between
5
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¹1
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,
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9
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126, 734.
We have developed a novel trigonal glutathione which takes
regulated conformation. This conjugate was self-assembled
into hard nanospheres by forming hydrogen bonds and/or
electrostatic interaction between glutathione moieties, and the
morphology depended on the concentration and pH. It is
notable that spherical assemblies were selectively formed
without the formation of fibrous assemblies often observed for
¢-sheet analogs. The spherical assemblies formed from CRTG
have narrow size distribution compared to those of conforma-
tionally non-regulated trigonal glutathiones assemblies,26 and
would be applicable as pH-responding and biodegradable
molecular carriers. The present molecular design based on the
conformation-regulated C3-symmetric strategy provides a novel
guideline for the design of peptide-based molecular assemblies.
Studies of the capability of these nanoassemblies to include
guest molecules are currently in progress.
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The present work is supported by PRESTO, Japan Science
and Technology Agency, by The Sumitomo Foundation, and by
a Grant-in-Aid for the Global COE Program, “Science for
Future Molecular Systems” from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
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