Size-selective olefin hydrogenation by a Pd nanocluster provided in an apo-ferritin cage

Article abstract of DOI:10.1002/anie.200353436

Deep penetration by substrates through the size-restricted channels of an apoferritin cage results in size-selective olefin hydrogenation at the Pd nanocluster core (see picture). The encapsulated zero-valent cluster is synthesized in situ by chemical reduction of Pd^II ions in the apoferritin cage.

Full text of DOI:10.1002/anie.200353436

Angewandte
Chemie
Hydrogenation Catalysis
Size-Selective Olefin Hydrogenation by a Pd
Nanocluster Provided in an Apo-Ferritin Cage**
Takafumi Ueno,* Masako Suzuki, Toshiaki Goto,
Tomoharu Matsumoto, Kuniaki Nagayama, and
Yoshihito Watanabe*
There has been much interest lately in chemical transforma-
tions in single capsules of supramolecular assemblies.[1–4]
Well-designed capsule structures could provide increased
concentration of substrates in the cavities with high selectivity
to allow transformation of the substrates. Proteins are very
attractive building components for the construction of self-
assembled cage structures.[1,5–12] For example,Mann and co-
workers have reported in their pioneering works the prepa-
ration of size-restricted metal oxides and sulfides by using the
biosupramolecular cage of ferritin.[5–7] In addition,protein
assemblies of viruses and the chaperonin protein GroEL were
also utilized for the encapsulation of metal nanoclusters.[10–12]
However,difficulties still remain in controlling reactions
catalyzed by metal clusters in protein cages.
Ferritin is known as an iron-storage protein and comprises
24 subunits that assemble to form a hollow cagelike structure
of 12 nm in diameter as shown in Figure 1. Iron atoms are
stored as a cluster of ferric oxyhydroxide within a cavity of
diameter 8 nm formed by the protein subunits (Fig-
ure 1b).[13,14] One of the most important consequences of
this protein cage is the perforation of the protein shell by
small channels that locate at the junctions of the subunits
(Figure 1c and d). The channels are required for the transport
of several metal ions and other organic molecules.[15] In
addition,penetration studies of organic molecules have
[*] Dr. T. Ueno
Research Center for Materials Science
Nagoya University
Nagoya 464-8602 (Japan)
Fax: (+81)52-789-2953
E-mail: taka@mbox.chem.nagoya-u.ac.jp
M. Suzuki, Prof. Dr. Y. Watanabe
Department of Chemistry
Graduate School of Science
Nagoya University
Nagoya 464-8602 (Japan)
Fax: (+81)52-789-2953
E-mail: yoshi@nucc.cc.nagoya-u.ac.jp
T. Goto
Department of Physics
Graduate School of Science
Nagoya University
Nagoya 464-8602 (Japan)
Dr. T. Matsumoto, Prof. Dr. K. Nagayama
National Institute for Physiological Science
Okazaki 444-8585 (Japan)
[**] This work was supported by Advanced Technology Institute and
Grant-in-Aid for Scientific Research from the Ministy of Education,
Culture, Sports, Science and Technology of Japan (Grants 14209019,
Y.W.; 15036229, T.U.).
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DOI: 10.1002/anie.200353436
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Communications
Figure 1. Schematic representation of horse-spleen ferritin: a) the com-
plete 24-meric assembly; b) the inner cavity; c) threefold axis hydro-
philic channel; d) fourfold axis hydrophobic channel taken from PDB
code: 1IER.[14]
confirmed the size-selective insertion of them into the holo-
ferritin interior through the channels.[16,17] These results could
imply that holo-ferritin could accommodate organic foreign
compounds in its cavity even in the presence of metal clusters.
Herein we describe a novel strategy for the construction of a
size-selective hydrogenation biocatalyst: a Pd nanocluster
encapsulated in the apo-ferritin cavity. The Pd cluster is
synthesized by in situ chemical reduction of PdII ions con-
centrated in the interior of apo-ferritin (Scheme 1) and the
size selectivity of the olefin hydrogenation is given by the
penetration of substrates through the channels.
Figure 2. a) UV/Vis spectra of Pd·apo-ferritin (solid line), and apo-ferri-
tin (dashed line); b) size-exclusion chromatography elution profile of
Pd·apo-ferritin monitored at 280nm (solid line) and 400nm (broken
line); c) native PAGE of marker (lane 1), Pd·apo-ferritin (lane 2), apo-
ferritin (lane 3). The gel (7.5%) was stained by Coomassie brilliant
blue.
clear brown solution. These results suggest specific formation
of Pd clusters within the protein cavities. Finally,the Pd·apo-
ferritin composite was isolated by size-exclusion chromatog-
raphy (Superdex G-200). The product elution was monitored
either at 280 nm (protein) or at 400 nm (zero-valent Pd
cluster),and the coelution of the protein and metallic
components through the column is the clear indication of
the composite nature of the material (Figure 2b). In addition,
a polyacrylamide gel electrophoresis (PAGE) experiment
with the Pd·apo-ferritin and apo-ferritin under native con-
ditions also indicates that the protein cage remains unchanged
during the reduction as shown in Figure 2c.
Scheme 1. Preparation of Pd·apo-ferritin.
The composite materials in aqueous solution were exam-
ined by transmission electron microscopy (TEM). The TEM
images clearly show that 1) the metal clusters are almost
monodispersed particles,2) their shape is roughly spherical
(Figure 3a),and 3) an intact protein shell surrounds the
metallic core (Figure 3b). The diameter of the metal particles
of Pd·apo-ferritin is 2.0 Æ 0.3 nm as shown in the histogram in
Figure 3a. We have also confirmed by electron-energy-loss
spectroscopy that the metal core contains only palladium but
not iron (data not shown).
Apo-ferritin is treated with 500 equivalents of K2PdCl4 to
afford a homogeneous pale yellow aqueous solution at pH 8.5
adjusted by 0.01n NaOH (aq). The PdII ions are reduced by
5 equivalents of NaBH4 to yield zero-valent Pd clusters
(Figure 2a). According to the reduction,the solution spec-
trum has been changed significantly; there is a broad intense
absorption at a longer wavelength (Figure 2a). A similar
spectroscopic change was observed for the reduction of PdII
ions in dendrimers.[18] Under the same conditions,black
precipitates were observed in a protein-free control experi-
ment,whereas the solution containing apo-ferritin remained a
The catalytic hydrogenation of olefins by Pd·apo-ferritin
was evaluated in aqueous medium. The turnover frequencies
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Chemie
aggregation of the composites during the reactions. Thus,
Pd·apo-ferritin is stable before,during,and after the catalytic
reaction. Pd·apo-ferritin also catalyzes the hydrogenation at
reasonable rates,for example,the TOF of Pd·apo-ferritin for
the N-isopropyl acrylamide reduction is almost comparable to
that of a dendrimer-encapsulated Pd catalyst in water.[18] On
the other hand,the TOF for acrylic acid ( 2) catalyzed by
Pd·apo-ferritin was eight times smaller than that of 3 though
TOF of 2 in the absence of apo-ferritin is almost identical to
that of 3 (Table 1). When the threefold channels in Pd·apo-
ferritin (Figure 1c) were blocked with TbIII,[17,19,20] the TOF of
1 was decreased to 7% of the TOF of 1 by Pd·apo-ferritin
without TbIII. Previous diffusion studies have also shown that
4-amino-TEMPO (TEMPO is 2,2,6,6-tetramethyl piperidine-
N-oxyl) could penetrate through the channels.[17] These
results,therefore,suggestive that the anionic threefold
hydrophilic channels (Figure 1c) are the pathway for sub-
strates. In comparison with neutral substrates,the TOFs of 5
and 6 by Pd·apo-ferritin are smaller than those of 1, 3,and 4
although the electron-deficient alkenes 1, 5,and 6 are
expected to be reduced faster than 3 and 4 if the electronic
substituent effect on the hydrogenation is considered.
Actually,the order of TOFs by Pd particle without apo-
ferritin was 1 > 5 > 6 > 3 > 4 (Table 1). Molecular-modeling
Figure 3. TEM images of the Pd·apo-ferritin: a) An ice-embedded
unstained sample and 4”image magnification (inset); b) a sample
negatively stained with uranyl acetate; c) histogram of the cluster size.
The temperature of the specimen stage was 77 K. The electron micro-
scopes were operated at acceleration potentials of 200 kV for (a) and
300 kV for (b). Scale bars are 50 nm.
calculations of the substrates indicate that the volumes of the
3
substrates are 54,97,110,106,and 120 Š
for 1, 3, 4, 5,and 6
respectively.[21] The order of the substrate size roughly
correlates to the experimental data for the hydrogenation
by Pd·apo-ferritin (Table 1). Thus,the substrate size is
discriminated by the threefold channels. While we have
attempted chiral discrimination in the hydrogenation of
racemic 6 at 158C,no enantioselectivity in the product was
observed.
In conclusion,we have demonstrated in situ synthesis of a
Pd nanocluster in the protein cage and size-selective olefin
hydrogenation by the Pd·apo-ferritin. The results provide us
with a method for the preparation of metal nanoclusters in
restricted interiors of a protein assembly. Further design of
the ferritin cage and channels to improve the reactivity is
currently under progress.
(TOF = [product (mol)] per atom of Pd per hour) of hydro-
genation for acrylamide derivatives are listed in Table 1. As
estimated by atomic-absorption and protein quantitative
Table 1: Hydrogenation activity of Pd·apo-ferritin in water.
Olefins
TOF for
TOF for Pd
particles[b],[c]
Pd·apo-ferritin[a],[b]
=
CH2 CHCONH2 (1)
72Æ0.7
58Æ5.9
12Æ2.6
15Æ0.3
6.1Æ0.6
28Æ2.6
23Æ0.3
=
CH2 CHCOOH (2)
6.3Æ1.1
51Æ6.5
=
CH2 CHCONH-iPr (3)
=
CH2 CHCONH-tBu (4)
31Æ5.9
=
CH2 CHCO-Gly-OMe (5)
6.3Æ3.8
Not detected
=
CH2 CHCO-d,l-Ala-OMe (6)
[a] Hydrogenation reactions catalyzed by Pd·apo-ferritin were carried out
at 78C (pH 7.5) with 30 mm of Pd. [b] TOFs were calculated on the basis
of the ratio of the product to substrate by analyzing the 1H NMR spectra.
[c] The same conditions were used except without apo-ferritin.
Experimental Section
Horse spleen ferritin was obtained as an 85 mgmLÀ1 0.15m NaCl
solution (Sigma). Substrates 4 and 5 were synthesized as reported.[22]
Preparation of Pd·apo-ferritin: Horse spleen ferritin was demin-
eralized with thioglycolic acid to prepare apo-ferritin by a reported
procedure.[23] The protein concentration was determined by using the
reported extinction coefficient at 280 nm.[24] An apo-ferritin solution
(9.3 mg,0.02 mmol in 0.15m NaCl/H2O) was adjusted to pH 8.5 with
0.01n NaOH,and aliquots of K 2PdCl4 (40 mm, 250 mL in H2O) were
added and this solution was stirred for 30 min at room temperature.
To the resulting solution,a solution of NaBH 4 (50 mmol in H2O) was
added and the mixture was stirred for 30 min at room temperature.
The reaction mixture was dialyzed against 0.15m NaCl/H2O for 12 h.
The protein was isolated by gel chromatography (Superdex 200,
eluent: 0.15m NaCl aq) and the purity was checked by native PAGE.
The proportion of Pd atoms to protein was determined by atomic
absorption analysis and Bicichonianate (BCA) method.
analyses each apo-ferritin contains 460 atoms of Pd0,thus,
the TOF of 72 for 1 implies a TOF of 33000 per Pd·apo-
ferritin molecule. The products of these hydrogenation
reactions were confirmed by NMR spectroscopy in D2O
solutions. According to the reduction of each substrate by H2
in the presence of Pd·apo-ferritin,the signals in the region of
vinylic protons (5–6 ppm) decreased and concomitant appear-
ance of new signals assignable to the hydrogenation of the
À
C C double bond in the aliphatic region (1–2 ppm) were
observed. These results indicate that the hydrogenation by
Pd·apo-ferritin proceeds without forming any other products.
After the catalytic reactions,the solution of Pd·apo-ferritin
remained clear and the native PAGE results showed no
Hydrogenation reactions: Hydrogenation reactions were carried
out in a 10 mL reaction tube sealed with a septam. A D2O solution
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containing the substrate (6 mmol,1 mL) was added to the reaction
Chemicaland BiochemicalPerspectives (Eds.: S. Mann,J. Webb,
R. J. P. Williams),Wiley-VCH,Weinheim, 1989,p. 257 – 294.
[20] To prepare TbIII blocked Pd·apo-ferritin,Pd·apo-ferritin
(0.5 mm) was incubated with TbCl3 (20 mm) for 24 h at 48C
and then unbound TbIII ions were removed by ultrafiltration
(microcon YM-10).
[21] Volumes of substrates were calculated by the Discovery Studio
program,ver 1.0 (Accelrys).
[22] M. P. Bueno,C. A. Cativiela,J. A. Mayoral, J. Org. Chem. 1991,
56,6551 – 6555.
tube and purged with H2 gas for 15 min. The catalytic reaction was
initiated by adding a Pd·apo-ferritin solution (1 ” 10À4 mmol,1 mL)
and stirred for 1 h under an H2 atmosphere with a flow rate of
50 mLminÀ1 at 78C. After the removal of Pd·apo-ferritin from the
resulting solution,the products were identified by 1H NMR spectros-
copy. Pd particles for the blank experiments (Table 1) were prepared
by the reaction of K2PdCl4 and NaBH4 in D2O. The mixtures were
used for the hydrogenation reactions without further purification. The
enantiomeric excess for the reaction of 6 at 158C was determined by
HPLC analysis by using a DAICEL CHIRALCEL OD column
[23] K. K. W. Wong,T. Douglas,S. Gider,D. D. Awschalom,S. Mann,
Chem. Mater. 1998, 10,279 – 285.
(0.46 cm ” 25 cm) at
a
flow rate of 0.5 mLminÀ1 (isopropanol/
hexane = 5:95).
[24] N. D. Chasteen,E. C. Theil, J. Biol. Chem. 1982, 257,7672 – 7677.
Transmission electron microscopy: A solution of Pd·apo-ferritin
(2 mL) was applied to a copper grid covered with a thin amorphous
carbon film,and the excess solution was removed with a filter paper.
For the cryo observation,a sample grid was rapidly plunged into
liquid propane (À1908C). The images of the ice-embedded and the
negatively stained samples were recorded on a JEM-2010HC micro-
scope (200 kV) equipped with a CT-3500 cryoholder and a JEM-
4010N microscope (300 kV),respectively.
Received: December 1,2003
Revised: February 23,2004 [Z53436]
Keywords: hydrogenation · nanoparticles · palladium · proteins ·
.
supramolecular chemistry
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