Synthesis and self-assembly of organoclay-wrapped biomolecules

Article abstract of DOI:10.1002/anie.200453868

Gift wrapped: Biomolecules can be enveloped within an ultrathin shell of an aminopropyl-functionalized magnesium (organo)phyllosilicate to produce aqueous dispersions of discrete protein-inorganic nanoparticles. Similar procedures but with organoclay oligomers that have pendent long-chain hydrophobic moieties result in self-assembly of the protein-inorganic nanoparticles into higher-order superstructures (see picture).

Full text of DOI:10.1002/anie.200453868

Communications
applications. Although there are numerous reports on the
enhanced thermal and chemical stability of proteins by
immobilization on surfaces[4–8] or within matrices such as
amorphous gels[9–11] and layered solids,[12,13] the wrapping of
individual protein/enzyme molecules with inorganic materials
to produce functionally isolated hybrid nanoparticles has not
been reported. Herein we describe investigations that
strongly suggest that individual molecules of met-myoglobin
(Mb),haemoglobin (Hb) or glucose oxidase (GOx) can be
wrapped with an ultrathin shell of an aminopropyl-function-
alized magnesium (organo)phyllosilicate to produce aqueous
dispersions of discrete protein–inorganic nanoparticles. Sim-
ilar procedures but with organoclay oligomers that have
pendent long-chain hydrophobic moieties result in self-
assembly of the protein–inorganic nanoparticles into higher-
order superstructures. In each case,the encapsulated proteins
are structurally and functionally intact and show enhanced
thermal stability up to temperatures of 858C.
In general,“armour-plated” protein/enzyme molecules
were prepared by mixing solutions of Mb,Hb,or GOx with
aqueous solutions containing oligomers of a positively
charged exfoliated organoclay (Figure 1). The organoclay
was prepared by chemical synthesis[14,15] (see Experimental
Section) and consisted of a highly disordered talclike 2:1
trioctahedral smectite structure with a central brucite sheet of
octahedrally coordinated MgO/OH chains overlaid on both
sides with an aminopropyl-functionalized silicate network to
give an approximate unit cell composition of
[H2N(CH2)3]8Si8Mg6O16(OH)4. Protonation of the amino
groups by dispersion of the clay in water resulted in
exfoliation and partial disintegration of the organoclay
layers into cationic oligomers that were fractionated by gel
chromatography to produce stable transparent sols that were
subsequently added to protein/enzyme solutions.
Protein-Inorganic Conjugates
Synthesis and Self-Assembly of Organoclay-
Wrapped Biomolecules**
For each protein/enzyme investigated,TEM studies
showed the presence of discrete electron-dense nanoparticles
randomly arranged across the support film of the grid
(Figure 2a–c). EDX analysis for samples prepared in the
presence of Mb or Hb,confirmed that the nanoparticles
comprised both protein (Fe,S) and organoclay (Si,Mg,Cl)
species (Figure 2d). In general,the nanoparticles were
spheroidal and monodisperse in size with mean dimensions
of 4.0 nm (s = 0.6 nm),7.8 nm ( s = 0.8) and 6.4 (s = 0.9 nm)
for Mb,Hb and GOx samples,respectively. The variation in
nanoparticle size showed a direct correlation with the
respective molecular dimensions of the different proteins/
enzyme (Mb,4.5 ” 3.5 ” 2.5 nm; Hb,6.5 ” 5.4 ” 5.3 nm; GOx,
6.0 ” 5.2 ” 3.7 nm),which suggests that each nanoparticle
consisted of a single biomolecule wrapped by a continuous
sheet of condensed organoclay oligomers. Significantly,no
organoclay nanoparticles were observed in the absence of the
proteins or enzyme,suggesting that condensation of the
magnesium (aminopropyl)phyllosilicate oligomers was spe-
cifically promoted by interactions with the biomolecule
surface.
Avinash J. Patil, Eswaramoorthy Muthusamy, and
Stephen Mann*
Protein-based nanostructures are expected to play a key role
in the development of multifunctional materials and devices
for bio-nanotechnological applications.[1–3] Although proteins
excel in functional specificity,their structural and chemical
sensitivity to ambient conditions can seriously compromise
the use and integration of such macromolecules in diverse
[*] A. J. Patil, Dr. E. Muthusamy, Prof. S. Mann
School of Chemistry
University of Bristol
Bristol BS81TS (UK)
Fax: (+44)117-929-0509
E-mail: s.mann@bristol.ac.uk
[**] We thank Dr. T. Ikoma (National Institute for Materials Science,
Biomaterials Center, Tsukuba, Japan), Dr. D. Scott, Mrs G. Shaw and
Dr. A. M. Seddon (University of Bristol) for help with CD spectros-
copy, AUC, mass spectroscopy, and enzyme kinetics, respectively.
The EPSRC (UK), Max Planck Society, and University of Bristol (ORS
award to A.J.P.) are acknowledged for their financial support.
Analytical ultracentrifugation of the Mb–organoclay
nanoparticles showed a single peak with a sedimentation
coefficient (s*) value of 1.5 S,thus indicating a narrow
distribution in size and shape of the hybrid structures. The
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
4928
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200453868
Angew. Chem. Int. Ed. 2004, 43, 4928 –4933

Angewandte
Chemie
of carbon monoxide under argon to the
dexoy–Mb(Hb)–organoclay conjugate
resulted in a shift in the soret band from
433 nm to 422 nm,which was further shifted
after exposure to dioxygen to a value of
416 nm (Figure 3c and Supporting Infor-
mation),consistent with the formation of
oxy–Mb or oxy–Hb.[10] Characteristic
changes in the a and b absorption bands
at around 580 and 545 nm were also
observed. The enzymatic activities of
native GOx and GOx–organoclay nano-
particles were monitored spectrophotomet-
rically at 414 nm to determine the initial
rates (V) for a range of substrate (S)
concentrations (see Experimental Section).
In both cases,plots of 1/ V against 1/[S]
showed characteristic linear plots consistent
with Michaelis–Menton kinetics (Support-
ing Information). The corresponding values
for Vmax were similar (free GOx,2.7 mmol-
minꢀ1; conjugated GOx,2.95 mmolminꢀ1),
thus indicating that the turnover rate at
substrate saturation was not significantly
affected by interactions with the organo-
clay. In contrast,the Km constant associated
with the activity of the GOx–organoclay
Figure 1. Scheme showing proposed molecular wrapping of Mb molecules by cationic orga-
noclay oligomers with an about 1.6-nm-thick layer structure. Tessellation of the entire pro-
tein by binding of the organoclay sheet surface (ab face, a=0.53 nm, b=0.91 nm, c=908)
requires approximately 30 unit cells. Wrapping with aminopropyl-functionalized organoclay
oligomers produces dispersed protein–organoclay nanoparticles, whereas hydrophobic inter-
actions between the exposed C16 chains of the aminopropyl/hexadecyl-functionalized oligo-
mers result in nanoparticle self-assembly into partially ordered superstructures.
sedimentation coefficient was reduced compared with native
Mb (s* = 2.5 S),which suggests a larger friction coefficient
(viscous drag) due to an increase in surface roughness. This
was consistent with the proposed core–shell structural model
for the organoclay–protein nanoparticles. MALDI-TOF mass
spectrometry showed primary peaks for intact Mb (16.9 k) as
well as a distribution of organoclay oligomers principally in
the mass/charge range of 400 to 700. Studies by using circular
dichroism (CD) and FTIR spectroscopies indicated that the
secondary structures of Mb,Hb,or GOx molecules were
preserved within the organoclay–protein conjugates. For
example,CD spectra of solutions of native met-Mb and
Mb–organoclay nanoparticles showed characteristic bands[10]
for the p–p* amide transitions at 192 nm and 209 nm,as well
as an a-helical n–p* amide transition at 220 nm (Figure 3a).
Similarly,FTIR spectra of the nanoparticles obtained by
solvent evaporation showed no changes in the protein amide I
nanoparticles was higher (500 mm) compared with free
enzyme (370 mm),which suggests that glucose binding was
partially inhibited at intermediate substrate concentrations
possibly by restricted diffusion through the organoclay shell
of the hybrid conjugate. Measurements of relative enzyme
activities at different pH values showed a nonlinear depend-
ence with a small (5–10%) enhancement for the GOx
nanoparticles in acid or alkaline conditions compared with
the free enzyme in solution (Figure 3d).
The thermal stabilities of organoclay-conjugated met-Mb
or Hb molecules were assessed by temperature-dependent
UV/Vis spectroscopy measurements of the intensity of the
soret band.[16] Compared with room-temperature values,
native Mb and Hb showed,respectively,a 49% or 25%
reduction in the intensity of the band at 408 nm after 5 min at
858C,which is consistent with significant unfolding of the
polypeptide chains.[13] In contrast,a decrease of only 10% or
5% in the intensity of the soret band were observed over this
temperature range for the met-Mb– or Hb–organoclay nano-
particles,respectively,(Figure 3e),thus indicating a marked
increase in thermal stability of the encapsulated protein
molecules. Similarly,an increased retention in the activity of
GOx–organoclay nanoparticles compared with the native
enzyme in solution was observed at elevated temperatures
(Figure 3 f). Although in both cases,the relative activities
were compromised at temperatures above 608C,the relative
activity of the GOx nanoparticles was at least 10% higher
even at 858C.
=
ꢀ
ꢀ
(C O str) and amide II (N H def,C N str) bands at
1653 cmꢀ1 and at 1554 cmꢀ1,respectively. The spectra also
showed absorption bands for CH2 (2800 cmꢀ1),Si
C
ꢀ
ꢀ1
ꢀ1
ꢀ ꢀ
ꢀ
ꢀ
ꢀ ꢀ
O
(1150 cm ),Si O Si (1025 cm ),and Mg O,Si O,Si
Mg (564–482 cmꢀ1) vibrations,confirming that condensed
magnesium (aminopropyl)phyllosilicate moieties were asso-
ciated with the protein/enzyme molecules.
The functional integrity and accessibility of the nano-
particle conjugates to small molecules and ions were assessed
by UV/Vis spectroscopy. Solutions of the Mb– or Hb–
organoclay nanoparticles showed a distinct p!p* soret
band at 408 nm associated with an intact haem prosthetic
group,which showed a characteristic shift to 433 nm on
formation,for example,of deoxy-Mb by dithionite reduction
(Figure 3b and Supporting Information). Reversible binding
The above results indicate that individual protein/enzyme
molecules interact in solution with magnesium (aminopro-
pyl)phyllosilicate oligomers to produce hybrid nanoparticles
with preserved protein structure and function,and enhanced
Angew. Chem. Int. Ed. 2004, 43, 4928 –4933
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4929

Communications
the positively charged organoclay oligomers (+ 12 mV
(pH 8.5). This mechanism was consistent with the observed
absence of hybrid nanoparticles in the presence of cyto-
chrome c (zeta potential = ++ 4 mV at pH 8.5),as well as a
reduction in the zeta potentials for Mb,Hb,and GOx-
containing nanoparticles to values of ꢀ18, ꢀ0.5 and
+ 1.8 mV,respectively. A similar electrostatic model has
recently been proposed to account for the wrapping of
viologen polymer molecules within a sheath of partially
condensed sodium silicate.[17,18]
Finally,self-assembled superstructures of the Mb–organo-
clay nanoparticles were produced by replacing about 50% of
the aminopropyl groups in the clay structure with covalently
attached hydrophobic hexadecyl groups (see Experimental
Section). Although the extent of exfoliation was reduced
compared with the 100% aminopropyl-functionalized clay,
TEM and EDX analysis showed the presence of self-
organized protein–organoclay superstructures that consisted
of close packed layers of discrete 4.5 nm-sized nanoparticles
separated by an interparticle spacing of 4 to 5 nm (Figure 4).
This spacing is commensurate with a bilayer of hexadecyl
chains as shown from XRD studies of the aminopropyl/
hexadecyl magnesium (organo)phyllosilicate clay that
revealed an interlayer d001 spacing of 4.6 nm (data not
shown). The results are consistent with the above structural
model in which individual biomolecules are encapsulated by
an organoclay shell with pendent organic functionalities,and
indicate that macroscopic assemblies of protein–organoclay
nanoparticles can be prepared by using interparticle hydro-
phobic interactions to drive the self-assembly process. Pres-
ervation of protein structure and function in these organized
hybrid materials was confirmed by using the above exper-
imental procedures (see the Supporting Information).
In conclusion,we have demonstrated that novel organo-
clay–protein conjugates can be prepared in the form of
discrete electron-dense nanoparticles. On the basis of our
results,we propose a core–shell structural model in which
individual biomolecules are wrapped by an inorganic shell of
condensed organoclay oligomers. The procedure is facile and
could lead to new types of stabilized biomolecules that can be
functionally isolated as discrete soluble units,or assembled
and integrated into nanostructured materials. In particular,
the use of “armour-plated” proteins should circumvent
problems arising from the loss of functional isolation asso-
ciated with adverse intermolecular interactions within inter-
connected networks of biomolecules. In addition,the
enhanced thermal and chemical stability of inorganically
wrapped proteins should markedly increase the scope for
using protein-based nanostructures in areas such as tissue
engineering and biomolecular sensing.
Figure 2. TEM images showing discrete protein-organoclay nanoparti-
cles. a) Mb, b) Hb, and c) GOx.; scale bars=50, 100, and 100 nm,
respectively. d) corresponding EDX analysis for Mb-organoclay nano-
particles. Peaks for Cu and Cr in the EDX spectrum are from the
sample holder.
thermal stability. Although further work is required to
confirm the proposed structural model and putative wrapping
mechanism,electrostatic interactions appear to be in part
responsible for conjugation as hybrid nanoparticles were only
produced when negatively charged native biomolecules [zeta
potential measurements; Mb, ꢀ25 mV (pH 8.5); Hb, ꢀ26 mV
(pH 8.5); GOx, ꢀ20 mV (pH 6)] were used in the presence of
Experimental Section
Organoclay synthesis and exfoliation: Typically,an aminopropyl-
functionalized magnesium (organo)phyllosilicate clay was prepared
at room temperature by dropwise addition of 3-aminopropyltriethoxy-
silane (1.3 mL,5.85 mmol) to an ethanolic solution of magnesium
chloride (0.84 g,3.62 mmol) in ethanol (20 g). The white slurry
obtained after 5 min was stirred overnight and the precipitate isolated
4930
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 4928 –4933

Angewandte
Chemie
Figure 3. a) CD spectra of solutions of native Mb and Mb–organoclay nanoparticles that show p–p* amide a-helical n–p* amide transitions corre-
sponding to intact secondary structures. b) UV/Vis spectra of met-Mb–organoclay conjugates that show soret band absorptions for as-prepared
nanoparticles and after dithioinite reduction and formation of deoxy-Mb. c) UV/Vis spectra of Mb–organoclay nanoparticles after CO binding to
&
the deoxy-protein and subsequent O2 binding and formation of oxy-Mb. d) Plot of relative activity (RA, %) against pH for native GOx ( ) and
*
*
GOx–organoclay nanoparticles ( ). e) Plots of absorbance intensity at 408nm ( A408) with temperature for solutions of native Hb ( ) and Hb–
&
organoclay nanoparticles ( ), showing increased thermal stability of the wrapped protein molecules. Samples were allowed to stand for 5 min at
&
each temperature prior to analysis. f) Plot of relative activity (%) against temperature for native GOx ( ) and GOx–organoclay nanoparticles.
by centrifugation,washed with ethanol (50 mL) and dried at 40 8C.
Exfoliation of the clay was undertaken by dispersing 10 mg of the
dried clay in distilled water (10 mL) followed by ultrasonication for
5 mins. The resulting cloudy dispersion was passed through a
sephadex G-25/75 column (Aldrich),and the clear eluate collected
and used for experiments with a range of biomolecules.
Organoclays consisting of covalently linked aminopropyl and
long-chain hexadecyl groups were prepared by addition with constant
Angew. Chem. Int. Ed. 2004, 43, 4928 –4933
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4931

Communications
native GOx oxidase or GOx–organoclay nanoparticles (100 mL,
[GOx] = 10 mm] to sodium phosphate buffered solutions (1.6 mL,
0.1m) containing 100 mL of ABTS (50 mm)/HRP (2.6 mm,
25 unitsmLꢀ1) and b-d-Glucose (concentration range,3.5 mm to
50 mm). Changes in A414 with time were converted to units of
enzyme specific activity (mmolminꢀ1 mgꢀ1) by using an extinction
coefficient of 3.6 ” 104 mꢀ1 cmꢀ1. Double reciprocal plots of 1/V (V=
initial rate) against 1/[glucose] showed linear behavior consistent with
Michaelis–Menton kinetics,and the Michaelis constant ( KM) and
maximal rate (Vm) were determined directly from the plots. The
above procedure was also carried out with native and organoclay-
wrapped GOx under a range of pH values (3 to 10) and temperatures
(25 and 858C). In each case,the specific activities of free GOx or
GOx-organoclay nanoparticles were converted into percentage
relative activities based on corresponding values obtained at pH 6
and 258C.
The thermal stabilities of native Mb/Hb and Mb/Hb–organoclay
nanoparticles were determined by temperature-dependent UV/Vis
spectroscopy (Perkin Elmer Lambda II) by using an attached Peltier
temperature-control system and monitoring changes in the absorb-
ance intensity of the 408 nm soret band of samples allowed to stand
for 5 mins at temperatures between 35 and 858C. Temperature-
dependent enzyme activities of free GOx and GOx–organoclay
conjugates were also determined by using the Peltier control system.
Samples were incubated for 5 mins at temperatures between 25 to
858C before measuring changes in absorbance changes at 414 nm.
Samples were characterized by TEM (JEOL 1200EX),EDX
analysis (Oxford Instruments,ISIS300),and XRD (Bruker-Nonius
Figure 4. a) TEM image showing self-organized layers of Mb molecules
wrapped by magnesium (aminopropyl/hexadecyl)phyllosilicate oligo-
mers. The lamellar structure is viewed side-on such that the interlayer
spacing of the superstructure is clearly revealed (arrow); scale
bar=100 nm. b) Corresponding image of a single layer viewed from
above showing the presence of superstructural ordering of the pro-
tein–organoclay hydrophobic nanoparticles; scale bar=50 nm.
stirring of a 1:1 molar ratio mixture of hexadecyltrimethoxysilane
(1.80 mmol) and 3-aminopropyltriethoxysilane (1.80 mmol) in etha-
nol (10 g) to magnesium chloride (0.25 g,1.08 mmol) dissolved in
ethanol (10 g),followed by addition of aqueous sodium hydroxide
(0.5m,20 mL). The reaction mixture was stirred for 24 h at room
temperature,after which a finely divided white precipitate was
collected by vacuum filtration and washed repeatedly with water and
ethanol then dried at 408C in air for 24 h. Exfoliation of the
bifunctional clay was undertaken by dispersion of 10 mg of a finely
ground sample in a water/ethanol (15 mL/5 mL) mixture by ultra-
sonication for 2 min. (Exfoliation was not successful in pure water due
to the highly hydrophobic character of the organoclay particles).
Protein-organoclay nanoparticles: A purified met-Mb aqueous
solution (1 mL,12.4 mm,horse skeletal muscle (sigma), Mr = 16.9k,
sephadex G-25/75) was added dropwise to the amino-functionalized
organoclay eluate (2 mL) to give a clear reddish-brown solution of
pH ꢁ 8.5 of the clay-wrapped protein molecules,which became
slightly turbid when left at room temperature for up to 24 h. Similar
procedures were also undertaken with purified Hb (1 mL,47 mm,
bovine (sigma), Mr = 64k,sephadex G-25/75) and GOx (1 mL,10 mm,
Aspergillus niger,type X-S,EC 1.1.3.4, Mr = 140k,pH 6) aqueous
solutions.
D8 diffractometer,Cu
radiation, l = 0.15405 nm). FTIR spectros-
Ka
copy (Perkin Elmer Spectrum 1) was carried out by using KBr discs.
CD spectra were measured at 298 K by using a JASCO model 725
spectrometer with a quartz cell and path length of 10 mm at a scan
speed of 50 nmminꢀ1. Zeta potential measurements were undertaken
by using a Zetaplus analyser. Analytical ultracentrifugation was
carried out by using a Beckman Analytical Centrifuge at 40000 rpm.
MALDI-TOF mass spectrometry (PE Biosystems Voyager-DE STR)
was undertaken using a nitrogen laser operating at 337 nm. Samples
and matrix (0.5 mL each) were spotted onto a sample plate and
calibrated against “Calmix 3” (PE,Biosystems) as external standard.
The matrix solution was freshly prepared sinapinic acid (Fluka) at a
concentration of 1 mg/100mL in a 50:50 mixture of acetonitrile/0.1%
TFA.
Received: January 27,2004
Revised: April 26,2004 [Z53868]
Alternatively,an Mb aqueous solution (1 mL,10 mgmL ꢀ1) was
added to the exfoliated dispersion of an aminopropyl/hexadecyl-
functionalized organoclay and stirred at room temperature for 5 days
at pH ꢁ 8.5. The precipitate obtained was centrifuged,washed with
water and dried at room temperature for two days.
Keywords: biological activity · nanoparticles · organic-inorganic
hybrid composites · self-assembly
.
Reduction of met-Mb (or met-Hb) to the deoxy form was
undertaken by treating native Mb (10 mL,0.10 mgmL ꢀ1) or Mb-
organoclay nanoparticles with sodium dithionite (1 mg) under an
argon atmosphere. Binding of carbon monoxide to dexoxy-Mb or
deoxy-Mb–organoclay conjugate solutions prepared by dithionite
reduction was undertaken by purging CO gas through the sample
solutions for one hour at room temperature. Subsequent exchange of
bound CO by dioxygen was undertaken by passing solutions of the
carbonyl-Mb through a sephradex gel column in air.
[1] T. O. Yeates,J. E. Padilla, Curr. Opin. Struct. Biol. 2002, 12,464 –
470.
[2] T. C. Holme, Trends Biotechnol. 2002, 20,16 – 24.
[3] J. D. Hartgerink,E. Beniash,S. I. Stupp, Proc. Natl. Acad. Sci.
USA 2002, 99,5133 – 5138.
[4] M. Jiang,B. Nolting,P. S. Stayton,S. G. Sligar, Langmuir 1996,
12,1278 – 1283.
[5] Y. Zhou,N. Hu,Y. Zeng,J. F. Rusling, Langmuir 2002, 18,211 –
219.
The rates of oxidation of b-d-glucose to d-gluconolactone and
H2O2 by native and organoclay-wrapped GOx were determined
spectrophotometrically.[19] Decomposition of H2O2 in the presence of
horse radish peroxidase (HRP,type II,sigma) and an electron donar
dye,2, ’-azino-bis(3-ethylbenzethiazoline-6-sulfonic acid (ABTS,
[6] V. Panchagnula,C. V. Kumar,J. F. Rusling, J. Am. Chem. Soc.
2002, 124,12515 – 12521.
[7] M. Onda,A. Katsuhiko,T. Kunitake, J. Biosci. Bioeng. 1999, 87,
69 – 75.
[8] F. Caruso,C. Schuler, Langmuir 2000, 16 9595 – 9603.
[9] I. Gill,A. Ballesteros, Trends Biotechnol. 2000, 18,282 – 296.
[10] L. M. Ellerby,C. R. Nishida,F. Nishida,A. Yamanaka,B. Dunn,
J. S. Valentine,J. Zink, Science 1992, 255,1113 – 1115.
sigma) was monitored by increases in absorption at 414 nm (A414
)
associated with formation of oxidized ABTS. Typically,reactions
were carried out at 258C in 3 mL glass cuvettes at pH 6 by addition of
4932
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 4928 –4933

Angewandte
Chemie
[11] Q. Gao,S. L. Suib,J. F. Rusling, Chem. Commun. 2002,2254 –
2254.
[12] C. V. Kumar,A. Chaudhari, J. Am. Chem. Soc. 2000, 122,830 –
837.
[13] C. V. Kumar,A. Chaudhari, Chem. Commun. 2002,2382 – 2383.
[14] S. L. Burkett,A. Press,S. Mann, Chem. Mater. 1997, 9,1071 –
1073.
[15] N. T. Whilton,S. L. Burkett,S. Mann, J. Mater. Chem. 1998, 8,
1927 – 1932.
[16] G. Acampora,J. Hermans, J. Am. Chem. Soc. 1967, 89,1543 –
1547.
[17] I. Ichinose,T. Kunitake, Chem. Rec. 2002, 2,339 – 351.
[18] I. Ichinose,T. Kunitake, Adv. Mater. 2002, 14,344 – 346.
[19] R. C. Bateman,J. A. Evans, J. Chem. Educ. 1995, 72(12),A240 –
A241.
Angew. Chem. Int. Ed. 2004, 43, 4928 –4933
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4933