Tailored synthetic polyamines for controlled biomimetic silica formation

Article abstract of DOI:10.1021/ja9061163

Organic compounds isolated from diatoms contain long-chain polyamines with a propylamine backbone and variable methylation levels and chain lengths. These long-chain polyamines are thought to be one of the important classes of molecules that are responsible for the formation of the hierarchically structured silica-based cell walls of diatoms. Here we describe a synthetic route based on solid-phase peptide synthesis from which well-defined long-chain polyamines with different chain lengths, methylation patterns, and subunits can be obtained. Quantitative silica precipitation experiments together with structural information about the precipitated silica structures gained by scanning and transmission electron microscopy revealed a distinct dependence of the amount, size, and form of the silica precipitates on the molecular structure of the polyamine. Moreover, the influence of the phosphate concentration was elucidated, revealing the importance of divalent anions for the precipitation procedure. We were able to derive further insights into the precipitation properties of long-chain polyamines as functions of their hydrophobicity, protonation state, and phosphate concentration, which may pave the way for better control of the formation of nanostructured silica under ambient conditions.

Full text of DOI:10.1021/ja9061163

Published on Web 12/30/2009
Tailored Synthetic Polyamines for Controlled Biomimetic
Silica Formation
Anja Bernecker,† Ralph Wieneke,‡ Radostan Riedel,‡ Michael Seibt,§
Armin Geyer,*,‡ and Claudia Steinem*,†
Institute of Organic and Biomolecular Chemistry, UniVersity of Go¨ttingen, Tammannstr. 2,
37077 Go¨ttingen, Germany, Philipps-UniVersita¨t Marburg, Hans-Meerwein-Strasse, 35032
Marburg, Germany, and IV. Physikalisches Institut, UniVersity of Go¨ttingen,
Friedrich-Hund-Platz 1, 37077 Go¨ttingen, Germany
Received July 22, 2009; E-mail: geyer@staff.uni-marburg.de; claudia.steinem@chemie.uni-goettingen.de
Abstract: Organic compounds isolated from diatoms contain long-chain polyamines with a propylamine
backbone and variable methylation levels and chain lengths. These long-chain polyamines are thought to
be one of the important classes of molecules that are responsible for the formation of the hierarchically
structured silica-based cell walls of diatoms. Here we describe a synthetic route based on solid-phase
peptide synthesis from which well-defined long-chain polyamines with different chain lengths, methylation
patterns, and subunits can be obtained. Quantitative silica precipitation experiments together with structural
information about the precipitated silica structures gained by scanning and transmission electron microscopy
revealed a distinct dependence of the amount, size, and form of the silica precipitates on the molecular
structure of the polyamine. Moreover, the influence of the phosphate concentration was elucidated, revealing
the importance of divalent anions for the precipitation procedure. We were able to derive further insights
into the precipitation properties of long-chain polyamines as functions of their hydrophobicity, protonation
state, and phosphate concentration, which may pave the way for better control of the formation of
nanostructured silica under ambient conditions.
design of their silica-based cell walls.5 Hence, considerable
research has been directed toward identifying the mechanisms
Introduction
Silica is one of the most abundantly used materials in the
world. The chemical synthesis of silica-based materials such
as molecular sieves, resins, and catalysts relies on extreme
conditions of temperature, pressure, and pH. Under these
conditions, control of complex three-dimensional structures on
the nanometer to micrometer scale remains tedious. However,
for example, nanometer-sized mesoporous silica particles have
attracted much interest in nanotechnology in recent years
because they have many attractive features, such as high pore
volume, large surface area, and ease of functionalization.1 The
highly porous materials can be utilized in the storage and
delivery of small molecules and thus can be used in catalysis,
drug delivery, and imaging.
involved in the biosilification process that results in hierarchi-
cally ordered siliceous diatom shells (frustules) in order to
understand this procedure and as a result to control and tailor
silica formation. Using mild conditions for silification might
offer the possibility of protecting sensitive molecules and
creating biochemical sensors, as proteins6 and even cells7 are
not harmed by encapsulation. Two main types of organic
compounds are intimately associated with the diatom shell. One
of these types consists of (poly)peptides with propylamino-
functionalized lysine side chains and phosphorylated serine side
chains, which are called silaffins.8-11 The other major group is
a series of long-chain polyamines12-14 that have also recently
In comparison with these processes, control of the formation
of hierarchically ordered, well-defined three-dimensional silica
structures under mild synthetic conditions is very well realized
in organisms such as sponges, diatoms, and radiolaria.2-4
Diatoms, ubiquitous unicellular photosynthetic organisms in the
marine ecosystem, are very well known for the spectacular
(5) Round, F. E.; Crawford, R. M.; Mann, D. G. The Diatoms; Cambridge
University Press: Cambridge, U.K., 1990.
(6) Dickerson, M. B.; Sandhage, K. H.; Naik, R. R. Chem. ReV. 2008,
108, 4935–4978.
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Angew. Chem., Int. Ed. 2009, 48, 9160–9163.
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1132.
† Institute of Organic and Biomolecular Chemistry, University of
Go¨ttingen.
(9) Kro¨ger, N.; Deutzmann, R.; Sumper, M. J. Biol. Chem. 2001, 276,
26066–26070.
‡ Philipps-Universita¨t Marburg.
§ IV. Physikalisches Institut, University of Go¨ttingen.
(1) Liong, M.; Angelos, S.; Choi, E.; Patel, K.; Stoddart, J. F.; Zink, J. I.
J. Mater. Chem. 2009, 19, 6251–6257.
(10) Poulsen, N.; Sumper, M.; Kro¨ger, N. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 12075–12080.
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Chem., Int. Ed. 2004, 43, 5933–5936.
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Nat. Prod. Rep. 2008, 25, 455–474.
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Acad. Sci. U.S.A. 2000, 97, 14133–14138.
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J. AM. CHEM. SOC. 2010, 132, 1023–1031 1023

A R T I C L E S
Bernecker et al.
been found in marine sponges.15 These polyamines have been
characterized as poly(propyleneimine) chains with up to 20
repeat units, attached to putrescine or 1,3-diaminopropane.12,15,16
The complex mixture of long-chain polyamines that diatoms
synthesize appears to be composed in a species-specific manner,
suggesting its important function in structure formation of the
silica shell. In vitro, these polyamines have been shown to
rapidly precipitate silica spheres with diameters of several
hundreds of nanometers.12,17 Sumper18 proposed a templating
mechanism based on a phase separation model that explains
the hierarchy of hexagonal silica structures producing the highly
symmetric valve patterning. The basic principle is the formation
of polyamine-containing droplets that consecutively segregate
into smaller droplets upon silica precipitation at the surface of
the droplet. Species-specific patterns result from variations in
the polyamine droplet size established during the very first phase
separation process.16 This, in turn, defines the wall-to-wall
distance of the largest hexagonal framework. Control of the
polyamine droplet size during phase separation is the crucial
point of the model. Sumper and Brunner16 have suggested that
divalent anions such as phosphate play a major role in
determining the size of these nanodroplets.
Scheme 1. General Procedure for the Solid-Phase Synthesis of
Long-Chain Polyamines
In previous work, long-chain polyamines synthesized by
polymerization, leading to molecules with distributed chain
lengths,19-21 or short alkylamines such as spermine, spermidine,
and their analogues22-24 as well as tripropylenetetramine and
pentapropylenetetramine25 have been investigated in terms of
their precipitation capability. They induce rapid precipitation
of silica spheres from silicic acid solutions on a short time scale.
However, well-defined long-chain polyamines with more than
seven amino group-containing units have not yet been synthe-
sized and investigated.
Here we provide a general strategy for synthesizing well-
defined long-chain polyamines with a number of nitrogen atoms
similar to that found in polyamines of diatoms. The silica-
precipitating properties of such polyamines were investigated,
with particular emphasis on their molecular structure and the
influence of phosphate anions. From the obtained results, we
expect to gain new insights into in vitro silica formation that
may pave the way for control of nanostructured silica.
precipitation capabilities. In this work, we have developed a
strategy based on solid-phase peptide synthesis (SPPS) that
enables the generation of well-defined long-chain polyamines
with more than seven amino group-containing units. The strategy
is outlined in Scheme 1. SPPS with temporary Fmoc protection
on a trityl resin yielded the resin-bound oligoamide precursors.
Reduction of oligoamides with borane at elevated temperatures
is the most popular method for the synthesis of secondary
amines in solution,26-28 and Hall and co-workers29-31 as well
as Houghten and co-workers32-35 have described reductions on
solid supports. A disadvantage of the method, however, is the
formation of relatively stable aminoborane complexes under the
reaction conditions, which require special workup.31,36 Most of
the published protocols are not compatible with the trityl resin.35
Thus, an adaption of basic conditions for the release of the
Results
Synthetic Strategy. To date, only long-chain polyamines
synthesized by polymerization, leading to molecules of distrib-
uted chain lengths,19–21 have been investigated in terms of their
(14) Sumper, M.; Brunner, E.; Lehmann, G. FEBS Lett. 2005, 579, 3765–
3769.
(15) Matsunaga, S.; Sakai, R.; Jimbo, M.; Kamiya, H. ChemBioChem 2007,
8, 1729–1735.
(26) Brown, H. C.; Heim, P. J. Org. Chem. 1973, 38, 912–916.
(27) Brown, H. C.; Narasimhan, S.; Choi, Y. M. Synthesis 1981, 441–
442.
(16) Sumper, M.; Brunner, E. AdV. Funct. Mater. 2006, 16, 17–26.
(17) Sumper, M.; Lorenz, S.; Brunner, E. Angew. Chem., Int. Ed. 2003,
42, 5192–5195.
(28) Roeske, R. W.; Weitl, F. L.; Prasad, K. U.; Thompson, R. M. J. Org.
Chem. 1976, 41, 1260–1261.
(18) Sumper, M. Science 2002, 295, 2430–2433.
(19) Brunner, E.; Lutz, K.; Sumper, M. Phys. Chem. Chem. Phys. 2004, 6,
854–857.
(29) Hall, D. G.; Laplante, C.; Manku, S.; Nagendran, J. J. Org. Chem.
1999, 64, 698–699.
(20) Patwardhan, S. V.; Mukherjee, N.; Steinitz-Kannan, M.; Clarson, S. J.
Chem. Commun. 2003, 1122–1123.
(30) Wang, F.; Manku, S.; Hall, D. G. Org. Lett. 2000, 2, 1581–1583.
(31) Manku, S.; Laplante, C.; Kopac, D.; Chan, T.; Hall, D. G. J. Org.
Chem. 2001, 66, 874–885.
(21) Behrens, P.; Jahns, M.; Menzel, H. In Handbook of Biomineralization;
Behrens, P., Ba¨uerlein, E., Eds.; Wiley-VCH: Weinheim, Germany,
2007; Vol. 2, pp 3-18.
(32) Nefzi, A.; Giulianotti, M. A.; Ong, N. A.; Houghten, R. A. Org. Lett.
2000, 2, 3349–3350.
(22) Belton, D. J.; Patwardhan, S. V.; Perry, C. C. J. Mater. Chem. 2005,
15, 4629–4638.
(33) Nefzi, A.; Ostresh, J. M.; Giulianotti, M.; Houghten, R. A. J. Comb.
Chem. 1999, 1, 195–198.
(23) Belton, D. J.; Patwardhan, S. V.; Annenkov, V. V.; Danilovtseva, E. N.;
Perry, C. C. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5963–5968.
(24) Annenkov, V. V.; Patwardhan, S. V.; Belton, D.; Danilovtseva, E. N.;
Perry, C. C. Chem. Commun. 2006, 1521–1523.
(25) Noll, F.; Sumper, M.; Hampp, N. Nano Lett. 2002, 2, 91–95.
(34) Nefzi, A.; Ostresh, J. M.; Houghten, R. A. Tetrahedron 1999, 55, 335–
344.
(35) Nefzi, A.; Ostresh, J. M.; Meyer, J.-P.; Houghten, R. A. Tetrahedron
Lett. 1997, 38, 931–934.
(36) Lane, C. F. Aldrichimica Acta 1973, 6, 51–58.
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A R T I C L E S
Table 1. Molecular Structures of the Polyamines under Investigation and Corresponding Mass Spectrometric Analysis Data and Yields
a Obtained using matrix-assisted laser desorption ionization (MALDI) mass spectrometry. b Obtained using electrospray ionization (ESI) mass
spectrometry.
Scheme 2. Solid-Phase Synthesis of C3N12Me
secondary amines was needed in our case.37,38 The best results
were obtained after repeated washing with piperidine, as
described in the experimental section in the Supporting Informa-
tion.
The synthesis of C3N12Me (Table 1) required a methylation
step before the oligoamide synthesis, as outlined in Scheme 2.
Aliquots of the polyamide precursors were cleaved from the
resin during SPPS and analyzed by mass spectrometry and
spectroscopic methods. All of the analytical data were in full
agreement with the supposed structures. The compounds
together with their yields and mass spectrometric characteriza-
tion data are summarized in Table 1.
Variation of the N-to-N Distance in Polyamines. The major
components of the polyamines found in diatoms are composed
of propyleneimine units. We wondered whether the distance
between two adjacent amino groups, the catalytically active sites
of silica formation, is a decisive parameter for SiO2 precipitation.
Thus, we performed precipitation experiments in the presence
of three different synthetic polyamines, termed C2N7, C3N7,
and C4N7 (Table 1). All three of these polyamines contain the
same number of nitrogen atoms but vary in the number of C
atoms between the amino groups. The amount of precipitated
silica, as determined by the ꢀ-silicomolybdate method,39 varied
considerably with the N-to-N distance. While for C2N7 only a
very small amount of precipitation was found, with a silicon
mass (mSi) of 1.4 ( 0.6 µg (n ) 7), mSi increased significantly,
even longer spacer of four C atoms, C4N7, only doubled the
by almost a factor of 8, to 10.6 ( 3.7 µg (n ) 8) for C3N7. An
mass in comparison with C3N7, affording mSi ) 21.6 ( 2.2 µg
(n ) 8). The fact that C2N7 precipitated only a very small
(37) Baldwin, R.; Washburn, R. J. Org. Chem. 1961, 26, 3549–3550.
amount of SiO2 is also reflected in the scanning electron
(38) Young, D. E.; McAchran, G. E.; Shore, S. G. J. Am. Chem. Soc. 1966,
microscopy (SEM) images showing sparse aggregates of small
silica spheres with an average diameter of dC2N7 ) 38 ( 15 nm
88, 4390–4396.
(39) Iler, R. K. The Chemistry of Silica; Wiley: New York, 1979.
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Figure 1. (top) SEM images and (bottom) histogram analyses of the silica structures of the precipitates formed in the presence of (a) C2N7, (b) C3N7, (c)
C4N7, (d) C3N6, (e) C3N12, and (f) C3N18. All of the precipitates were obtained in 30 mM phosphate buffer (pH 6.8) with cN ) 1 mM and cSi(OH) ) 100
4
mM. The reaction time was 10 min. Scale bars are 1 µm.
(Figure 1a). Spheres were also found for C3N7- and C4N7-
induced precipitates (Figure 1b,c). However, in case of C3N7,
two different populations of silica spheres could be distin-
guished. A small fraction of the spheres had the same diameter
(d1-C3N7 ) 36 ( 8 nm) as those formed in the presence of C2N7,
while the majority of the spheres were significantly larger (d2-
C3N7 ) 122 ( 34 nm). Even larger were the silica spheres
precipitated in the presence of the polyamine C4N7, with dC4N7
) 172 ( 35 nm. From these results, it becomes obvious that
the distance between the amino groups significantly alters not
only the amount of precipitated silica but also the size of the
silica spheres.
Chain Lengths of the Polyamines. In all diatoms, polyamines
with rather long chains of 7-18 amino groups are found. Here
we investigated how the chain length of the polyamine influ-
ences the amount and structure of the silica precipitate. Three
different polyamines were investigated. In accordance with the
propyl spacer found in natural polyamines and the results
obtained in the previous section, all of the compounds were
composed of propyleneimine units, the shortest having four
(C3N6), the intermediate 10 (C3N12), and the longest 16 units
(C3N18) (Table 1). As the amino groups are the catalytically
active sites for precipitation, the number of nitrogen atoms was
chosen to be identical in each sample (cN ) 1 mM). The amount
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A R T I C L E S
Figure 2. (a-c) Characteristic TEM bright-field micrographs of a silica precipitate induced by C3N6. (d) STEM annular dark-field micrograph of a silica
precipitate. (e, f) EDX element maps showing overlays of (e) the silicon (red) and carbon (green) signals and (f) the silicon (red) and oxygen (blue) signals.
The precipitates were obtained in 30 mM phosphate buffer (pH 6.8) with cN ) 1 mM and cSi(OH) ) 100 mM. The reaction was stopped after 10 min. Scale
4
bars are 200 nm.
of precipitated silica was found to be a linear function of the
number of propyleneimine units. While for C3N6 an amount
of 9.0 ( 2.6 µg (n ) 32) of silicon was found, C3N12
precipitated mSi ) 14.0 ( 1.5 µg (n ) 20). For C3N18, mSi
was determined to be 22.0 ( 1.7 µg (n ) 32). For C3N6, SEM
images demonstrated that silica spheres with a rather broad size
distribution (d2-C3N6 ) 130 ( 48 nm) were formed (Figure 1d).
A minor fraction of small spheres with d1-C3N6 ) 27 ( 5 nm
was also visualized by means of SEM. In the case of C3N12-
induced silica precipitates, a single much more narrow distribu-
tion of silica spheres was found, with an average diameter of
dC3N12 ) 169 ( 23 nm (Figure 1e). The morphology of the silica
structures changed considerably when C3N18 was used. Here,
on the one hand, silica spheres were formed with an average
diameter of dC3N18 ) 163 ( 36 nm (Figure 1f) with a shoulder
to smaller sizes on one side. On the other hand, several
micrometer long filament-like structures with a mean diameter
of dF-C3N18 ) 33 ( 17 nm were visualized. Some of the filaments
appeared as a chain of spheres fused together to form tubules
(Figure 1f, zoom-in view).
From the SEM images, it was unclear whether the formed
silica spheres are solid or hollow in nature. To be able to analyze
the interior of the spheres, transmission electron microscopy
(TEM) was performed in conjunction with energy dispersive
X-ray spectroscopy (EDX). Figure 2 shows TEM bright-field
images of silica spheres produced in the presence of C3N6. The
spheres appear to be partly solid and partly hollow (Figure 2a,b).
However, Figure 2c strongly suggests that the material density
is smaller in the interior of the spheres. The thickness of the
shells was determined to be 32 ( 2% (n ) 14) of the diameter
of the spheres. To be able to determine the distribution of the
compounds from which the silica spheres were made, an EDX
analysis was performed. According to Kro¨ger et al.,8,12 it is
expected that SiO2 and polyamine coprecipitate to form a
composite material that is stable even after washing with water.
Hence, the precipitate was expected to be composed of four
elements, namely, carbon, nitrogen, silicon, and oxygen. Figure
2d shows a scanning TEM (STEM) annular dark-field image
of silica spheres produced in the presence of C3N6 with
apparently hollow and solid spheres. However, silicon (red) and
oxygen (blue), reflecting the position of SiO2, are restricted only
to the shells of the spheres, even in the case where the spheres
appear to be solid in the annular dark-field image (see particle
1 in Figure 2d-f). The oxygen signal in line scans and X-ray
maps strictly follows the silicon signal with a ratio corresponding
to that of SiO2, showing that there is no significant oxygen signal
from any material in the interior of the hollow spheres. The
overlay depicted in Figure 2e demonstrates that in addition to
silicon (red), carbon (green) is also found in the shells. Spheres
that appear solid in the annular dark-field image exhibit an
additional carbon signal in their interiors. The small nitrogen
signal in the EDX spectra, however, does not significantly
exceed the bremsstrahlung background (3σ criterion), so only
the carbon content of some hollow spheres can be concluded
from the X-ray analyses. These results suggest that the silica
spheres are composed of a shell with a thickness of 20-60 nm
that consists of SiO2, while the interior might still contain some
polyamine. A similar result was obtained for silica precipitates
catalytically formed by C3N12. TEM and STEM images
revealed SiO2 shells with a thickness of 25-50 nm, which is
on average 24 ( 4% (n ) 37) of the sphere diameter (see the
Supporting Information). Interestingly, the spheres appear to
be hollow in all cases, and the carbon signal in the interior of
the spheres is missing. Precipitates induced by the polyamine
C3N18 are characterized by two different morphologies. Besides
spheres having a SiO2 shell with a thickness of 20-50 nm [23
( 3% (n ) 20) of the spheres’ diameter; see the Supporting
Information], filamentous structures were found that were also
composed of SiO2, as proven by EDX analysis.
N-Methylation of Polyamines. Long-chain polyamines of
diatoms contain nonmethylated as well as methylated amino
groups.14 Recently, for a short-chain C3N3 molecule, it has been
demonstrated that the methylation pattern alters the kinetics of
the first condensation reaction of silicic acid.23 Here we chose
the long-chain polyamine C3N12, as this compound was proven
to precipitate very well defined hollow silica spheres. We
methylated all of the secondary amino groups (C3N12Me) and
compared the obtained results with those of the nonmethylated
molecule. The amount of silica precipitated in the presence of
C3N12Me was determined by the ꢀ-silicomolybdate method
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Bernecker et al.
amount of silica, mSi ) 23.6 ( 0.7 µg, was even further
reduced to 2.7. The sensitivity of the precipitation reaction
with respect to the phosphate concentration, given as the
linear increase in mSi as a function of the cP /cN ratio with a
i
value of 7.0 ( 0.8 µg, is further increased by a factor of 2
relative to the slope obtained for C3N12.
For all three compounds, the precipitates obtained at three
different phosphate concentrations (3, 10, and 30 mM Pi) were
investigated by means of SEM, and the size of the resulting
silica spheres was analyzed by histograms. The results are
presented in Figure 4. Only a small amount of silica was
precipitated with C3N6 (Figure 4a) at 3 mM phosphate. As a
result, besides some spherical structures with diameters of d1-
C3N6 ) 58 ( 17 nm (3 mM Pi) and d2-C3N6 ) 121 ( 26 nm (3
mM Pi), some rather nondefined silica structures were also
formed. An increase in the phosphate concentration to 10 mM
generated more well-defined silica structures; in particular,
smooth silica spheres with a mean diameter of dC3N6 ) 111 (
15 nm (10 mM Pi) were formed. An even further increase in
the phosphate concentration then only slightly increased the
average sphere diameter to dC3N6 ) 128 ( 42 nm (30 mM Pi),
with an overall broader size distribution. Interestingly, the
influence of the phosphate concentration on the silica morphol-
ogy was very small in case of C3N12 (Figure 4b). Here, silica
spheres having average diameters of very similar sizes were
formed, with dC3N12 ) 93 ( 22 nm (3 mM Pi), dC3N12 ) 101 (
21 nm (10 mM Pi), and dC3N12 ) 106 ( 24 nm (30 mM Pi).
This did not hold for the longest polyamine that was investigated
in this study, C3N18 (Figure 4c). From the experiments at 30
mM phosphate, we already know that besides silica spheres,
filament-like structures with diameters of 30-40 nm were also
formed. This was not the case at 3 mM phosphate. Under these
conditions, only spheres with a diameter of dC3N18 ) 80 ( 18
nm (3 mM Pi) were formed. Filament-like structures were,
however, formed at 10 mM phosphate. Besides these filaments,
two different spherical distributions could be distinguished, with
mean diameters of d1-C3N18 ) 25 ( 7 nm and d2-C3N18 ) 130 (
19 nm (10 mM Pi). Interestingly, the size of the small spheres
was in the same range as the diameter of the filaments and might
be interpreted as a prestate of the filaments or vice versa. The
same kind of size distribution, but with slightly larger diameters,
was found for 30 mM phosphate. Here, filaments were the
predominant structures. Average sphere diameters of d1-C3N18
) 30 ( 10 nm and d2-C3N18 ) 154 ( 46 nm (10 mM Pi) were
measured. In comparison with the result obtained for C3N18
in the presence of 30 mM phosphate (Figure 1f), here two
different size distributions were found as a result of the slightly
different experimental conditions. However, in principle, the
formation of small silica spheres might already be indicated in
the shoulder of the histogram in Figure 1f. In summary, the
combination of polyamine chain length with propyleneimine
units and the phosphate concentration significantly determines
the amount and structure of the resulting silica precipitates.
However, a correlation between the droplet size found in
solution prior to silica precipitation and the resulting structure
of the precipitates was not found. Independent of the polyamine,
the droplets were considerably larger than the resulting silica
particles (see the Supporting Information).
Figure 3. Mass of precipitated silica (mSi) as a function of the phosphate
to nitrogen ratio cP /cN for the three different long-chain polyamines C3N6,
i
C3N12, and C3N18. Precipitates were obtained in 30 mM acetate buffer
(pH 6.8) with cN ) 1 mM and cSi(OH) ) 100 mM. The reaction time was
4
set to 10 min.
to be mSi ) 31.0 ( 1.5 µg (n ) 2), which is a factor of 2 larger
than the mSi obtained in the case of C3N12. More interesting
was the fact that the SEM images of the precipitates revealed
two well-defined populations of silica spheres formed in the
presence of C3N12Me (see the Supporting Information). One
distribution exhibited an average diameter of d1-C3N12Me ) 59
( 24 nm, but the majority of spheres had an average diameter
of d2-C3N12Me ) 183 ( 27 nm.
Influence of Phosphate Ions on Precipitation. Long-chain
polyamines from diatom biosilica are extracted from aqueous
solution by chloroform/methanol and thus behave like
amphiphilic substances. It is expected that long-chain
polyamines in aqueous solutions form aggregates with
positively charged surfaces, with the tendency to form larger
aggregates in the presence of multivalent anions.17 To prove
this idea, we performed dynamic light scattering (DLS)
experiments with C3N6, C3N12, and C3N18 in aqueous
solution. The DLS results verified the presence of particles
exhibiting hydrodynamic diameters of several hundred nano-
meters with a rather broad distribution (see the Supporting
Information) in the presence of 30 mM phosphate. For
C3N12, the influence of the phosphate concentration was
further investigated, and it was found that a larger phosphate
concentration leads to larger hydrodynamic diameters, i.e.,
larger droplet sizes (see the Supporting Information). These
results support the notion that the polyamines form emulsions
of microdroplets in aqueous solution and that droplet
formation is a function of the number of multivalent anions.
We further investigated the influence of the phosphate
concentration on the precipitation products of C3N6, C3N12,
and C3N18. Independent of the polyamine, the amount of
precipitated silica increased with increasing phosphate con-
centration (Figure 3) until saturation occurred. Two distinct
regions were defined for each plot, a linear one and a constant
one. For C3N6, linear behavior of the mass of precipitated
silica as a function of the cP /cN ratio with a slope of 0.9 (
i
0.5 µg was found. The amount of precipitated silica became
independent of the phosphate concentration at a cP /cN ratio
i
of 7.5 with an mSi value of 9.1 ( 0.7 µg. The slope for
C3N12, 3.5 ( 0.2 µg, was almost a factor of 4 larger than
that of C3N6. At the much smaller cP /cN ratio of 3.2, the
i
Discussion
precipitation mass became independent of the phosphate
concentration in solution and levels off at a value of mSi
)
To date, no synthetic route has been described for obtaining
large quantities of well-defined long-chain polyamines, which
are one class of important compounds involved in silica
14.8 ( 0.7 µg. In the case of C3N18, the phosphate
concentration (i.e., the cP /cN ratio) to precipitate the maximum
i
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1028 J. AM. CHEM. SOC. VOL. 132, NO. 3, 2010

Polyamine-Induced Silica Formation
A R T I C L E S
Figure 4. Histogram analyses and corresponding scanning electron micrographs of the silica precipitates obtained in the presence of (a) C3N6, (b) C3N12,
and (c) C3N18 using three different phosphate concentrations (3, 10, and 30 mM). Silica precipitates were generated in 30 mM acetate buffer (pH 6.8) with
cN ) 1 mM and cSi(OH) ) 100 mM. Scale bars are 1 µm.
4
production in diatoms and sponges. The synthesis of oligoamides
on a solid support using well-established peptide synthesis
protocols followed by reduction and cleavage of the oligoamines
from the resin proved to be superior to the stepwise assembly
of oligoamines in solution. Deletion sequences from incomplete
couplings of oligoamides are easily identified and can be
discarded, while the uniformity of the chain length of oligoam-
ines can be verified by mass spectrometry. The presented
protocol avoids the analytical characterization of oligoamines
until the final reduction step.
With the well-defined compounds in hand, it was the aim of
this study to gain new insights into in vitro silica formation to
facilitate the synthesis of distinct silica nanostructures under
mild conditions. It was originally recognized by Mizutani et
al.40 that amines and polyamines are capable of accelerating
silicic acid polymerization. The group proposed that the
polyamine acts as an acid-base catalyst, thus facilitating
condensation of two monosilicic acid molecules. Accordingly,
the transition state may be stabilized by two appropriately
arranged amino groups within the polyamine backbone. Indeed,
we found that when the spacer between two adjacent amino
groups was composed of only two C atoms, only a very small
number of silica spheres were precipitated. Through the use of
propyleneimino subunits instead of ethyleneimino subunits, the
amount of silica was greatly increased by a factor of ∼8.
Notably, long-chain polyamines isolated from diatom species
have also been characterized as poly(propyleneimime) chains
with up to 20 repeat units. Coradin et al.41 proposed that the
protonated amino groups serve as binding sites for silicic acid.
The protonation state was calculated, and the charge distribution
and hydrophobicity of each polyamine were estimated (see the
Supporting Information). The results are summarized in Figure
5 along with the respective precipitated silicon masses. While
only 56% of the amino groups are protonated in C2N7, 73%
are protonated in the case of C3N7 at pH 6.8, which reflects
the greater distance between two amino groups. Separating the
amino groups by four C atoms (C4N7) results in protonation
(40) Mizutani, T.; Nagase, H.; Fujiwara, N.; Ogoshi, H. Bull. Chem. Soc.
Jpn. 1998, 71, 2017–2022.
(41) Coradin, T.; Durupthy, O.; Livage, J. Langmuir 2002, 18, 2331–2336.
9
J. AM. CHEM. SOC. VOL. 132, NO. 3, 2010 1029

A R T I C L E S
Bernecker et al.
found for the variation of the N-to-N distance. The silica mass
increased by a factor of 1.5 with increasing chain length. From
C3N6 to C3N18, the hydrophobicity of the molecules increases,
while the relative protonation state slightly decreases (Figure
5). This promotes aggregation of the molecules, as hydrophobic
attraction counteracts electrostatic repulsion, which influences
the amount and size of the silica structures. This may also
explain the different cP /cN ratios that are necessary to obtain
i
the maximum amount of precipitated silica, as discussed below
in more detail. The change in diameter of the observed silica
spheres with increasing chain length was rather small, while
drastic changes in the structure from spheres to filaments were
observed. Silica fibers have been described in other studies after
application of shear stress or electrostatic fields,44,45 but these
were not applied in our experiments. Van Bommel et al.46
observed silica with a pearl-necklace-like morphology similar
to the structures found in Figure 1f when fibrous organogel
networks were used as templates for silica deposition. They
proposed a surface mechanism according to which silica
formation starts at some random points on a fiber, after which
silica grows in a spherical manner and later merges into fibers.
A transition between spherical and filament-like structures has
also been observed in our experiments and might be an
indication of the existence of spherical and filamentous C3N18
polyamine aggregates.
A different level of methylation of the amino groups is
another structural characteristic of long-chain polyamines found
in different diatom species. While polyamines extracted from
Coscinodiscus granii cell walls do not exhibit any methylation
at all, a higher degree of methylation is for example found in
polyamines from Coscinodiscus wailesii. Polyamines of Cos-
cinodiscus concinnus are permethylated.43,47 These different
methylation patterns suggest that methylation per se is not a
requirement for silica formation. It has, however, been shown
that increased methylation of the amino groups enhances the
reaction rate48 and that the degree of methylation in polyamines
affects their ability to promote silica condensation.49 Hence, it
might be conceivable that different methylation patterns can
affect the rate of silica formation and therefore play a role in
creating species-specific silica nanostructures. Relative to C3N12
(70% protonation), the degree of protonation of C3N12Me is
reduced (61%; see the Supporting Information) while the
hydrophobicity in turn is increased, which is in line with the
trend that increased hydrophobicity leads to larger silica mass
(Figure 5). This observation emphasizes the importance of
attractive forces between polyamine molecules. Comparing the
silica structures produced by the polyamines C3N12 and
C3N12Me also demonstrates that by changing only the methy-
lation pattern, a different structure is obtained. While C3N12
produces a well-defined narrow distribution of silica nanospheres
(dC3N12 ) 169 nm), precipitation in the presence of the
methylated compound C3N12Me results in two well-defined
populations of silica nanospheres with different diameters (d1-
C3N12Me ) 59 nm and d2-C3N12Me ) 183 nm).
Figure 5. Relationship between the amount of precipitated silica (given
as mSi), the protonation state z, and the hydrophobic character h of the
polyamines C3N6, C3N12, C3N18, C2N7, C3N7, C4N7, and C3N12Me.
of 97% of the amino groups. However, the hydrophobicity is
independent of the N-to-N distance and was estimated to be
78-79%. Figure 5 shows that an almost linear relationship
between the relative protonation state and precipitated silica
mass is found. One has to keep in mind that the calculation is
only a rough approximation, as the formation of emulsions of
microdroplets as well as phosphate anions were not taken into
account.
It is known that each diatom species exhibits characteristic
silica structures on the micro- and mesoscale as well as very
specific patterns on the nanoscale,42 which among other factors
is concomitant with the observation that each species is equipped
with specific polyamine structures. Structural variations include
the overall chain length, the degree of methylation, and the
positions of secondary and tertiary amino groups.43 These
findings may imply that the polyamine structures are a deter-
minant for the species-specific nanopatterning of diatom bio-
silica. Here we found that the structure (i.e., the size of the silica
spheres) is significantly increased when the distance between
two amino groups is increased, even though the number of
nitrogen atoms is constant. This result might reflect the interplay
between charge repulsion between ammonium cations and
electrostatic attraction with phosphate anions, which results in
a particular supramolecular assembly acting as the template for
silica precipitation, as proposed by Sumper16 and discussed
below.
When the distance between the amino groups was kept
constant at three C atoms, as is found in natural polyamines,43
but the chain length was increased stepwise to a length that is
typically found in diatoms, a mixture of protonated and
unprotonated amino groups was found (see the Supporting
Information) with rather similar average protonation states of
75% (C3N6), 70% (C3N12), and 69% (C3N18) at pH 6.8. For
these three polyamines, the amount of precipitated silica as well
as the diameter of the silica spheres is influenced by the chain
length, although the differences are less pronounced than those
(44) Patwardhan, S. V.; Mukherjee, N.; Clarson, S. J. J. Inorg. Organomet.
Polym. 2001, 11, 117–121.
(45) Rodriguez, F.; Glawe, D. D.; Naik, R. R.; Hallinan, K. P.; Stone, M. O.
Biomacromolecules 2004, 5, 261–265.
(46) van Bommel, K. J. C.; Shinkai, S. Langmuir 2002, 18, 4544–4548.
(47) Sumper, M.; Lehmann, G. ChemBioChem 2006, 7, 1419–1427.
(48) Robinson, D. B.; Rognlien, J. L.; Bauer, C. A.; Simmons, B. A. J.
Mater. Chem. 2007, 17, 2113–2119.
(42) Hildebrand, M.; York, E.; Kelz, J. I.; Davis, A. K.; Frigeri, L. G.;
Allison, D. P.; Doktycz, M. J. J. Mater. Res. 2006, 21, 2689–2698.
(43) Sumper, M.; Brunner, E. ChemBioChem 2008, 9, 1187–1194.
(49) Menzel, H.; Horstmann, S.; Behrens, P.; Ba¨rnreuther, P.; Krueger, I.;
Jahns, M. Chem. Commun. 2003, 2994–2995.
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Polyamine-Induced Silica Formation
A R T I C L E S
Independent of the exact molecular structure of the long-chain
polyamine, these amphiphilic molecules are thought to undergo
phase separation in aqueous solution to form an emulsion of
microdroplets.16 By means of DLS, we also found droplets with
a size of several hundred nanometers in the aqueous phase in
the presence of phosphate. Silica precipitation is supposed to
occur at the interface of the polyamine droplets. As silica is
produced on a short time scale, i.e. within minutes, it hinders
the silicic acid from diffusing deeper into the polyamine droplet,
and thus, the interior of the produced silica spheres is expected
to be “hollow” instead of solid. Such “hollow” silica shells were
indeed unambiguously revealed by the EDX element maps
(Figure 2). The silica shells observed here can be discussed in
terms of the phase separation model proposed by Sumper.18 He
suggested that a two-dimensional monolayer of polyamine-
containing droplets in the silica deposition vesicle guides silica
deposition, with polymerization of silica at the surface of the
droplets leading to a honeycomb-like framework. The hollow
silica shells observed in this study can be rationalized in a similar
way. Three-dimensional droplets of polyamines guide the
polymerization at the surface of the droplets, resulting in silica
spheres.
same was observed when the polyamine chain length was
elongated at a constant phosphate concentration of 30 mM.
The intrinsic tendency of polyamines to form aggregates in
aqueous solution as a function of chain length is also reflected
by the cP /cN ratio necessary to obtain the maximum silica mass
i
that can be precipitated under these conditions. The longer the
polyamine, the less phosphate is required for maximum silica
precipitation, probably because of enhanced hydrophobicity, a
decreased protonation state, and hence increased attractive
interactions between the molecules.
Conclusions
The molecular requirements for long-chain polyamines to
form well-defined silica precipitates have been elucidated. By
means of solid-phase peptide synthesis, it became possible to
tailor these molecules and obtain them in large quantities. This
is the prerequisite for controlling the growth of an inorganic
material such as silicon dioxide on a short time scale and under
ambient conditions. We were able to show that formation of
hollow silica spheres with a narrow size distribution as well as
filaments becomes possible with such well-defined long-chain
polyamines, which might be attractive structures for nanotech-
nological applications.
The phase separation model also suggests that multivalent
anions control the silica precipitation process by modulating
the polyamine aggregates. It has been shown that the silica
sphere size depends on the phosphate concentration in case of
polyamines from Stephanopyxis turris, which consist of 15-21
N-methylpropyleneimine repeating units.17 We found that for
C3N6, 3 mM phosphate is not sufficient to form well-defined
silica aggregates, which is probably a result of the small chain
length. With an increase in the phosphate concentration, slightly
larger silica spheres were obtained. A similar trend was found
for C3N12, where a tendency to form larger sphere diameters
with increasing phosphate concentration was found. A significant
influence of the phosphate concentration was observed for
C3N18. Interestingly, for this polyamine, the silica structure
changes from spheres to filaments with increasing phosphate
concentration, even though the silicon mass is constant. The
Acknowledgment. We thank Hans-Jo¨rg Langer for experimental
assistance. Financial support by the VW-Stiftung (I/82 042) is
gratefully acknowledged.
Supporting Information Available: Experimental details,
TEM and STEM micrographs and EDX element maps (Figures
S1 and S2), SEM micrographs and histogram analysis for
C3N12Me (Figure S3), hydrodynamic diameters of the polyamine
droplets (Table S1), space-filling models with charge distribu-
tions of polyamines (Figure S4), and the procedure for calculat-
ing the polyamine protonation state (Figures S5 and S6). This
material is available free of charge via the Internet at http://
pubs.acs.org.
JA9061163
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