Silica precipitation with synthetic silaffin peptides

Article abstract of DOI:10.1039/c1ob05406f

Silaffins are highly charged proteins which are one of the major contributing compounds that are thought to be responsible for the formation of the hierarchically structured silica-based cell walls of diatoms. Here we describe the synthesis of an oligo-pr

Full text of DOI:10.1039/c1ob05406f

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PAPER
Silica precipitation with synthetic silaffin peptides†
Ralph Wieneke,a Anja Bernecker,b Radostan Riedel,a Manfred Sumper,c Claudia Steinem*b and Armin Geyer*a
Received 15th March 2011, Accepted 5th May 2011
DOI: 10.1039/c1ob05406f
Silaffins are highly charged proteins which are one of the major contributing compounds that are
thought to be responsible for the formation of the hierarchically structured silica-based cell walls of
diatoms. Here we describe the synthesis of an oligo-propyleneamine substituted lysine derivative and its
incorporation into the KXXK peptide motif occurring repeatedly in silaffins. Ne-alkylation of lysine
was achieved by a Mitsunobu reaction to obtain a protected lysine derivative which is convenient for
solid phase peptide synthesis. Quantitative silica precipitation experiments together with structural
information about the precipitated silica structures gained by scanning electron microscopy revealed a
dependence of the amount and form of the silica precipitates on the peptide structure.
silica precipitation in diatoms. Chemical synthesis circumvents
Introduction
the microheterogeneity of natural silaffins and the solid-phase
Diatoms are unicellular eukaryotic organisms whose silica shells
protocol presented here allows for the straightforward assembly of
belong to the most outstanding nanostructures found in nature.1
systematically diversified polycationic peptides. The biosilification
The two valves of their silica shell are precisely reproduced
studies identify a notable influence of the amino acid composition
in every cell cycle. How the formation of highly symmetrical
on the nanostructure of the precipitated silica.
shell shapes is controlled on the nano- and micrometre scale is
a fascinating question for chemists, biochemists, and materials
scientists. Nanofabrication of silica in diatoms results from specific
interactions between silicic acid and long-chain polyamines2 or
Results and discussion
In 2004, the entire genome of the diatom Thalassiosira pseudonana
was sequenced.11 Silaffins and the recently described cingulins
from T. pseudonana contain more than 18% phosphorylated
serine residues and above 10% lysine residues.12 Lysines are either
post-translationally dimethylated at the e-nitrogen or even more
frequently these are extended with oligo-propyleneamine repeats.
Nearly all lysines (K) are assembled in KXXK tetrapeptide
domains. The 105-mer peptide Silaffin 3 (Sil-3) has mainly A,
G, S, or E at either position X, whereas Q and H occur only once
in the altogether 15 domains (Fig. 1a).13
5 out of the 15 domains of Sil-3 bear the w-dimethylated
dipropyleneamino group at the N-terminal position and e-
dimethyllysine at the C-terminal position. In the title compounds
of this work (1–3, Fig. 1b), these two lysine derivatives flank
three dipeptides of increasing polarity. The systematic variation
of the isolated KXXK domains shall identify the sequence
specificity of post-translationally modified lysine residues on silica
precipitation.
Synthesis started with the reductive amination of the hydrochlo-
ride of a-Fmoc-protected lysine 4 (Scheme 1) with formaldehyde
solution and NaBH4 in dioxane yielding e,e-dimethyllysine 5
in 88% yield after simple extraction with CHCl3. The pH was
adjusted between 3 and 6 with acetic acid to avoid cleavage of the
Fmoc group. The synthesis of w,w-dimethyl-dipropyleneamino-
lysine starts from completely protected lysine derivative 6.
The a-N-protecting group Dde [Dde = 4,4-dimethyl-2,6-dioxo-
cyclohexylidene)-ethylamino-] is stable under basic conditions
polar proteins, so-called silaffins which promote the formation
of SiO2 from silicic acid.3–5 Silaffins are characterized by a large
number of phosphorylated serine residues and post-translationally
modified lysines which carry oligo-propyleneamine functionalized
side chains.6 Both modifications increase the tendency of silaffins
to form large aggregates which guide the nanopattering of silica
matrices at ambient temperature and pH.7,8 The protein self-
assembly is driven by ionic interactions and is a major prerequisite
for the silica precipitation in vivo. Although any amine induces
silica precipitation in vitro, nature employs complex gene products
which allow for the fine tuning of charges and countercharges in
the peptide side chains. We observed a structural dependence for
synthetic polyamines9,10 and expected therefore the local amino
acid composition to also have a distinguishable influence on the
precipitation process. The systematic variation of peptide com-
position can give a deeper insight into the secrets of hierarchical
aFaculty of Chemistry, Philipps-Universita¨t Marburg, 35032, Marburg,
Germany. E-mail: geyer@staff.uni-marburg.de; Fax: +49 (0)6421 282021;
Tel: +49 (0)6421 282030
bInstitute of Organic and Biomolecular Chemistry, University of Go¨ttingen,
Tammannstr. 2, 37077, Go¨ttingen, Germany. E-mail: Claudia.Steinem@
chemie.uni-goettingen.de; Fax: +49-(0)551-39 3228; Tel: +49-(0)551-39
3294
cBiochemistry, University of Regensburg, 93040, Regensburg, Germany.
E-mail: manfred.sumper@vkl.uni-regensburg.de
† Electronic supplementary information (ESI) available: Scanned copies
of the 1H and 13C NMR spectra. See DOI: 10.1039/c1ob05406f
5482 | Org. Biomol. Chem., 2011, 9, 5482–5486
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acid (TFA) and triethylsilane as a scavenger gave the orthogonally
protected lysine derivative 10. Both derivatives 5 and 10 were then
used in the solid phase peptide synthesis.
Fmoc-protected 5 (Scheme 2) was attached to 2-chlorotrityl
resin preloaded with glycine using Fmoc solid phase peptide syn-
thesis (SPPS) protocols. O-Benzotriazole-N,N,N¢,N¢-tetramethyl-
uronium-hexafluoro-phosphate (HBTU) and 1-hydroxy-1H-
benzotriazole (HOBt) were used as coupling reagents throughout
the synthesis and the Fmoc-group was removed using 25%
piperidine in DMF. The a-Dde-protected lysine derivative 10 was
coupled under standard Fmoc-SPPS-protocols without compli-
cations. Selective removal of the Dde-group was achieved by a
repeated titration of the resin with 2% hydrazine. A final coupling
with Fmoc-glycine and deprotection with piperidine followed
by mercaptoethanol and DBU (1,8-diazabicyclo[5.4.0]undec-7-
ene) yielded the hexapeptides still bound to the resin. Liber-
ation from the solid support was achieved with trifluoracetic
acid–triisopropylsilane and water (TFA : TIPS : H2O, 95 : 2.5 : 2.5).
Analysis of the products by MALDI-TOF showed one major
peak corresponding to 1, 2, and 3, respectively (Scheme 2). Other
analytical methods showed the homogeneity of the peptides, too.
1, 2, and 3 are the first peptides with a uniform composition,
in contrast to the silaffins from natural origin, which exhibit
microheterogeneity.
Fig. 1 a) The 10 different KXXK domains as observed in the silaffin
polypeptide Sil-3 with increasing polarity (from top to bottom). The side
chains of the amino acids are shown on the right with maximum charges.
Serine is uncharged while phosphoserine can even bear two negative
charges. KAEK occurs twice and KASK even three times in Sil-3. The
three post-translationally modified lysine species as observed in the silaffins
together with serine phosphate are shown on the right. b) Charge pattern
of the three GKAXKG hexapeptides 1, 2, and 3 which contain the KXXK
motif as derived from silaffin polypeptides and which were synthesized to
study their silica precipitation properties.
Scheme 2 Solid-phase synthesis of peptides 1, 2, and 3.
In order to investigate the influence of peptides 1, 2, and 3 on the
biosilification process, silica precipitation assays were undertaken.
In the presence of all three compounds silica precipitates were
found. However, the amount was dependent on the molecular
structure. Fig. 2 shows the amount of precipitated SiO2, given as
mSi, as a function of different peptide concentrations at pH 6.8.
While in the presence of compound 2 only small SiO2 masses are
found, mSi is larger if compound 3 is added to the Si(OH)4 solution.
The largest amount of SiO2 is found in case of compound 1. Almost
no SiO2 is generated in the presence of all three compounds if the
pH is shifted to 5.5.
Scheme 1 Synthesis of orthogonally protected lysine derivatives 5 and 10
for peptide synthesis.
and the carboxylate was protected as tert-butylester in order to
avoid side reactions during the Mitsunobu reaction.14 The first
chain elongation was done according to a protocol invented
by Fukuyama and co-workers.15,16 N-alkylation with 3-bromo-1-
propanol at the nosylated nitrogen under basic conditions deliv-
ered a primary alcohol 7 which was subjected to a second chain
elongation with 8 under Mitsunobu conditions (DIAD/PPh3) to
afford 9. Cleavage of the tert-butylester (9) with trifluoroacetic
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the assumed increase of the size of the microdroplets from the
postulated phase separation process during silica precipitation.17,18
This phase separation was characterized for biogenic and synthetic
polyamines without covalently linked counter ions like Glu in
peptide 2.9 This study shows that already isolated KXXK domains
can determine the morphology of silica precipitates. The chemical
methods introduced here will give access to longer expansions
or even the entire silaffin peptides for nanopattern formation
studies.
Fig. 2 Amount of precipitated silica given as mSi in the presence of
various concentrations of compound 1 (ꢀ), 2 (᭡), and 3 ( ), respectively.
᭹
Conclusion
Precipitation was carried out at pH 6.8. At pH 5.5, even at 1 mM of the
corresponding compound, only very little precipitate was found (striped
symbols).
The self-assembling process of silaffins is guided by ions, which
are provided by cationic side chain functionalities and by an-
ionic modifications like serine phosphate. Polyamines are post-
translationally attached to lysines in order to promote and catalyze
the silica precipitation. This study identifies a correlation between
the amino acid composition of synthetic KXXK domains and the
character of the silica precipitates. The biosilification process is a
finely balanced interaction between several organic components
present in diatoms. We could show that a small change in the
primary structure in the peptide side chains are directly expressed
in the amount of precipitated silica as well as its morphology.
The SPPS protocol for the assembly of poly-cationic peptides
will allow the synthesis of even more complex peptides and thus
accelerate the elucidation of structure-function relationships in the
biosilification process of diatoms. Currently, longer sections of the
silaffin peptidic backbone together with other post-translational
modifications found in diatoms are being synthesized.
To further elucidate the structure of the silica precipitates
scanning electron microscopy images were taken. Fig. 3 shows
scanning electron microscopy (SEM) images of silica structures
obtained in the presence of 1 mM of the three different compounds
at pH 6.8. In the presence of compound 1, silica spheres of the size
of hundreds of nanometres are found together with amorphous
silica. Compound 2 generates only few silica spheres but rather
grainy silica structures, which demonstrates that the change in one
amino acid influences the silica structure considerably. In case of
compound 3, mainly silica spheres in the range of a few 100 nm
can be observed. Decreasing polarity of the side chains in the
order Glu, Ser, Ala increases the size of the detected spheres
of the silica precipitate. This observation is in accordance with
Experimental
(S )-6-Dimethylamino-2-(9H -fluoren-9-ylmethoxycarbonyl-
amino)-hexanoic acid (5). To a solution of Fmoc-L-lysine (4,
5.0 g, 10.3 mmol), 37% formaldehyde solution (4.2 mL, 51.7 mmol)
and acetic acid (1.8 mL) in 1,4-dioxane (30 mL) were added NaBH4
(3.5 g, 93.1 mmol) in portions at 0 ◦C. The pH of the reaction
mixture was maintained between 3 and 6 by addition of acetic
acid. After half of the NaBH4 was added, another portion of
37% formaldehyde-solution (4.2 mL, 51.7 mmol) was added. The
reaction mixture was warmed to room temperature and stirred
for 12 h. The mixture was diluted with H2O (30 mL) and the pH
was adjusted to 6. The organic solvents were removed in vacuo
and the remaining water phase was extracted with CHCl3 (8 ¥
50 mL). The collected organic phases were dried over MgSO4,
filtered and concentrated in vacuo. Titration with Et2O and drying
under high vacuum delivered 5 (3.9 g, 88%) as a white foam. (Rf
0.40 on SiO2, 95 : 5 CH3OH–H2O); [a]1D8 +2.9 (c 1.21, EtOH); 1H
NMR (DMSO-d6, 300 MHz, 300 K) d 7.87 (d, 2H, J = 7.5 Hz,
CH), 7.72–7.67 (m, 2H), 7.40 (t, 2H, J = 7.0 Hz, CH), 7.30 (t,
2H, J = 7.3 Hz, CH), 7.22 (d, 1H, J = 7.5 Hz, NH), 4.26–4.17 (m,
3H, OCH2, CH), 3.79–3.86 (m, 1H, Ha), 2.44 (t, 2H, J = 7.5 Hz,
He), 2.30 (s, 6H, 2 ¥ NCH3), 1.73–1.54 (m, 2H, Hb), 1.50–1.37 (m,
2H, Hd), 1.33–1.27 (m, 2H, Hg ); 13C NMR (DMSO-d6, 75 MHz)
d 173.7 (C O), 156.2 (C O), 143.8 (C), 140.7 (C), 127.6 (CH),
127.1 (CH), 125.2 (CH), 120.1 (CH), 65.6 (CH2), 56.0 (CH), 53.7
(CH2), 46.6 (CH), 41.8 (CH3), 30.1 (CH2), 23.1 (CH2), 22.7 (CH2);
HRMS (ES) Calc. for C23H27N2O4 395.1976, found 395.1972.
Fig. 3 Scanning electron microscopy images of the silica precipitates
obtained in the presence of 1 mM of the indicated peptide 1, 2, or 3
at pH 6.8. The silica obtained from peptide 1 exhibits spheres together
with grainy precipitate. Peptide 2 delivers grainy silica only, while spheres
dominate the silica precipitated with 3.
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yellow oil. (Rf 0.41 on SiO2, 5 : 1, CHCl3 : MeOH); [a]1D8 -0.2 (c
1.23, CHCl3); H NMR (CHCl3, 300 MHz, 300 K) d 13.66 (d,
(S)-2-[1-(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-ethylamino]-
6-[(3-hydroxy-propyl)-(2-nitrobenzenesulfonyl)-amino]-hexanoic
acid tert-butyl ester (7). Dde-L-Lys(Ns)-OtBu (6, 10.5 g, 19.0
mmol), K2CO3 (11.8 g, 85.7 mmol) and TBAI (0.1 g, 0.5 mmol)
were dissolved in DMF (75 mL) and heated up to 70 ◦C. At this
temperature 3-bromopropanol (2.1 mL, 24.8 mmol) was added
slowly. The mixture was stirred for 8 h at this temperature. H2O
(300 mL) was added and then the mixture was extracted with
EtOAc (2 ¥ 200 mL). The collected organic solvents were again
washed with brine (3 ¥ 150 mL). After drying over MgSO4 and
filtration, the organic solvents were removed in vacuo. The crude
product was purified by flash chromatography (SiO2, 10 : 0 → 4 : 1,
EtOAc–MeOH) to yield 7 (9.0 g, 78%) as a yellow oil. (Rf 0.25
1
1H, J = 7.5 Hz, 1H, NH), 7.95–7.89 (m, 2H, CH), 7.65–7.60 (m,
4H, CH), 7.57–7.53 (m, 2H, CH), 4.17 (app. q, 1H, J = 6.8 Hz,
Ha), 3.29–3.18 (m, 8H, CH2, He), 2.41 (s, 3H, CH3), 2.34 (s, 2H,
CH2), 2.28 (s, 2H, CH2), 2.23 (t, 2H, J = 7.2 Hz, CH2), 2.14
(s, 6H, CH3), 1.85–1.75 (m, 4H, CH2, Hb), 1.70–1.61 (m, 2H,
t
CH2), 1.55–1.46 (m, 2H Hd), 1.39 (s, 9H, Bu), 1.33–1.23 (m,
2H, Hg ), 0.96 (s, 6H, CH3); 13C NMR (CHCl3, 75 MHz) d 199.3
(C O), 196.8 (C O), 172.9 (C O), 169.1 (C), 147.9 (C), 133.6
(C), 132.9 (C), 131.8 (CH), 130.8 (CH), 130.6 (CH), 124.2 (CH),
124.1 (CH), 108.2 (C), 83.2 (C), 56.7 (CH2), 56.3 (CH), 53.6 (CH2),
52.2 (CH2), 47.4 (CH2), 46.0 (CH2), 45.3 (CH2), 45.1 (CH3), 45.0
(CH2), 32.2 (CH2), 30.0 (C), 28.4 (CH3), 28.2 (CH3), 27.9 (CH3),
27.6 (CH2), 26.0 (CH2), 22.2 (CH2), 18.2 (CH3); HRMS (ES) Calc.
for C40H59N6O12S2 879.3627, found 879.3632.
1
on SiO2, EtOAc); [a]D26 +2.5 (c 0.64, CHCl3); H NMR (CHCl3,
300 MHz, 300 K) d 13.63 (d, 1H, J = 7.5 Hz, NH), 7.93–7.89
(m, 1H, CH), 7.66–7.60 (m, 1H, CH), 7.58–7.54 (m, 1H, CH),
4.23–4.15 (m, 1H, Ha), 3.59 (t, 2H, J = 5.9 Hz, CH2OH), 3.34
(t, 2H, J = 7.0 Hz, NCH2), 3.22 (t, 2H, J = 7.3 Hz, He), 2.41 (s,
3H, CH3), 2.41 (bs, 2H, CH2), 2.28 (bs, 2H, CH2), 1.86–1.78 (m,
2H, CH2), 1.75–1.67 (m, 2H, Hb), 1.59–1.47 (m, 2H, Hd), 1.40 (s,
(S)-6-[{3-[(3-Dimethylamino-propyl)-(2-nitrobenzenesulfonyl)-
amino]-propyl}-(2-nitrobenzenesulfonyl)-amino]-2-[1-(4,4-dime-
thyl-2,6-dioxo-cyclohexylidene)-ethylamino]-hexanoic acid (10).
9 (615 mg, 0.69 mmol) was dissolved in CH2Cl2 (4.3 mL). TFA
(2.0 mL, 27.3 mmol) and Et3SiH (0.84 mL, 5.3 mmol) were added
and the resulting solution was stirred for 12 h at room temperature.
After evaporation of the solvent in vacuo and extraction of the
residue with CHCl3 (3¥) the product was dried under high vacuum
to yield 10 (655 mg, quant.) as a white foam. The product was
directly submitted to SPPS to avoid loss of the Dde-group.
t
9H, Bu), 1.37–1.27 (m, 2H, Hg ), 0.96 (s, 6H, CH3); 13C NMR
(CHCl3, 75 MHz) d 199.5 (C O), 197.1 (C O), 173.2 (C O),
169.1 (C), 148.2 (C), 133.7 (C), 133.2 (CH), 131.8 (CH), 130.7
(CH), 124.3 (CH), 108.5 (C), 83.4 (C), 59.3 (CH2), 56.9 (CH), 53.6
(CH2), 52.4 (CH2), 47.8 (CH2), 44.9 (CH2), 32.3 (CH2), 31.3 (CH2),
30.2 (C), 28.4 (CH3), 28.2 (CH2), 28.0 (CH3), 22.5 (CH2), 18.4
(CH3); HRMS (ES) Calc. for C29H43N3O9SNa 632.2612, found
632.2615.
General procedure for Fmoc Solid Phase Peptide Synthesis
N-(3-Dimethylamino-propyl)-2-nitrobenzenesulfonamide
(8).
To a solution of N,N-dimethyl-1,3-diaminopropane (6.3 mL,
50.0 mmol) and Et3N (6.7 mL, 50 mmol) in CH2Cl2 (100 mL) was
added Ns-Cl (5.54 g, 25.0 mol) in several portions. After addition
the resulting solution was stirred for 24 h. The mixture was
extracted with half saturated NaCl-solution (50 mL) and H2O
(50 mL). The organic phase was dried over Na2SO4, filtered and
the solvent was removed under high vacuum. The crude product
was purified by flash chromatography (SiO2, 5 : 1, CHCl3 : MeOH)
to yield 8 (6.21 g, 86%) as a yellow solid. (Rf 0.23 on SiO2, 5 : 1,
CHCl3 : MeOH); 1H NMR (CHCl3, 300 MHz, 300 K) d 8.05–8.00
(m, 1H, CH), 7.76–7.72 (m, 1H, CH), 7.59 (bs, 1H, NH), 3.11 (t,
2H, J = 5.9 Hz, CH2), 2.33 (t, 2H, J = 5.9 Hz, CH2), 2.16 (s, 6H,
CH3), 1.63 (app. qn, 2H, J = 5.9 Hz, CH2); 13C NMR (CHCl3,
75 MHz) d 148.1 (C), 133.8 (CH), 133.3 (CH), 132.4 (CH),
130.9 (CH), 125.0 (CH), 58.9 (CH2), 45.2 (CH3), 44.4 (CH2),
25.3 (CH2); HRMS (ES) Calc. for C11H18N3O4S 288.1013, found
288.1006.
Fmoc-protected amino acid (4.0 eq to resin loading), HOBt (4.0
eq) and HBTU (4.0 eq) were dissolved in DMF (5 mL) and
DIPEA (10.0 eq) was added. After five minutes pre-activation the
solution was transferred to the pre-swelled resin. 2-Chlorotrityl
chloride resin with a loading of 0.68 mmol g-1 was used for the
synthesis. The reaction was swirled for 2 h. Double coupling was
applied throughout the synthesis. After completion the solution
was drained and the resin washed with DMF (3¥), MeOH (3¥)
and CH2Cl2 (3¥). The completion of couplings was ascertained
by TNBS test. The Fmoc group was removed by treatment of the
pre-swelled resin with 20% piperidine in DMF (1 ¥ 10 min, 1 ¥
20 min).
Removal of Dde, Ns and final cleavage from the resin
A solution of 2% hydrazine in DMF (3 mL) was added to the
resin and swirled for 5 min. The washing step was repeated two
more times (1 ¥ 5 min, 1 ¥ 10 min). After deprotection the resin was
washed with DMF (3¥), MeOH (3¥) and CH2Cl2 (3¥). Coupling
of the following residue and Fmoc removal was done as described
above. Ns-cleavage was done by using 2-mercaptoethanol (20 eq)
and DBU (10 eq) for 30 min (3¥). After washing with DMF
(3¥), MeOH (3¥) and CH2Cl2 (3¥) the resin was dried under high
vacuum for 12 h. Final cleavage of the peptide from the resin was
done with 95 : 2.5 : 2.5 (TFA : TIPS : H2O) for 2 h. Filtration and
concentration of the filtrate in vacuo and precipitation with Et2O
yielded 1 (43 mg, 98%), 2 (44 mg, 97%), and 3 (37 mg, 86%),
respectively, as slightly yellow solids. 1: HRMS (ES) Calc. for
C32H65N10O8 717.4981, found 717.4984. 2: HRMS (ES) Calc. for
(S)-6-[{3-[(3-Dimethylamino-propyl)-(2-nitrobenzenesulfonyl)-
amino]-propyl}-(2-nitrobenzenesulfonyl)-amino]-2-[1-(2,4,4,6-
tetramethyl-cyclohexylidene)-ethylamino]-hexanoic acid tert-butyl
ester (9). PPh3 (2.51 g, 9.59 mmol) and DIAD (1.9 mL, 9.59
mmol) were dissolved in abs. THF (80 mL) at 0 ◦C. The mixture
was stirred until a precipitate was formed (15 min). To this
suspension was added 8 (2.43 g, 8.46 mmol) and stirred for another
20 min. After that time 7 (3.44 g, 5.64 mmol) in abs. THF (10 mL)
was slowly added via syringe. The mixture was warmed to room
temperature and stirred for 3 d. After evaporation of the solvent
in vacuo the crude product was purified by flash chromatography
(SiO2, 5 : 1, CHCl3 : MeOH). 9 (4.60 g, 93%) was obtained as a
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C34H67N10O9 759.5087, found 759.5088. 3: HRMS (ES) Calc. for
C32H65N10O7 701.5032, found 701.5032.
References
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Acknowledgements
This work was supported by the Volkswagenstiftung.
5486 | Org. Biomol. Chem., 2011, 9, 5482–5486
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