Bioorganic hybrid OMS by straightforward grafting of trialkoxysilyl peptides

Article abstract of DOI:10.1039/c3tb20122h

A novel strategy for the synthesis of peptide-bioorganic hybrid silica materials is reported. It relies on the design of hybrid peptides bearing a trialkoxysilane group. These reactive monomers are covalently attached to silica without requiring any prior surface modification. As an example, a bioorganic-inorganic OMS aldolisation catalyst is prepared using the peptide sequence Pro-Pro-Asp-Lys.

Full text of DOI:10.1039/c3tb20122h

Journal of
Materials Chemistry B
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PAPER
Bioorganic hybrid OMS by straightforward grafting of
trialkoxysilyl peptides†
Cite this: J. Mater. Chem. B, 2013, 1,
2921
a
a
b
a
Said Jebors,ab Christine Enjalbal, Muriel Amblard, Ahmad Mehdi, Gilles Subra
*
*
and Jean Martineza
Received 25th January 2013
Accepted 17th April 2013
A novel strategy for the synthesis of peptide–bioorganic hybrid silica materials is reported. It relies on the
design of hybrid peptides bearing a trialkoxysilane group. These reactive monomers are covalently
attached to silica without requiring any prior surface modification. As an example, a bioorganic–
inorganic OMS aldolisation catalyst is prepared using the peptide sequence Pro-Pro-Asp-Lys.
DOI: 10.1039/c3tb20122h
www.rsc.org/MaterialsB
Organic–inorganic hybrid materials based on silica and lengths have been successfully anchored onto silica, greatly
obtained by the sol–gel process have attracted considerable limiting the scope of potential applications. Moreover, in all the
attention during the last decade. They constitute a fascinating cases, graing a bioorganic moiety on silica surfaces is achieved
class of materials combining the properties of organic moieties via ligation chemistry which required two mutually reactive
and the inorganic matrix.1–3 Indeed, silica can easily be func- functional groups on the material and on the graed
tionalized through trialkoxysilane chemistry, providing func- compound.
tional groups used as anchoring points for covalent
In this study, we rst report the preparation of hybrid bio-
immobilization of organic moieties.4,5 This strategy has been organic–OMS material by straightforward anchoring of peptides
used to anchor organic groups not only on silica nanoparticles6 derivatized with a trialkoxysilane moiety onto the pores of a
but also onto the inner pore surface of ordered mesoporous ‘blank’, non-functionalized, silica matrix.
silicas (OMS).7 The latter are of special interest because their
synthesis proceeds in the presence of surfactants, enabling the
tuneable control of this structure on the nanometric scale,
yielding pores from 2 to 30 nm in diameter.8 Compared to other
materials of undened porosity such as silica gels, the use of
OMS greatly rationalizes not only the inclusion of organic
1.5). The obtained solution was then added to 9.40 g (44.9 mmol)
compounds, but also the inclusion of larger biomolecules such
Experimental
Preparation of large pore SBA-15
4.0 g (0.067 mmol) of triblock copolymer EO20PO70EO20 called
P123 were dissolved in 160 mL of an acidic aqueous solution (pH
as enzymes and heme proteins.9 Recently, homopeptide nano-
composite materials were prepared by polymerization of various
amino acid N-carboxyanhydrides (NCAs) on the pore's amino-
functionalized OMS obtained either by post synthetic graing10
or by direct synthesis.11 Such OMS, combining structural
features of mesoporous silica and peptide properties, constitute
a new class of hybrid bioorganic–inorganic materials that could
nd applications in catalysis, separation or molecular recogni-
tion. So far, only homo-oligomers of amino acids of random
of TEOS. The resulting mixture was vigorously stirred for 3 hours
at room temperature until a clear solution appeared. In order to
start the polymerization process, 76 mg (1.8 mmol) of NaF were
added and the mixture was heated at 60 C. Aer aging under
regular stirring for 3 days at 60 ꢀC, the resulting material was
ꢀ
ltered off and the surfactant (P123) was removed by soxhlet
ꢀ
extraction over ethanol 95ꢀ for 24 hours. Aer drying at 100 C
under vacuum, 2.75 g of SBA-15 were obtained as white powder.
Preparation of triethoxysilyl peptides (compounds 1, 3 and 4)
To the solution of peptide (0.1 mmol) in 100 mL of DMF were
added DIEA (2.1 eq.) and 3-isocyanatopropyltriethoxysilane
(1.2 eq.). The reaction mixture was allowed to stir for 2 hours at
room temperature. Aer 120 min, the reaction was monitored
by HPLC. Ether (30 mL) was poured into the reaction mixture to
cause precipitation. The precipitate was suspended in ether
again and recollected. This procedure was repeated three times
to remove TICPS and DIEA. All crude compounds were analyzed
by analytical HPLC, LC/MS and NMR and used without further
purication.
a
´
´
Institut des Biomolecules Max Mousseron (IBMM), UMR5247 CNRS, Universites
Montpellier 1 et 2, 15 avenue Charles Flahault, 34093, Montpellier, France. E-mail:
gilles.subra@univ-montp1.fr; Fax: +33 4 11759641
b
´
Institut Charles Gerhardt (ICG), UMR5253 CNRS, CMOS, Universite Montpellier 2,
`
Place Eugene Bataillon, 34095 Montpellier, France. E-mail: ahmad.mehdi@
univ-montp2.fr; Fax: +33 4 6714 3809
† Electronic supplementary information (ESI) available: Materials and methods;
peptide and hybrid peptide synthesis; hybrid peptide mediated aldolisation;
NMR LC/MS data; preliminary experiments of direct cleavage of trialkoxysilyl
peptides from solid support; chiral HPLC chromatograms. See DOI:
10.1039/c3tb20122h
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Preparation of triisopropoxysilyl peptide (compound 2)
To a solution of 5-oxo-5-((3-(triisopropoxysilyl)propyl)amino)
pentanoic acid in DMF were added DIEA (2.2 eq.) and BOP
(1.1 eq.) as the coupling reagent. Aer 15 min, a solution of
peptide and DIEA was added. The reaction mixture was stirred
for 2 h at room temperature and monitored by HPLC. Ether (30
mL) was poured into the reaction mixture to induce precipita-
tion of the hybrid peptide compound. The precipitate was sus-
pended in ether again and recollected. This procedure was
repeated three times to remove the organotrialkoxysilane and
DIEA. Crude compounds were analyzed by analytical HPLC, LC/
MS and NMR and used without further purication.
Preparation of hybrid peptide OMS by graing
General procedure. To a suspension of SBA-15 (500 mg) in
20 mL of dry DMF was added a hybrid peptide block (50–
100 mg). The reaction mixture was allowed to stir for 1 hour at
room temperature and 24 h at 80 ꢀC. The functionalized SBA-15
(SBA–Pep) was ltered off and washed with each 10 mL of DMF
(3Â), DCM (3Â), diethylether (3Â), before being dried under
vacuum conditions.
In the case of graing a protected hybrid peptide block, the
general procedure is followed by treatment of hybrid silica in
5 mL of TFA–DCM (2/1 v/v) for 1 h. The functionalized depro-
tected SBA-15 (SBA–Pep) was ltered off and washed with 10 mL
of DMF–DIEA (0.5%) (3Â), DMF (3Â), DCM (3Â), diethylether
(3Â), before drying in vacuo.
Scheme 1 Synthesis of trialkoxysilyl peptides.
Hybrid trialkoxysilyl peptide synthesis
while trialkoxysilylated peptides were not, which allowed easy
purication of the desired compounds. For the rest of this
study, we choose the isocyanate route to functionalize peptides.
One other hybrid tripeptide block was prepared with this
strategy (yield 80%) (Table 1, compound 3).
First of all, we had to set up a general procedure for the prep-
aration of trialkoxysilyl peptides. The synthesis of the peptides
is well established on a solid support by highly efficient proto-
cols allowing automation of ltration, addition of reactants for
coupling and intermediate deprotection steps.12 Cleavage of the
peptide from the support, usually with concomitant removal of
side chains protecting groups, is usually performed in an acidic
medium (i.e. triuoroacetic or uorhydric acid).
Our rst attempts to synthesize hybrid trialkoxysilated-
peptides consisted in the functionalization of a peptide still
attached on its solid support. Aer TFA-mediated cleavage, the
target hybrid peptide was not obtained, but peptide oligomers
formed by polycondensation of trialkoxysilane groups through
Si–O–Si bond formation were observed (see ESI†). As most of the
trialkoxysilane groups were hydrolyzed and condensed during
cleavage and subsequent work-up, we decided to introduce such
a moiety onto the peptide sequence aer removal of the peptide
from the solid support. This was achieved by reacting free
amino groups onto peptide sequences via isocyanate or acti-
vated carboxylic acid derivatives. Both strategies were used on
the N-terminus of a model tripeptide H-Gly-Phe-Glu-NH2
(Scheme 1, pathway A). Hybrid peptides 1 and 2 were obtained
in 65% and 70% yield, respectively (Table 1). Interestingly, the
Peptides present a large diversity of reactive chemical groups
on their side chains that are not all compatible with the strategy
presented in pathway A (Scheme 1). In particular, when peptides
contain more nucleophilic functions, they have to remain
masked during the reaction with ICPTS. In this case, mild
cleavage treatment was required to release peptides from the
resin while keeping acid labile protecting groups intact
(pathway B, Scheme 1). As an example, the peptide Boc-Pro-Pro-
Asp-Lys-NH2 was chosen to prepare a hybrid trialkoxysilyl
peptide block 4, which allowed to obtain the supported aldoli-
sation catalyst hybrid peptide silica materials 8–10 (Scheme 2).13
As it has been shown previously, the peptide N-terminus has to
be free to promote aldolisation catalysis.13 For this reason, peptide
anchoring on the solid support for synthesis was performed via
the side chain of a lysine added at the C-terminus of the sequence.
Grafting of hybrid peptide blocks on non-
functionalised silica
reaction between tripeptide H-Gly-Phe-Glu-NH2 and 3-iso- Hybrid trialkoxysilylated peptides 1–4 were graed in a
cyanatopropyltriethoxysilane (ICPTS) did not require the use of straightforward way onto the pore surfaces of non-functional-
coupling agents. Moreover, ICPTS was soluble in diethylether, ized large pore SBA-15 silica (see Experimental section), to yield
2922 | J. Mater. Chem. B, 2013, 1, 2921–2925
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Table 1 Hybrid trialkoxysilylated peptide blocks prepared according to pathway A (1–3) or pathway B (4)
Hybrid peptide
trialkoxysilane block
[M + H]+ m/z
found/calc.
[RSi(OH)2]+ m/z
Peptide–silica
material
Surface
areab
Loadingc
found/calc.a
Pore volumeb
0.92
%Nc
0.67
(mmol gÀ1
)
1
2
3
597.7/598.2
—/710.4
496.2/496.5
566.2/566.6
528.3/528.2
5
6
7
540
490
590
0.10
0.85
0.94
0.99
0.30
0.15
0.05
—/630.3
8a, 8b
9
430, 427
466
0.72, 0.70
0.79
1.2, 1.8
2.0
0.13, 0.19
0.21
10a, 10b
357, 341
0.62, 0.61
1.1, 1.7
0.11, 0.18
4
858.6/858.1
756.4/756.9
a
Siliconium ions [peptide–Si(OH)2]+ are the most abundant species detected during LC/MS analysis (see ESI). b Calculated by BET. c Calculated by
elemental analyses.
treatment was performed on hybrid silica 8a and 8b prior to TFA
deprotection, to hydrophobize the silanol groups to produce
hybrid silica 10a and 10b.
All these new bioorganic hybrid materials were characterized
by infra-red spectroscopy, elemental analysis and N2 adsorp-
tion–desorption isotherm (Table 1). N2 adsorption–desorption
measurements at 77 K showed type IV isotherms with a clear
H1-type hysteresis loop at relatively high pressure (Fig. 1C
and D), characteristic of mesoporous materials. XRD patterns
exhibited three low-angle reections, d100, d110 and d200,
characteristic of well-ordered SBA-15 type materials (Fig. 1B).
Further evidence for an ordered hexagonal structure was
provided by transmission electron microscopy (TEM) (Fig. 1A).
It is worth noting that the TEM and XRD patterns were
similar to those obtained before peptide graing (see ESI†).
Taken together, these results proved that the properties of OMS
were conserved aer chemical modication with peptide units.
The efficiency of peptide loading of hybrid OMS was determined
from elemental analysis ranges (Table 1). Depending on the
nature of the hybrid peptide, loading ranges from 0.05 to
Scheme 2 Synthesis of bioorganic hybrid peptide silica materials.
hybrid bioorganic–inorganic materials 5–8, respectively 0.21 mmol gÀ1, which corresponds to a molecule of peptide for
(Scheme 2). Briey, SBA-15 in suspension in a DMF solution about 60 Si atoms. Slight loading differences were observed with
containing the hybrid peptide blocks (1/10 w/w hybrid peptide– materials 5 and 6, which were obtained with the same peptide
SBA-15) was stirred for 1 hour at r.t., and then for 24 hours at sequence coupled to different spacers (hybrid peptides blocks
ꢀ
80 C. This two-step procedure was designed to obtain a more 1 and 2). The longer the spacer, the higher the loading and the
homogeneous grating. The rst step performed at r.t. is slower smaller the pores. As expected, OMS 8b, obtained with a three-
than the one performed at 80 ꢀC, enabling the peptides to reach fold higher ratio of the hybrid peptide, had a higher loading
a deeper location within the pores without reacting quickly on than 8a (0.19 vs. 0.13 mmol gÀ1).
the most accessible surface. Two different ratios of hybrid
Loadings were in accordance with the BET surface areas
peptide 4 (1/10 or 3/10, w/w, hybrid peptide–SBA-15) were used (Table 1). In all cases and as expected, graing induced a
for the synthesis of protected hybrid silica 8a and 8b. 8a was signicant decrease in the pore size (6.1 nm), surface and
treated with TFA to remove tert-butyl and Boc protecting groups, volume, compared to the starting non-functionalized SBA-15
yielding unprotected hybrid silica 9. Alternatively, a HMDS silica (7 nm, 674 m2 gÀ1, 1.18 cm3 gÀ1). It is worth noting that
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Fig. 1 TEM (A) and XRD patterns (B) of bioorganic hybrid OMS 10a. N2 adsorption–desorption isotherms of SBA 15 and hybrid OMS 5–7 (C) and 8–10 (D).
protecting group removal (materials 8a to 9) resulted in a pore
Overall, aldolisation proceeded more slowly than in the
size increase (0.72 to 0.79 cm3 gÀ1) of the hybrid material. In homogeneous system. Using acetone as both reactant and
contrast, silylation with HMDS resulted in a signicant pore solvent, total conversion was achieved within 24 hours with all
size reduction of the hybrid materials as shown by comparison the hybrid bioorganic inorganic catalysts. It is worth noting that
with 9 and 10a (0.79 cm3 gÀ1 vs. 0.62 cm3 gÀ1).
no reaction was observed with non-functionalized SBA-15.
The enantioselectivity of the reaction was evaluated by
Remarkably, organic contents (weight%) and diminution of
pore volume measured in these new hybrid OMS are very close to chiral HPLC (see ESI†). As already described for this aldolisa-
those commonly observed either by direct graing of small tion reaction, water was not favourable for enantioselectivity.19
organotrialcoxysilane into unfunctionalized SBA-15 (ref. 14) or by This was supported by comparing materials 9 and 10a. Using
adsorption15 or postmodication of already functionalized silica.16 acetone as solvent, hydrophilic OMS 9, which displayed free
In addition, the obtained graing density is comparable to this silanol groups yielded only 9% enantiomeric excess, while
obtained by postmodication of azido-functionalized mesoporous hydrophobic OMS 10a, which had been treated by HMDS
silica nanoparticles with poly-L-arginine using click chemistry.17
yielded 55% ee. Enantiomeric excesses obtained in the aldo-
lisation reaction using HMDS-treated hybrid OMS 10a and 10b
were very close. These results suggested that loading had a
limited inuence on enantioselectivity induction by the
catalyst.
The solvent used in the aldolisation reaction was described
to be of particular signicance. Thus, hybrid OMS 10a was
selected to perform a solvent screening (Table 2). In all solvents
tested, the aldolisation reaction proceeded slowly compared to
acetone; completion was not observed even aer 24 hours of
stirring. Reaction in water led to a complete loss of selectivity. In
contrast, a good selectivity (>95%) was achieved in DMSO. This
result was in accordance with the reaction performed in solu-
tion with other similar catalysts.20,21
Mesoporous peptide hybrid silica as an
aldolisation catalyst
As an example of the application of hybrid bioorganic materials
and to demonstrate that they retain the peptide's original prop-
erties in solution, materials 9, 10a and 10b were used as a support
catalyst (2 mol%) for the enantioselective aldolisation reaction of
p-nitrobenzaldehyde with acetone.13 On the other hand, the
peptide H-Pro-Pro-Asp-Lys-NH2 was synthesized and used as a
soluble catalyst (2 mol%)18 and non-functionalized SBA silica was
used as a negative control. Reactions were monitored by HPLC.
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Table 2 Aldolisation of p-nitrobenzaldehyde with acetone catalysed by hybrid
peptide–OMS
Notes and references
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H-PDDK–NH2
9
Acetone
Acetone
Acetone
Acetone
Chloroform
DMF
4
24
24
24
24
24
24
24
100
100
100
100
54
61
59
47
80
10
55
58
46
52
>95
2
10a
10b
10a
10a
10a
10a
DMSO
Water
Hybrid peptide OMS were compatible with a wide range of
solvents (displaying various ee proles) and could be removed
from the reaction medium by a simple ltration step. This is a
key issue when catalyst recycling is envisaged.
Conclusions
We describe in this study a simple and general approach for the
synthesis of hybrid bioorganic silica materials containing
peptides. Instead of relying on surface modication chemistry
to immobilize a bioorganic moiety on silica, we synthesized
hybrid peptides selectively functionalized with a trialkoxysilane
group at a suitable position. As demonstrated in this paper,
these bioorganic–inorganic hybrid building blocks could be
used to gra ‘blank’ silica surfaces by a straightforward che-
moselective reaction. It required no specic functionalization of
the silica surface and could be performed in the presence of
other functional groups (i.e. peptide side chains). Combining
peptide chemistry and sol–gel inorganic ‘so’ polymerisation,
this new strategy opens a broad avenue for the design of bio-
inspired hybrid materials with tunable properties and for the
direct synthesis of bio-inspired materials.
20 M. Messerer and H. Wennemers, Synlett, 2011, 499–
502.
Acknowledgements
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122, 2395–2396.
This study was supported by the University Montpellier1 Post-
Doc Program.
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