Immobilization of an artificial imine reductase within silica nanoparticles improves its performance

Article abstract of DOI:10.1039/c6cc04604e

Silica nanoparticles equipped with an artificial imine reductase display remarkable activity towards cyclic imine- and NAD⁺ reduction. The method, based on immobilization and protection of streptavidin on silica nanoparticles, shields the bioti

Full text of DOI:10.1039/c6cc04604e

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Immobilization of an artificial imine reductase
within silica nanoparticles improves its
performance†
Cite this:DOI: 10.1039/c6cc04604e
Received 1st June 2016,
Accepted 28th June 2016
Martina Hestericova,a M. Rita Correro,b Markus Lenz,c Philippe F.-X. Corvini,c
´
Patrick Shahgaldian*b and Thomas R. Ward*a
DOI: 10.1039/c6cc04604e
www.rsc.org/chemcomm
Silica nanoparticles equipped with an artificial imine reductase display environments.15 With the aim of improving its catalytic perfor-
remarkable activity towards cyclic imine- and NAD+ reduction. The mance, we set out to shield an artificial transfer hydrogenase
method, based on immobilization and protection of streptavidin on (ATHase) in a protective organosilica layer on silica nano-
silica nanoparticles, shields the biotinylated metal cofactor against particles (SNPs) and evaluate its catalytic performance towards
deactivation yielding over 46000 turnovers in pure samples and the reduction of imines and the regeneration of NAD+.16
4000 turnovers in crude cellular extracts.
Working on the development of nanomaterials endowed with
virus recognition properties,17,18 we have designed a chemical
Catalysis plays a crucial role in a variety of scientific fields strategy that allows embedding enzymes in a biomimetic and
including synthetic organic chemistry. Given this importance, a soft organosilica layer.19,20
wide range of synthetic systems, including heterogeneous
In the present work, we have expanded this shielding strategy to
solids, organocatalysts, metal complexes and enzymes, have artificial metalloenzymes and tested the resulting nanocatalysts for
been developed in the past century.1 Transition metal catalysts the reduction of cyclic imine 1 in the presence of various cellular
and enzymes possess complementary properties, which can be extracts. As shown previously with soluble ArMs, embedding the
exploited in the synthesis of various enantiopure compounds.2 biotinylated pianostool iridium complex [Cp*Ir(biot-p-L)Cl] within
The industrial use of enzymes is partially limited by their various streptavidin mutants (Sav hereafter) allows to access either
operational instability and, in some cases, high price.3 Therefore, (R)- and (S)-salsolidine 2 (Fig. 1), depending on the Sav mutant
significant efforts have been invested in developing immobilization (Table 1). This observation ensures that the biotinylated cofactor
strategies that may contribute to stabilize enzymes, while allowing [Cp*Ir(biot-p-L)Cl] is indeed incorporated within Sav, as the bare
their recycling.4
cofactor yields (rac)-salsolidine 2.
An artificial metalloenzyme (ArM) results from the incorporation
of a synthetic metal complex cofactor within a host protein.5 The
biotin–streptavidin technology has been widely exploited to ensure
the localization of a biotinylated cofactor within the host protein.6
As ArMs often bear a soft metal, cellular metabolites, including
organosulfur compounds present in the E. coli cellular debris, fre-
quently deactivate the metal.7 Such hybrid catalysts can be viewed as
complementary to both homogeneous catalysts and enzymes.2,8–14
d6-piano stool complexes have shown great potential for
in vivo catalysis as they display promising stability in biological
a Department of Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel,
Switzerland. E-mail: thomas.ward@unibas.ch
b Institute of Chemistry and Bioanalytics, School of Life Sciences,
University of Applied Sciences and Arts Northwestern Switzerland,
¨
Fig. 1 (a) Immobilization and protection of ATHase in a protective organosilica
layer yielding active and protected nanoparticles. The ATHase is covalently
anchored (1) on the surface of SNPs (dark grey), followed by self-assembly and
polycondensation of silanes (2), which eventually build a protective layer (3).
(b) ATHase of cyclic imine and structure of the iridium cofactors.
Grundenstrasse 40, CH-4132 Muttenz, Switzerland
c Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences
¨
and Arts Northwestern Switzerland, Grundenstrasse 40, 4132 Muttenz, Switzerland
† Electronic supplementary information (ESI) available: Experimental details,
methods, assays and spectra. See DOI: 10.1039/c6cc04604e
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Table 1 Reactions performed using purified proteins
and [Cp*Ir(biot-p-L)Cl]ÁSav@prot-SNP respectively only led to a
modest variation of the enantioselectivity of the hybrid catalyst
towards the reduction of imine 1 (Table 1, entries 6 and 10).
The activity (i.e. total turnover number, TON) of the SNPs was
established based on the iridium concentration. This latter was
determined by inductively coupled plasma mass spectrometry
(ICP-MS) for each SNP batch. Dried SNPs contain approximately
0.04 ng Ir per mg SNPs. Salsolidine reduction using [Cp*Ir(biot-
p-L)Cl]ÁS112A Sav SNPs yielded total TON of 4294 after 2 days
(Table 2, entry 1).
Entry Sav mutant
eea,b (%) Conv.a (%) TON
1
2
3
4
5
6
[Cp*Ir(biot-p-L)Cl]ÁSav WT
38
87
À70
À35
79
70
89
86
99
98
99
140
178
173
198
196
198
[Cp*Ir(biot-p-L)Cl]ÁS112A
[Cp*Ir(biot-p-L)Cl]ÁS112K
[Cp*Ir(biot-p-L)Cl]ÁK121A
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A
[Cp*Ir(biot-p-L)Cl]ÁR84A-S112A-K121A 68
Reactions were performed in 200 ml of reaction mixture. a Enantiomeric
excess and conversion were determined by HPLC analysis on a
b
Chiralpak-IC column. Positive ee values correspond to (R)-salsolidine
and negative ee values correspond to (S)-salsolidine.
In line with the performance of the soluble ArM, the
immobilized [Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav@prot-SNP
yielded the best results, with TON of 12885 and almost full
conversion (entry 11). Upon increasing the substrate concen-
tration (200 mM), a TON of 46 747 was obtained using SNPs
with [Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav@prot-SNP (entry 14).
Compared to the homogeneous ArMs (entries 5, 10 and 15
respectively), the TONs are significantly increased, at the cost
of slight erosion in selectivity. We hypothesize that the increased
rate results from the protective effect of the organosilane layer
combined with the increased hydrophobicity around the active
site. The preferential formation of enantioenriched salsolidine 1,
depending on the Sav mutant, unambiguously demonstrates
that the SNPs indeed contain the intact ArMs with preserved
quaternary structure. Importantly, the SNPs can be recycled at
least twice, at the cost of an erosion in conversion (entries 2, 3, 7,
8, 12 and 13), yet with virtually the same enantiomeric excess.
Control experiments with native SNPs or SNPs without the
presence of Sav (e.g. empty SNPs) did not show any activity
towards NAD+ or imine reduction (entries 16 and 17). To address
the synergistic effect of the silica layer and the streptavidin
host on the catalytic performance of the iridium cofactor,
a non-biotinylated [Cp*Ir(4C-L)Cl] analog was synthesized,
immobilized and protected within SNPs ([Cp*Ir(4C-L)Cl]@prot-
SNP). In contrast to the ArM, upon immobilization, the activity of
[Cp*Ir(4C-L)Cl]@prot-SNP decreases upon mineralization within
SNPs, (Table 2, entries 19 and 20).
We have also demonstrated the stability of enantioselectivity
over time (see ESI†). The time-point assay showed only a slight
erosion of enantioselectivity over the course of 7 days (decrease
of 11%), which could be explained by disruption of the protective
layer and leakage of the catalyst caused by friction in solution.20
However, the ICP-MS measurements of the reaction medium
after the reaction revealed only traces of iridium. We thus argue
that the decrease in activity is predominantly caused by loss of
nanoparticles during centrifugation and washing cycles.
With biomedical applications23 in mind, we evaluated the
regeneration of NAD+ by [Cp*Ir(biot-p-L)Cl]ÁS112A Sav@prot-
SNP. For this purpose, the formation of NADH was monitored
by absorbance spectroscopy at 340 nm (for detailed data
see ESI†). The results confirm that the immobilization does
not significantly affect the catalytic properties of the hybrid
catalysts towards the NAD+ reduction. The protected catalyst
[Cp*Ir(biot-p-L)Cl]ÁSav@prot-SNP has a initial lower reaction rate,
but overall slightly improved TONs compared to [Cp*Ir(biot-p-
L)Cl]ÁSav.
Single mutants [Cp*Ir(biot-p-L)Cl]ÁS112A Sav and [Cp*Ir(biot-
p-L)Cl]ÁS112K Sav were selected for immobilization and protection.
These ArMs afford opposite enantiomers of amine 2. Additionally,
the double mutant [Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav was
selected for immobilization in view of its remarkable activity
as purified ArM.21
¨
Native SNPs (240 nm in diameter) were prepared by the Stober
method.22 Next, in order to covalently anchor Sav on the surface of
the nanoparticles, the SNPs were amino-modified by incubation
with (3-aminopropyl)-triethoxysilane (APTES) and further reacted
with glutaraldehyde. Sav isoforms preincubated with the iridium
cofactor, [Cp*Ir(biot-p-L)Cl]ÁS112A Sav, [Cp*Ir(biot-p-L)Cl]ÁS112K
Sav and [Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav, were immobilized
at the surface of SNPs by reacting with the free aldehyde
functions present at the surface of the SNPs. The synthesis of
a protective organosilica layer was achieved by incubating the
SNPs with tetraethyl orthosilicate and APTES for one- and four
hours respectively, yielding protected SNPs (prot-SNPs, Fig. 2).
SNPs were imaged using a field emission scanning electron
microscope (FESEM). The micrographs revealed monodisperse and
well-defined nanoparticles with an average diameter of 260 nm. As
illustrated in Fig. 2, the SNPs equipped with the additional protec-
tive silica layer (prot-SNPs) display a homogeneous size distribution
with a diameter increase of approx. 10 nm upon protection.
Gratifyingly, immobilization and additional protection of
the ATHase on the SNPs to yield [Cp*Ir(biot-p-L)Cl]ÁSav@SNP
Fig. 2 Scanning electron micrographs of the SNPs; (A) native amino-
modified SNPs, (B) [Cp*Ir(biot-p-L)Cl]ÁS112A@prot-SNP, (C) [Cp*Ir(biot-p-
L)Cl]ÁS112K@prot-SNP and (D) [Cp*Ir(biot-p-L)Cl]ÁS112A-K121A@prot-SNP.
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Table 2 Asymmetric imine reduction yielding salsolidine 2 using silica nanoparticles equipped with an artificial transfer hydrogenase
Entry
Sav sourcea
Ir concentrationb (mM)
eec,d (%)
Conv.c (%)
TON
4294
2172
1863
16 990
1154
1
2
3
4
5
[Cp*Ir(biot-p-L)Cl]ÁS112A@prot-SNP
2.6
2.6
2.6
2.6
2.6
90
83
80
88
79
56
28
24
22
15
[Cp*Ir(biot-p-L)Cl]ÁS112A@prot-SNP 1st recycling
[Cp*Ir(biot-p-L)Cl]ÁS112A@prot-SNP 2nd recycling
[Cp*Ir(biot-p-L)Cl]ÁS112A@prot-SNP 200 mM substratee
[Cp*Ir(biot-p-L)Cl]ÁS112A Sav purified protein f
6
7
8
9
[Cp*Ir(biot-p-L)Cl]ÁS112K@prot-SNP
1.2
1.2
1.2
1.2
1.2
À56
À53
À58
À48
À66
22
12
9
10
5
3775
2000
1580
16 667
833
[Cp*Ir(biot-p-L)Cl]ÁS112K@prot-SNP 1st recycling
[Cp*Ir(biot-p-L)Cl]ÁS112K@prot-SNP 2nd recycling
[Cp*Ir(biot-p-L)Cl]ÁS112K@prot-SNP 200 mM substratee
[Cp*Ir(biot-p-L)Cl]ÁS112K Sav purified protein f
10
11
12
13
14
15
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A@prot-SNP
1.5
1.5
1.5
1.5
1.5
70
68
67
76
89
97
56
50
36
19
12 885
7294
6570
46 747
2513
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A@prot-SNP 1st recycling
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A@prot-SNP 2nd recycling
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A@prot-SNP 200 mM substratee
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav purified protein f
16
17
18
19
20
Native SNPs
0
0
2.6
80
80
0
0
0
0
0
0
0
8
42
26
0
0
444
104
66
Empty prot-SNPs
[Cp*Ir(biot-p-L)Cl] freeg
[Cp*Ir(4C-L)Cl] free g
[Cp*Ir(4C-L)Cl]@prot-SNPs
a
Reactions performed in a reaction buffer using 1 mg of SNPs in 100 ml reaction mixture with 20 mM substrate concentration at RT for 48 hours.
b
c
Determined by means of ICP-MS. The enantiomeric excess and conversion were determined by HPLC analysis on a Chiralpak-IC column (see
d
e
ESI for details). Positive ee values correspond to (R)-salsolidine 2 and negative ee values correspond to (S)-salsolidine 2. Reactions performed in
a reaction buffer using 1 mg of SNPs in 100 ml reaction mixture with 200 mM substrate concentration for 48 hours. Reactions were performed with
free purified enzymes with the same iridium concentration as used with SNPs. Reactions performed without the presence of Sav using the
f
g
iridium ligand 10 [Cp*Ir(4C-L)Cl] in reaction buffer.
In order to test the compatibility of SNPs with complex erosion in ee), which results in protein denaturation without
cellular media, we performed experiments in the presence of the presence of protective organosilane layer and a complete
E. coli cell lysates.
loss of activity (Table 3, entries 9 and 10).
In stark contrast to the homogenous ArMs, which lose most of
The catalytic experiments reported herein reveal several
their activity in the presence of cellular debris solution, the SNPs noteworthy features:
partially maintained their catalytic activity. Up to 80 TONs were
(i) Immobilization of Sav isoforms on silica nanoparticles
achieved with [Cp*Ir(biot-p-L)Cl]ÁS112A Sav@prot-SNPs and 171 and their protection with organosilane layer yields catalysts
TONs with [Cp*Ir(biot-p-L)Cl]ÁS112K Sav@prot-SNPs (for detailed with higher turnover number rates compared to the use of
data see ESI†). Other complex media were evaluated with purified proteins.
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav@prot-SNPs (Table 3).
(ii) Such nanoparticles can be recycled up to three times with
Up to 4500 TONs were obtained upon performing the imine only a slight erosion in selectivity.
reduction in urine. SNPs maintain their catalytic activity also
(iii) Protected catalysts can withstand the presence of cellular
in the presence of methanol (4000 TONs, accompanied by an debris without the need of diamide pretreatment.7
Table 3 Asymmetric imine reduction yielding salsolidine 2 using [Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav@prot-SNP
Entry
SNP
Cellular debrisa
eeb,c (%)
Conv.b (%)
TON
1
2
3
4
5
6
7
8
9
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav@prot-SNP
Milk
41
37
25
35
27
89
33
30
14
0
24
1
11
1
6
3
2569
158
4554
392
862
1873
132
20
Yeast extract
Urine pH 6
Blood pH 7
Blood serum
Empty vector CFE
Empty vector CL
GSH 5 mM
MeOH 50%
MeOH 50%
1
0.15
30
0
4024
0
10
[Cp*Ir(biot-p-L)Cl]ÁS112A-K121A Sav purified proteind
Reactions were performed in a reaction buffer using 1 mg of SNPs in 100 ml reaction mixture with 20 mM substrate concentration at RT for
b
48 h. a Reaction mixture contained 48 ml of crude cellular extract, organic solvent or stock solution of glutathione. Enantiomeric excess and
c
conversion were determined by HPLC analysis on a Chiralpak-IC column. Positive ee values correspond to (R)-salsolidine 2 and negative ee values
d
correspond to (S)-salsolidine 2. Reaction performed with purified protein without any protection against the organic solvent.
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In summary, we have successfully developed an artificial
biocatalyst consisting of a pianostool iridium cofactor within
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