In order to exclude that the observed RCM activity is
caused by traces of non-(strept)avidin bound catalyst, the
affinity of the guest C host interaction was determined. At
pH 7.0, a HABA-substitution titration (HABA: 40-hydroxyazo-
benzene-2-carboxylic acid) was carried out:13 HABA C Sav
and HABA C Avi display an induced CD signal (lmax 506 nm,
log Ka = 3.5 and 5.5, respectively14) which, upon addition of
Biot-m-ABA-1 decreases up to four equivalents. The resulting
CD spectra were analyzed and fitted using specfit (See
Fig. S1w).15 Despite the tetrameric nature of the host protein
and the use of a diastereomeric mixture of biotinylated ligands,
the data could be fitted with a single binding constant (i.e. non-
cooperative binding events): log Ka (Biot-m-ABA-1 C Sav)
8.88 ꢀ 0.6 (pH 7.0, 0.5 M MgCl2) and log Ka (Biot-1 C Avi)
8.21 ꢀ 0.053 (pH 7.0). As HABA precipitates at pH 4.0, the
Biot-1 C Avi affinity was estimated using tryptophane fluores-
cence quenching16 (log Ka (Biot-1 C Avi) > 9, See Fig. S2w).
We conclude that, despite the presence of chaotropic agents
and under RCM conditions—using [host protein]monomer
1 mM, Biot-spacer-1 0.73 mM—the biotinylated metathesis
catalyst is quantitatively incorporated (i.e. > 99%) either
within Sav or Avi, thus confirming that the observed RCM
activity is indeed provided by the artificial metalloenzyme.
The results presented herein demonstrate that the biotin-
(strept)avidin technology offers a versatile scaffold for the
creation of artificial metalloenzymes that display multiple
turnovers for ring closing metathesis. Current efforts are
directed at harnessing the power of site directed mutagenesis
to improve the performance of the hybrid catalyst and to
address challenging enantio- and diastereoselectivity issues.
Financial support for this project comes from the SNF
(Grants FN 200020_126366), the Marie Curie Training Network
(FP7/2007-2013 grant agreement n1 238434), the International
Centre for Frontier Research in Chemistry (FRC), Strasbourg
and the University of Basel. We thank Dr B. Jung for help with
Specfit, Prof. C. R. Cantor for the Sav plasmid and Belovo Egg
Science and Technology for a generous gift of avidin.
Scheme 2 Synthesis of the biotinylated complexes and operating
conditions for ring-closing metathesis.
Table 1 Selected results for the ring-closing metathesis of N-tosyl
diallylaminea
No.
Complex
Protein
pH
MgCl2 [M]
Conv. (%)
1
2
3
4
5
6
7
8
Biot-1
Biot-1
—
7.0
7.0
7.0
7.0
4.0
4.0
7.0
7.0
7.0
4.0
4.0
4.0
—
—
—
0.5
—
0.5
—
—
0.5
—
0.5
0.5
74
o1
8
33
41
71
17
6
54
79
95
95
Sav
Sav
Sav
Sav
Sav
Avi
Avi
Avi
Avi
Avi
—
Biot-m-ABA-1
Biot-m-ABA-1
Biot-m-ABA-1
Biot-m-ABA-1
Biot-1
Biot-m-ABA-1
Biot-m-ABA-1
Biot-1
9
10
11
12
Biot-1
Biot-1
a
Reaction conditions: [Sav]tetramer 0.25 mM, [catalyst] 0.73 mM,
[substrate] 15.21 mM, Vtot 120 mL (VDMSO 20 mL), pH 7.0 no buffer;
pH 4.0: acetate (0.1 M), 16 h, 40 1C, reactions were carried out in
triplicate (See SI for a complete list of all catalytic experiments).
Reactions carried out under rigorous exclusion of oxygen gave very
similar results.
modest: 17% conversion (i.e. > 3 turnovers) with Biot-1 C
Avi. Upon lowering the pH and/or adding MgCl2, the
conversions increased significantly: up to 95% conversion with
Biot-1 C Avi at pH 4.0 in the presence of MgCl2. The
performance of the protein-free catalyst Biot-1 was also
slightly improved by the acidic pH and the presence of MgCl2,
affording the RCM product in up to 95% conversion. Overall,
Biot-1 performed better than Biot-m-ABA-1 in the presence of
Avi, Table 1 and Fig. 1.
Notes and references
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Fig. 1 Graphical summary of the catalytic results obtained for the
RCM of N-tosyl diallylamine.
c
12066 Chem. Commun., 2011, 47, 12065–12067
This journal is The Royal Society of Chemistry 2011