(η6-Arene) ruthenium(ii) complexes and metallo-papain hybrid as Lewis acid catalysts of Diels-Alder reaction in water

Article abstract of DOI:10.1039/c001630f

Covalent embedding of a (η⁶-arene) ruthenium(ii) complex into the protein papain gives rise to a metalloenzyme displaying a catalytic efficiency for a Lewis acid-mediated catalysed Diels-Alder reaction enhanced by two orders of magnitude in water.

Full text of DOI:10.1039/c001630f

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www.rsc.org/dalton | Dalton Transactions
(
g6-Arene) ruthenium(II) complexes and metallo-papain hybrid as Lewis acid
catalysts of Diels–Alder reaction in water†
a
a
a
a
b
c
Barisa Talbi, Pierre Haquette, Annie Martel, Fr e´ d e´ ric de Montigny, C e´ line Fosse, St e´ phane Cordier,
c
a
a
Thierry Roisnel, G e´ rard Jaouen and Mich e` le Salmain*
Received 25th January 2010, Accepted 22nd March 2010
First published as an Advance Article on the web 20th April 2010
DOI: 10.1039/c001630f
6
Covalent embedding of a (g -arene) ruthenium(II) complex
positioning of the complexes with respect to the protein host, i.e.
10,11
into the protein papain gives rise to a metalloenzyme display-
ing a catalytic efficiency for a Lewis acid-mediated catalysed
Diels–Alder reaction enhanced by two orders of magnitude
in water.
within its catalytic pocket.
enzymes were designed by Kaiser et al. from this protein.
recently, several metal complexes and a pyridoxamine cofactor
were attached to papain for the same purpose.
Historically, the first semi-synthetic
12,13
More
14
15
We synthesized complex 1a (Scheme 1) according to our
11
previously described procedure. This complex carried a chloroac-
etamide functional group on the arene ligand for covalent
Artificial metalloenzymes combining transition metal species and
biopolymers have attracted much attention during the last five
years as asymmetric catalysts operating under mild conditions.
Artificial Diels–Alderases resulting from the association of Cu(II)
ion with multi-dentate nitrogen ligands strongly interacting with
or covalently linked to DNA or albumins have been reported.
In these examples, the metal complex acted as a Lewis acid
coordinating the heteroatoms of the dienophile thus lowering the
energy of its LUMO. This very attractive approach is nonetheless
limited to two-point binding dienophile substrates that can chelate
to the Lewis acid. Several Lewis acids have been shown to
accelerate Diels–Alder (D–A) reactions of one-point binding
-
1
anchoring to papain. Crystals of 1a were obtained when the Cl
-
counter-anion was replaced by BF
4
. The molecular structure of
this molecule was solved by X-ray diffraction (Fig. 1). It shows a
three-legged piano stool structure typical of these complexes. The
dicationic aquo complex 1b was produced by chloride abstraction
2,3,4
+
in the presence of Ag ions. Furthermore, the non-functionalized
5
monocationic (2a and 3a) and dicationic (2b and 3b) complexes
were prepared as models for the catalytic studies.
4,6
7
dienophiles in water. However, the construction of artificial
metalloenzymes from these Lewis acids is not straightforward
because none of them can be readily associated with proteins.
6
Organometallic ruthenium(II) complexes, in particular [(h -
2
+
arene)RuL
2
(H
2
O)] with L being a bidentate P,P-, P,N-, N,O-
2
or N,N-ligand have been shown to catalyse D–A reactions in
8,9
organic solvents thanks to their Lewis acid character. Catalysis
involves transient coordination of the dienophile carbonyl group
9
to the metal by displacement of a coordinated solvent molecule.
Scheme 1 Numbered complexes.
As these complexes are air- and water-tolerant, we reasoned that
they should be suitable to construct artificial Diels–Alderases
through their association with a protein host. We chose the
cysteine endoproteinase papain (PAP) for this purpose because
this protein conveniently contains a single free cysteine residue
to which complexes may be anchored by covalent attachment
via one of the ligands coordinating the metal, allowing a precise
a
Chimie ParisTech (Ecole Nationale Sup e´ rieure de Chimie de Paris),
Laboratoire Charles Friedel, CNRS UMR 7223, 11 rue Pierre et Marie
Curie, 75231, Paris cedex 05, France. E-mail: michele-salmain@chimie-
paristech.fr; Fax: +33 1 43260061; Tel: +33 144276732
b
Chimie ParisTech (Ecole Nationale Sup e´ rieure de Chimie de Paris),
Laboratoire de spectrom e´ trie de masse, 11 rue Pierre et Marie Curie, 75231,
Paris cedex 05, France
c
Universit e´ de Rennes 1, UMR CNRS 6226 “Sciences Chimiques de Rennes”,
Fig. 1 ORTEP drawing of the molecular structure of 1a. Ellipsoids are
shown at 50% probability.
avenue du G e´ n e´ ral Leclerc, 35042, Rennes cedex, France
†
Electronic supplementary information (ESI) available: Synthetical pro-
, kinetics data of aquation
cedures, X-ray crystallographic data of 1a·BF
4
The D–A reaction between cyclopentadiene and acrolein was
chosen as a test reaction to evaluate the catalytic ability of the
complexes (and the metalloenzymes) in water. The formation of
of 1a and anation of 1b and of Diels–Alder reactions. CCDC reference
number 742394. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c001630f
This journal is © The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 5605–5607 | 5605

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Table 1 Kinetic parameters for the reaction of CpH (0.46 M) with
time plot showed that the reaction was completed in ca. 120 min
and the yield reached 90%. More interestingly, the same result was
obtained with 0.8 and even 0.2 mol% metalloprotein (Fig. 2). By
comparison, it took 360 min to reach completion without catalyst
and 250 min with the complexes alone (Fig. S6). The TOF was
◦
acrolein (0.046 M) in water at 2 C
3
-1a
Yield (%) TOF/h-1c
b
Entry Catalyst
k
obs/10 min
1
2
3
4
5
6
7
8
No catalyst
5.1 ± 1.6
10 ± 2
17.0 ± 0.7
17.4 ± 1.4
15.1 ± 1.6
4.8 ± 1.0
19.0 ± 1.3
62
61
65
65
68
62
50
54
—
10 mol% 1a
1.4
3.6
3.4
3.4
20
130
65
-1
equal to 220 h at 30 min (entry 9). This is an improvement by a
10 mol% 1b
factor of 60 with respect to the dicationic complexes 1b–3b alone
and 150 when related to the monocationic complex 1a at 10 mol%.
Notice that no enantiomeric excess was detected.
10 mol% 2b
10 mol% 3b
0.2 mol% 1a
0.2 mol% PAP-NEM
0.2 mol% 1a + 0.2 mol% 15.8 ± 1.3
PAP-NEM
9
0.2 mol% PAP-1a
14.6 ± 1.4
88
220
a
Pseudo-first order rate constant calculated from the non-linear regression
b
analysis of the kinetic plots (see ESI†).c Determined by GC after 250 min
using decane as an internal standard. Turnover frequency calculated at
3
0 min reaction time after subtraction of product contribution resulting
from uncatalysed reaction.
the endo- and exo- cycloaddition adducts was monitored during
2
50 to 500 min by chiral GC analysis. The pseudo-first-order
rate constants of reaction kobs were extracted from the non-linear
regression analysis of the time plots (Table 1, Fig. S6†).
In the absence of ruthenium species (entry 1, Table 1), the
D–A reaction went to 62% conversion in 250 min with a rate
constant of 0.005 min- and the endo/exo ratio was equal to 92/8.
It was anticipated that this reaction should take place in these
conditions, owing to the well-described rate increase observed in
1
16,17
aqueous medium for cycloaddition reactions.
When 10 mol%
Fig. 2 Kinetic plots of the Diels–Alder reaction between CpH and
acrolein in the absence (¥) and presence of 1a, (ꢀ), PAP-NEM (᭹), 1a +
PAP-NEM (ꢀ) and PAP-1a (ꢁ) at 0.2 mol%. Curves result from non-linear
regression analysis of the data.
(
with respect to the dienophile) of the dicationic complexes 1b–3b
were added to the reaction medium (entries 3 to 5), an increase of
the reaction rate by a factor of ca. 3 was observed. The yield in
bicyclo[2.2.1]hept-5-ene-2-carboxaldehyde after 250 min together
with the endo/exo ratio were identical to the experiment carried
1
out without Ru species. H NMR monitoring of the reaction in
To delineate the mechanism by which the metalloenzyme
catalysed the D–A reaction, additional experiments were run in
parallel with 1a and papain reacted with the thiol-blocking agent
NEM (PAP-NEM). It appeared that 1a alone had no significant
effect on the rate and final yield at 0.2 mol% loading with respect to
control (Fig. 2 and Table 1, entry 6). Conversely, PAP-NEM at 0.2
mol% loading led to an increase of the reaction rate (kobs multiplied
by 4, entry 7) with respect to control but had a detrimental effect on
the yield that reached a maximum of only 50% after 200 min. This
initial rate enhancement could be due to a non covalent catalytic
effect occurring by binding of the diene and/or the dienophile to
D
2
2
O at r.t. confirmed the formation of bicyclo[2.2.1]hept-5-ene-
-carboxaldehyde together with dicyclopentadiene. Although the
tolerance of a salen Ru(II) Lewis acid activity to the presence
18
6
of water has been previously mentioned, the ability of (h -
arene)Ru(II) Lewis acids to catalyse a Diels–Alder reaction in pure
water is demonstrated for the first time.
More surprisingly, cycloaddition between CpH and acrolein
was also accelerated in the presence of 1a although this complex
is not a Lewis acid (entry 2). This may be explained by the
aquation reaction undergone by 1a in aqueous medium in the
same timescale, allowing in situ generation of the catalytic species
17,20
a protein cavity in a similar fashion as cyclodextrins operate.
19
1
b. ‡ The lower efficiency of 1a compared to 1b is due to a low
This process could affect the rate of both cycloaddition reactions
1
amount of catalytic species at the early stages of the reaction.
Affinity-purified papain was allowed to react with 1a in the
presence of 0.15 M NaCl (to avoid aquation at this stage).
Chemoselective attachment of 1a to papain’s single cysteine
residue (Cys25) was evidenced by a complete loss of hydrolytic
activity of the enzyme upon reaction. ESI-MS analysis of the
hybrid protein gave an observed molecular mass of 23 914 ± 4,
corresponding to the adduct resulting from nucleophilic substi-
tution at the chloroacetamide function of 1a and replacement
of the ancillary chloro ligand by a formato ligand during the
MS analysis (Fig. S2†). The metalloprotein was then tested as
a catalyst in the Diels–Alder reaction between acrolein and CpH
at an initial loading of 1.6 mol% with respect to dienophile. The
previously shown to take place by H NMR. Alternatively, it
could be due to covalent catalysis, the carbonyl dienophile being
activated by reversible formation of iminium adduct resulting from
the condensation with the protein’s amine groups, just as amines
21
were shown to catalyse D–A reactions. Finally, addition of both
1a and PAP-NEM at 0.2 mol% to the reaction mixture gave the
same trend as PAP-NEM regarding the yield and the rate (entry
8). Thus, only covalent binding of 1a to papain led to an efficient
catalyst at low loading. The high conversion rate measured in this
case may be due to the fact that the catalyst only accelerated the
cycloaddition of CpH and acrolein. The partners taken separately
or not covalently combined had low or even no catalytic effect at
the same loading on the reaction of acrolein and CpH in water.
5
606 | Dalton Trans., 2010, 39, 5605–5607
This journal is © The Royal Society of Chemistry 2010

View Article Online
Reaction rate accelerations were previously observed for artifi-
cial metalloenzymes resulting from the supramolecular or covalent
anchoring of metal complexes to biopolymers as compared to the
8 J. Faller and J. Parr, Curr. Org. Chem., 2006, 10, 151–163; D. Carmona,
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complex alone.
1
1
1
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Dalton Trans., 2010, 39, 5605–5607 | 5607