Efficient conversion of alkenes to chlorohydrins by a Ru-based artificial enzyme

Article abstract of DOI:10.1039/c6cc08873b

Artificial enzymes are required to catalyse non-natural reactions. Here, a hybrid catalyst was developed by embedding a novel Ru complex in the transport protein NikA. The protein scaffold activates the bound Ru complex to produce a catalyst with high regio- and stereo-selectivity. The hybrid efficiently and stably produced α-hydroxy-β-chloro chlorohydrins from alkenes (up to 180 TON with a TOF of 1050 h^?1).

Full text of DOI:10.1039/c6cc08873b

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Rondot, C. Cavazza, M. Iannello, E. Boeri Erba, N. Burzlaff, F. Strinitz, A. Jorge-Robin, C. Marchi-
Delapierre and S. menage, Chem. Commun., 2017, DOI: 10.1039/C6CC08873B.
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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
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Efficient conversion of alkenes to chlorohydrins by a Ru-based
artificial enzyme.
.Received 00th January 20xx,
Accepted 00th January 20xx
Sarah Lopez,a,b,c Laurianne Rondot,a,b,c Christine Cavazza,a,b,c Marina Iannello,a,b,c Elisabetta Boeri-
Erba,a,d Nicolai Burzlaff,e Frank Strinitz,e Adeline Jorge-Robin,a,b,c Caroline Marchi-Delapierrea,b,c
and Stéphane Ménagea,b,c
DOI: 10.1039/x0xx00000x
www.rsc.org/
these two catalytic methods; they take advantage of the wide
structural diversity of proteins in combination with the broad range
Artificial enzymes are required to catalyse non-natural reactions.
Here, a hybrid catalyst was developed by embedding a novel Ru
complex in the transport protein NikA. The protein scaffold of metal-catalysed reactions.10 However, up to now only one
example of electrophilic diaddition has been described.11 In this
activates the bound Ru complex to produce a catalyst with high
regio- and stereo-selectivity. The hybrid efficiently and stably
produced α-hydroxy-β-chloro chlorohydrins from alkenes (up to
180 TON with a TOF of 1050 hr-1).
⊂
case, osmylation was catalysed by streptavidin OsO4 with high
enantioselectivity but moderate efficiency (26 TONs at best).11
Here, we report an efficient bio-inspired design for metal-catalysed
synthesis of chlorohydrins (Scheme 1) with attractive turnover
numbers in a one-pot process. To improve the sustainability of
production, we chose to base our artificial metalloenzyme on a
ruthenium complex embedded in a nickel transport protein, NikA
from E. coli (EcNikA).12 As part of a bioinspired approach, we
selected the Ru scorpionate complex, Ru(bpza)(CO)2Cl (Ru1)13
[Hbpza = bis(pyrazol-1-yl)acetate scorpionate ligand] which mimics
the protein environment in the active sites of iron dioxygenases.
Ru1 analogues perform electrophilic addition in organic solvents13
and possess a carboxylate function, which has previously been
In organic synthesis, oxidative functionalisation of olefins is
frequently required.1 However, apart from epoxidation and
dihydroxylation, it remains challenging to catalyse selective
asymmetric (di)addition of halogen electrophiles to olefins.2
Vicinal halohydrins are attractive versatile building blocks3 and
valuable bioactive materials.4 Two procedures can be used for
their production. The first is a two-step sequence involving an
epoxide intermediate and subsequent nucleophilic attack.5 The
second approach is applied industrially to produce propene
oxide; it involves direct electrophilic addition to a double bond
using HOCl (generated by adding Cl2 to water).6 The reaction shown to be useful in protein binding.12 The EcNikA⊂Ru1 hybrid
mixtures for these procedures are corrosive, and in addition to
the desired products, byproducts, such as vicinal dihalogens
and diols, are also generated. Alternative processes are
displays higher potential for chloride insertion processes than
natural metallic haloperoxidases, and demonstrates that it is
possible to produce novel non-natural catalytic activity from
scratch.14
therefore
actively
sought.7
Metal-based
halogeno-
functionalisation has been developed, but was found to be
quite unselective; enantioselectivity can be enhanced, but
efficiency suffers (high catalyst loading).8 Biocatalysis
represents an attractive, “greener”, alternative and
chlorohydrins have been produced but only via
a
stereoselective reduction of chloroketones using Escherichia
coli cultures overproducing alcohol dehydrogenases.9
Recently, artificial enzymes have emerged as a combination of
Scheme 1. Oxychloration of trans-β-methylstyrene with EcNikA-Ru1. See Table 1 for
experimental conditions.
The X-ray structure of the new Ru1 complex (in C2/C) shows
that the ruthenium (II) atom is octahedrally coordinated (see ORTEP
representation in Figure S1; relevant distances and angles are listed
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in Tables S1-S4. All the distances are in the range of those reported
for the related complex Ru(L)(CO)2Cl, in which L corresponds to
molecule B. However, probably due to the very low solubility
of Ru1 in the crystallisation solution, in all the crystals tested in
scorpionate ligands with substituents on the pyrazolyl rings (Ru-N, this study, and whatever the soaking conditions, Fe-EDTA was
only replaced by Ru1 in molecule B. Consequently, only
molecule B will be discussed hereafter. The X-ray structure
shows a single enantiomer (∆) with Ru1 bound to the protein
in its nickel-binding site at an occupancy close to one. Two
main interactions are involved in this binding: a salt bridge
between Arg137 and the oxygen atoms in the carboxylate
moiety of the bpza ligand, and an interaction with a structural
water molecule. This water molecule is conserved in all the
2.073(7) Å to 2.116(8) Å; Ru-CO, 1.877(10) Å and 1.941(10) Å; Ru–Cl
2.369(3) Å).13 ATR-FTIR spectra for Ru1 as a solid and in buffered
solution (10 mM HEPES, pH 7.0) exhibit two distinct νCO vibrations
at 2076 and 2005 cm-1 (Figure S2). These values match those of the
analogous cis-Ru(L)(CO)2Cl (L=η-C5H5) and
κ3-HB(pz)3(Tp)
complexes, and were attributed to the combination of two sets of
CO stretches.13 Positive-mode ESI-MS analysis of the complex in
water revealed the presence of one major fragment at m/z 422.9 structures of EcNikA interacting with metal complexes
described so far.12a Compared to free Ru1, protein-bound Ru1
(100%) and several minor fragments, at 406.9 (15%), 384.9 (8%) and
366.9 (6%), which were attributed to [Ru(bpza)(CO)2Cl + K+]+,
undergoes a slight rearrangement. This rearrangement did not
[Ru(bpza)(CO)2(Cl)
+
Na+]+, [Ru(bpza)(CO)2Cl
+
H+]+ and
affect the ruthenium ion, which is hexacoordinated by the two
[Ru(bpza)(CO)2(OH2)]+, respectively. The complex therefore largely
retains its structure in aqueous medium (Figure S3), although some
chloride may exchange with water. Conductivity measurements
corroborated this result, indicating a molar conductivity of 110
S.cm2.mol-1, which is within the range of a 1:1 electrolyte.15
Onsager plots confirmed that Ru1 behaves as a 1:1 electrolyte (by
comparison with KCl), thus validating the presence of Cl- as a
counter-anion in buffered solution (Figure S4).
nitrogen atoms of the pyrazolyl rings, the chloride ion, an
oxygen atom from the carboxylate ion, and the CO trans to the
pyrazolyl nitrogen (CO 1). The major difference is the position
of the second CO (CO 2), which is no longer trans to the
oxygen in the carboxylate moiety (observed at a Ru distance of
1.94 Å for free Ru1). Depending on the structures analysed,
the electron density for this region can be modelled as i) a
water molecule, ii) an unknown ligand, or (in most cases) iii) a
bent CO with an occupancy of 0.8. The binding mode for this
CO group has a Ru-CO distance of 2.3 Å and a Ru-C-O angle
between 180° and 115° (Figure 1). A similar deviation from
linearity was observed for Ru(II) dicarbonyl complexes bound
to lysozyme.16 Although its origin remains to be established,
the Ru-CO bond could be weakened due to i) the
accommodation of the ligand’s carboxylate within the protein
via the salt bridge, ii) the change to overall charge in the
protein, and iii) hydrogen bonding between the CO and two
water molecules. In investigations into carbon monoxide
binding to myoglobin, CO was observed by X-ray diffraction to
be bent out of the normal haeme plane, but these results were
challenged by calculations.17 This discrepancy was explained by
rotation of the Fe-CO, i.e., if the ligand was visualised just prior
to its dissociation from the structure. Thus, the CO ligand may
be labile in EcNikA⊂Ru1, making a free position available
where the oxidant can bind to Ru.
ATR-FTIR spectra for EcNikA⊂Ru1 displayed two large CO
vibration bands at 2082 and 2017 cm-1 and a weaker one at 2005
cm-1, suggesting the presence of at least two Ru-CO containing
species. The vibration pair at 2005 and 2082 cm-1 was similar to the
pair observed with Ru1 (Figure S2). The relative intensities of these
two features suggested the presence of a second cis-Ru(CO)2
Figure 1: Crystal structure and Fo-Fc omit Fourier electron density maps (green) of Ru1
in the nickel-binding site of EcNikA at 1.8 Å resolution. Electron densities for the two
CO and Cl were mapped at the 3.5 σ level. Blue: EcNikA, brown: Ru, green: chloride ion,
grey: CO and bpza ligand. Water molecules (W) are shown as red spheres.
species, characterised by the νCO at 2082 and 2017 cm-1. The most
intense of these signals could correspond to a new species, while
the other is probably due to partial release of Ru1 from the hybrid.
The slight shift in the low-energy νCO signal for Ru1 bound to the
protein is linked to changes to the overall charge of the complex,16
due either to an altered coordination sphere or to the weaker Ru-
CO bond observed. Peaks corresponding to the apoprotein and the
holoprotein were detected by native ESI mass spectrometry of
EcNikA⊂Ru1 (Figure S5). The estimated mass difference between
EcNikA⊂Ru1 was prepared by incubating the inorganic
complex with EcNikA (see ESI). Binding of Ru1 to NikA in
solution was verified by ICP-AES. A Ru/EcNikA ratio of 0.9 ± 0.1
was found, thus one complex binds per protein. The crystal
structure at 1.8 Å resolution was solved for EcNikA⊂Ru1 by
soaking EcNikA Fe-EDTA with Ru1 (Figure 1, Table S5, PDB
⊂
code: 5L8D). As previously, two molecules (called A and B)
were identified in the asymmetric unit,12a The main difference
between them is the relative position of the two lobes of
EcNikA, with molecule A in a more open conformation than
2 | J. Name., 2012, 00, 1-3
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the two forms was ≈385, which is in line with binding of the intact
Interestingly, Ru1 and RuCl3 complexes also displayed almost
no activity under these conditions, producing only traces of
epoxides (maximum yield 3% even after two hours). Hence,
embedding Ru1 in the protein activates it. The role of the salt
bridge between the protein and Ru1 in this activation was
demonstrated by Ru(DMSO)2Cl2, which was inactive. The
Ru1 complex to EcNikA.
Table 1: Alkene oxidation by NikA⊂Ru1 in buffer solution.
Experimental
conditionsa)
Yield
(%)b)
TON
c)
Substrate
Product
d.r.c)
regioselectivity and chemoselectivity noted for
α-hydroxy-β-
chloro chlorohydrin was also observed when using styrene and
related electron-rich aromatic alkenes as substrates (Scheme
2) while Ru1 alone remains inactive for all substrates.
Trans-β-
Std cond.
99
67
6.5:1
methylstyrene
225 eq. Cl-
instead of 75
No catalyst
NikA alone as
catalyst
81
9
182
-
-
-
OH
Br
OH
-
-
9
Ru1 or RuCl3
alone as
Cl
Cl
3
0
-
-
-
-
Yield : 9%, TON 6
Yield : 78%, TON 53
catalyst
⊂
NikA Ru
HO
OH
-
(DMSO)2Cl2
Cis-β-
Std cond
91
62
1:2.6
Cl
methylstyrene
Cl
Yield : 39%, TON 33, d.r. 4.7 : 1
Yield : 73%, TON 49
a) Standard conditions: 1/500/600/75 ratio for catalyst/substrate/oxidant/chloride,
respectively, for a final Ru1 or NikA⊂Ru1 concentration of 37 µM in Tris.HCl (40 mM)
pH 7.5 and HEPES (10 mM); reaction at room temperature for 10 min. b) Yield
calculated relative to initial Cl- concentration. c) Calculated from HPLC traces.
OH
Cl
OH
Ru(L)(CO)2Cl is known to catalyse epoxidation with hypervalent
iodine compounds as oxidants in organic media.13 Therefore,
Cl
Yield : 79%, TON 54, d.r. 16.5 : 1, e.e. 19%
Yield : 28%, TON 21
oxidation of alkenes by EcNikA⊂Ru1 and Ru1 was investigated
Scheme 2. Product scope. See Table 1 for experimental conditions. Yield calculated
relative to initial Cl- concentration. d.r. calculated from HPLC traces unless for indene
chlorohydrin calculated from GC traces.
under identical conditions (Table 1). Diacetoxyiodobenzene
was used as oxidant as it is more soluble in aqueous solution
than iodosylbenzene. H2O2 and alkylhydroperoxides were
inefficient under these conditions, but NaOCl provided
moderate activity (20% yield, 15 turn over numbers (TONs)).
Yields ranged from 9% for 2-bromostyrene to over 50% for
several substrates (styrene or methylstyrene); indene achieved
39% conversion, and 1-methylcyclohexene 28% (Figure S6).
The hybrid preferentially catalysed the conversion of enriched
double bonds, emphasising the electrophilic character of the
addition; thus, 4-bromostyrene, cis-stilbene, trans-stilbene,
methylcinnamate and trans-2-heptene were not converted
under the standard conditions. The reaction was totally
Analysis of the products of trans-
EcNikA Ru1 revealed the exclusive formation of 2-chloro-1-
phenylpropanol (trans- -methylstyrene- -chlorohydrin); 20%
β-methylstyrene oxidation by
⊂
β
β
of the substrate was converted after 10 minutes (Figure S6).
The chloride source for this reaction was the HCl present in the
Tris.HCl buffer used during EcNikA purification and hybrid
synthesis, thus the final concentration of chloride anion was
estimated to be 2.5 mM (70 eq. relative to the catalyst). In
these conditions, the reaction yield was 99%. The reaction was
partially controlled by the free halide concentration. Upon
regiospecific for α-hydroxy-β-chloro chlorohydrin but
displayed moderate diastereoselectivity for unsymmetrical
disubstituted olefins. The diastereomeric ratio (d.r.) varied
from 16.5:1 for 1,2-dihydronaphthalene to 1:2.6 for cis-β-
methylstyrene (Scheme 2). The reaction is nevertheless
addition of extra Tris.HCl--up to 0.5 eq. chloride per trans-β-
methylstyrene (225 eq. catalyst)--the conversion yield reached
a plateau. The same β−chlorohydrin was exclusively produced
in these conditions (81% yield; 182 TONs), which corresponds
to a 3-fold increase of TONs (Table 1).‡ This last condition
stereospecific, as the d.r. values obtained from cis- and trans-
β-methylstyrene oxidation products attest ((1S,2S/1R,2R) in
the case of the cis configuration of the substrate) (Figure S7).
Finally, by chiral HPLC we observed an enantiomeric excess of
demonstrated the high efficiency of EcNikA⊂Ru1, underlined
19±5% only when
a
rigid substrate such as 1,2-
by a turn over frequency (TOF) of 1050 TON.h-1. These values
dihydronaphthalene was used (Figure S8).
surpassed those of Ru1 analogues in organic solvent (26 TON
and TOF of 1 TON.h-1)13 or even with streptavidin
⊂OsO4 (27
The integrity of the hybrid was assessed by ESI-MS under
denaturating conditions in the absence of substrate. One
heterogenic fragment was detected at m/z 56689.28 after 5
minutes, and at m/z 57170.04 after 15 minutes. A fragment
smaller than apo-EcNikA (m/z 56303.3) was also detected, but
TON with an estimated TOF around 1 TON.h-1 but for diol
formation).11 No product was detected by GC-MS in the
absence of EcNikA⊂Ru1 or in the presence of EcNikA alone.
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with a low peak intensity (Figure S9). The higher m/z was Centre (ISBG: UMS 3518 CNRS-CEA-UJF-EMBL) is supported by
linked, as expected, to multiple additions of oxygen to the FRISBI (grant ANR-10-INSB-05-02) and GRAL (grant ANR-10-
protein residues (estimated to maximum 50 after 15 minutes LABX-49-01). We thank Dr. Luca Signor for help with mass
for a total of 502 amino acids), and the lower m/z corresponds spectrometry under denaturing conditions.
to a minor proteolytic product. As the reaction was quasi
quantitative and in the presence of substrate, we assume that
Notes and references
oxidation of the protein does not interfere with catalysis for 10
minutes,. An additional reaction cycle was performed to
confirm the hybrid’s stability. After one 10-minute reaction
cycle, activity could not be restored by adding each
‡ A higher halide concentration results in uncatalysed chlorohydrin
build-up (10% yield with 225 eq.), and produces traces of
dichlorinated and dihydroxylated products. Chloride concentration
was therefore maintained below 200 eq. to ensure
chemoselectivity.
component (EcNikA, Ru1 or EcNikA
⊂
Ru1
)
separately.
However, activity was partially recovered after adding extra
chloride. 95 TON and a yield of 63% was obtained for two
reaction cycles; efficiency was further increased (86% yield at
130 TON) when extra equivalents of oxidant were added along
with the chloride.
1
J. E. Bäckvall, in Modern Oxidation Methods, 2nd Edition, ed.
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2
3
When corresponding epoxides were used as substrates, only
diol-derivatives were obtained, thus the reaction mechanism
must not involve a nucleophilic attack on a transient epoxide.
4
5
6
7
Consequently,
α-hydroxy-β-chloro chlorohydrin must be
produced as a result of electrophilic attack of the double bond
by a chloronium intermediate with exclusive Markovnikov
regioselectivity.18 The nature of the oxidising species is
currently under investigation but Cl2 was not detected and
NaOCl is excluded on its poor reactivity under our catalytic
conditions (see above and ESI). The precise nature of the
chloronium intermediate remains to be established, but
activation of the oxidant by the Ru1 embedded in the hybrid
plays a role in its production. Observation of the replacement
8
9
10 (a) Y. Lu, N. Yeung, N. Sieracki and N. M. Marshall, Nature,
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of CO by a water molecule in EcNikA
⊂ Ru1 suggests that the
resulting Ru(bpza)(CO)(Cl)(OH2) activates the hypervalent
iodine. The release of one equivalent of CO into the headspace
of the reaction flask upon completion of the catalytic reaction
supports this hypothesis (Figure S10). Conversely, the catalysis
was fully inhibited when performed under positive CO
atmosphere. No CO was produced by the oxidant alone, in the
presence of the protein and/or chlorohydrin; the presence of
the Ru1 complex, either in the hybrid or in solution in the
buffer, was absolutely required. A residual amount of CO (0.2
equiv. after 30 mins) was produced by Ru1. Consequently, Ru1
in aqueous medium is inactive because both CO ligands are
maintained in the coordination sphere during the catalytic run.
Chlorohydrin formation by
a stepwise metal-based
13 (a) G. Turkoglu, F. W. Heinemann and N. Burzlaff, Dalton
Trans., 2011, 40, 4678-4686. (b) G. Tuerkoglu, S. Tampier, F.
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mechanism is relatively unusual in catalysis, and our work
represents a novel example of how bioinspired strategies can
help develop new synthetic methods, competing with
biocatalysis (182 TONs and a TOF of 1050 h-1) . The impact of
the protein scaffold in the hybrid presented here is
demonstrated at two different levels: i) the protein’s activation
of the metal-based catalysis; ii) the (enantio)selectivity. These
results pave the way for the development of new catalytic
oxidation pathways with artificial metalloenzymes.
This work was supported by the French National Agency for
Research (grant ANR-14-CE06-0005-01), Labex ARCANE (ANR-
11-LABEX-0003-1), and recurrent funding from CEA, CNRS and
Univ. Grenoble-Alpes. The MS platform at Grenoble Instruct
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PhI(Y)2
Acꢀvaꢀon of a ruthenium complex by its inserꢀon into a protein scaffold leads to an
efficient non natural transformaꢀon of alkenes into α-hydroxy-β-chloro chlorohydrin