Lipase active site covalent anchoring of Rh(NHC) catalysts: Towards chemoselective artificial metalloenzymes

Article abstract of DOI:10.1039/c4cc09700a

A Rh(NHC) phosphonate complex reacts with the lipases cutinase and Candida antarctica lipase B resulting in the first (soluble) artificial metalloenzymes formed by covalent active site-directed hybridization. When compared to unsupported complexes, these

Full text of DOI:10.1039/c4cc09700a

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Lipase active site covalent anchoring of Rh(NHC)
catalysts: towards chemoselective artificial
metalloenzymes†
Cite this: Chem. Commun., 2015,
51, 6792
Received 4th December 2014,
Accepted 10th March 2015
M. Basauri-Molina, C. F. Riemersma, M. A. Wurdemann, H. Kleijn and
¨
R. J. M. Klein Gebbink*
DOI: 10.1039/c4cc09700a
www.rsc.org/chemcomm
A Rh(NHC) phosphonate complex reacts with the lipases cutinase and supported Ru(Cp)–protein bifunctional catalytic hybrids9 for
and Candida antarctica lipase B resulting in the first (soluble) lipase labeling and dynamic kinetic resolution, respectively, were
artificial metalloenzymes formed by covalent active site-directed developed. ASD hybrids promise robustness of the covalent
hybridization. When compared to unsupported complexes, these new hybrid for ease of manipulation with reduced leaching, char-
robust hybrids show enhanced chemoselectivity in the (competitive) acterization by mass spectrometry, and anchoring of the metal-
hydrogenation of olefins over ketones.
locatalyst in a naturally selective environment (i.e. the lipase
active site). Furthermore, phosphonate esters are not restricted
The embedding of synthetic metallocatalysts in protein scaffolds to a particular lipase thus potentially allow for the screening of
allows for the development of a so-called 2nd coordination sphere different protein environments.10
around the metallic center, which can result in catalytic selectivity
Our next goal was to further develop the catalytic applications
due to the intrinsically chiral and bulky character of the protein of the ASD method by the use of a versatile ligand (see Scheme 1).
macromolecules, blocking specific sterically demanding transi- To this end, N-heterocyclic carbenes (NHCs) are attractive
tion states in a catalytic reaction.1 This way, proteins as naturally water- and oxygen-tolerant spectator ligands that present robust
abundant supports represent an alternative to the development s-donating monodentate coordination towards a myriad of metal-
of chiral, bulky or enlarged ligands. Moreover, the solubility of lic centers without restriction of the remaining coordination sites
organometallic species, normally restricted to organic media, is for catalytic performance.11 Functionalization of the N-substituents
expanded by the nature of the protein scaffold, whose macro- has allowed for their immobilization12 and they have been applied
molecular nature additionally allows for a facilitated separation recently in the construction of Grubbs-catalyst/protein artificial
of the artificial enzymes.
Pioneering work from Whitesides,2 optimized and extended selective behavior of such hybrids has not yet been documented.
by Ward,3 on the supramolecular (strept)avidin–biotin system
Rhodium species were our first target given their relatively
enzymes,13 however, to the best of our knowledge, the plausible
has provided early examples of the development of artificial facile synthesis. The absence of this metal in biological systems
metalloenzymes. Other hybridization strategies include alkyla- makes it an attractive moiety to extend enzyme reactivity. The
tion of amino acid residues,4 the use of apomyoglobin with hydrogenation of acetophenone in water with Rh(NHC)-based
metallated artificial cofactors5 and metal–DNA hybrids.6 Enantio- catalysts has been studied by Herrmann and Ku¨hn and proves
and regioselective catalysis can be achieved or further optimized the reactivity of such catalytic centers in aqueous media.14 With
by mutagenic treatment of the proteomic scaffold.7
other types of ligands, e.g. arenes or bidentate phosphines, the
Recently, we have studied the active site-directed (ASD) reduction of ketones as well as olefins via transfer hydrogenation
hybridization strategy using organometallic phosphonate esters
that covalently and irreversibly bind to the active serine residue
of lipases. In this manner, protein–ECE metallopincer hybrids8
Organic Chemistry and Catalysis, Debye Institute for Nanomaterials Science,
Utrecht University, Universiteitsweg 99, 3584CG Utrecht, The Netherlands.
E-mail: r.j.m.kleingebbink@uu.nl
† Electronic supplementary information (ESI) available: Detailed experimental
Scheme 1 Covalent inhibition of lipases with P-functionalized phosphonate
esters towards metal(NHC)–protein hybrids.
procedures and spectroscopical characterization of new compounds. See DOI:
10.1039/c4cc09700a
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with artificial enzymes has been addressed by Ward15 and Reetz.16
When monodentate phosphites were addressed by de Vries,17
bulky protecting groups were needed to avoid their oxidation; yet
no selectivity was found in spite of satisfactory catalytic activity.
Without the use of protein scaffolds, Noyori’s ruthenium catalysts
are among the current state-of-the-art catalysts for hydrogena-
tions, which in function of their phosphine ligands achieve
excellent enantioselectivities in organic media.18
After our successful experience with cutinase from fusarium
solani pisi, a lipase that reacts with hydrophobic esters without
an initial activation step,19 as host in the formation of semi-
synthetic enzymes,8,9 we addressed the preparation of Rh(NHC)–
protein semi-synthetic hybrids to form (the first) catalytically
active cutinase metallohybrid.20 and studied its behavior in
hydrogenations to explore the (enantio)selectivity of the active
site-embedded Rh(NHC) fragment.
Accordingly, a phosphonate cofactor was designed for an
orthogonal metal-NHC–protein orientation. Allylphosphonamidate
1 and brominated imidazolinium salt 2 were synthesized and
cross-coupled to form N-tethered NHC ligand 3. Deprotonation
with the non-nucleophilic base potassium hexamethyldisilazide
(KHMDS), followed by treatment with [Rh(m-Cl)(cod)]2 (cod =
cycloocta-1,5-diene) led to rhodium compound 4 as a mixture of
isomers. Next, the dimethylamide group was substituted with
Fig. 1 ESI-MS analysis of Rh-cut hybrid (top, [M]10+ calc.: 2127.43, found:
2126.59) and the determination of residual hydrolytic activity of cutinase
(bottom).
p-nitrophenolate (pNP) towards complex Rh-pNP (Scheme 2). exterior of the enzyme. In addition, no residual hydrolytic
The mono p-nitrophenyl ethyl P-propylphosphonate motif used cutinase activity was found when p-nitrophenyl butyrate (pNPB)
here has proven an effective inhibitor of cutinase and CalB,8,9,21 was treated with Rh-cut, leading to a hydrolysis of pNPB only by
in contrast to dinitrophenyl phosphonates, which can lead to the buffer, again confirming full inhibition of cutinase (Fig. 1,
slow hydrolysis of the phosphorous–serine bond.16b Distinctive bottom). These observations demonstrate the covalent character
chemical shifts of the P-nucleus in NMR for 1 through Rh-pNP, and single site hybridization in forming Rh-cut.
facilitate its characterization in combination with mass spectro-
The catalytic performance of Rh-cut was evaluated in the
metry (for details on synthesis and characterization, see the ESI†). hydrogenation of acetophenone, a prochiral ketone, under a H2
Next, cutinase was treated with an excess of Rh-pNP for the atmosphere (40 bar) at room temperature for 20 h in aqueous
formation of the hybrid. A dialyzed and denatured aliquot (10% biphasic conditions with CH2Cl2 (5%, v/v) in TrisHCl buffer at pH
formic acid) was analyzed by ESI-MS, showing complete conver- 8.5. The organic solvent was chosen to promote the interaction of
sion of cutinase and formation of the desired Rh-cut hybrid the protein with organic substrates; CH2Cl2 also has low acidity
(Fig. 1, top). Secondary peaks with considerably lower intensity and low coordination behavior with organometallics. At these
were also observed. These originate from minor impurities in conditions no product was observed. The same was observed
Rh-pNP, which due to their smaller size have a high inhibitory when the non-supported catalyst analog [RhCl(SIMes)(cod)] 5
competence (see ESI†), and do not arise from decomposition of (where SIMes
= 1,3-dimesityl-4,5-dihydroimidazolin-2-ilydene)
the hybrid. Also, a single hybridization stoichiometry of 1 : 1 was used (Table 1, entries 1 and 2). When the reaction with 5
was found, which discarded the association of Rh-pNP with the was not buffered, 1-phenylethanol was produced in 12% yield and
four cysteine (all in cystine form) and six lysine residues at the this significantly improved to a yield of 90% using a NaH2PO4/
Na2HPO4 buffer at pH 8.5 (Table 1, entries 3 and 4). The difference
in conversion using different buffers is attributed to the inter-
ference of the high concentration of chlorides in TrisHCl buffer
with the Rh center. Accordingly, using phosphate buffer, Rh-cut
proved to be an active artificial enzyme for the hydrogenation of
acetophenone, albeit at a lower yield of 27% (Table 1, entry 5).
Next, we investigated the hydrogenation of a prochiral
olefin, methyl 2-acetamidoacrylate, using 5 and Rh-cut under
equivalent conditions as the previous reaction and found
excellent yields with both catalysts towards the desired methyl
acetylalaninate product (Table 1, entries 6 and 7).
Although a clear decrease of activity of the Rh(NHC) motif by
Scheme 2 Synthesis of lipase inhibitor Rh-pNP.
its anchoring in the protein was found for the hydrogenation of
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Table 1 Catalytic hydrogenation of acetophenone 6 and methyl 2-
acetamidoacrylate 7 with catalysts 5, Rh-cut and Rh-calba
enzymes shows the versatility of the ASD method in protein
scope applicability.
Studying Rh-calb in catalytic hydrogenations again showed
no product enantioselectivity. On the other hand, the difference
in reactivity between acetophenone and methyl 2-acetamido-
acrylate was larger than with the Rh-cut analog. The effect of
CalB over the Rh(NHC) motif seemed more pronounced allow-
ing for complete blocking of the ketone in contrast to quanti-
tative hydrogenation of the olefin (Table 1, entries 8 and 9),
suggesting full chemoselectivity by this artificial enzyme.
In order to obtain more insight in the apparent chemo-
selectivity gained with the hybrids, we performed a series of
competition experiments using both substrates in the reaction
mixture with Rh-cut and 5 (Table 1, entries 10 and 11). The product
yields were slightly lower than in the separate reactions. This
decrease is attributed to the distribution of the catalyst’s turn-
over over the two substrates. The olefin was hydrogenated in 93%
and 78% by 5 and Rh-cut, respectively; in the same manner, the
reduction of the ketone yielded 71% and 15%. These results
again show an accentuated discrimination of the ketone when
the metalloenzyme is used (Fig. 2), supporting that the protein
backbone increases the difference between the reaction rates
of the susbtrates. At shorter reaction times (40 min, Table 1,
entries 12 and 13) the ketone was reduced in some 15% by 5,
whereas Rh-cut afforded no ketone reduction at all (at 63%
Entry
Catalyst
Substrate
Buffer
Product formationb (%)
1
2
3
4
5
6
7
8
Rh-cut
5
5
5
Rh-cut
5
Rh-cut
Rh-calb
Rh-calb
5
6
6
6
6
6
7
7
7
TrisHCl
TrisHCl
0
0
12
90
No buffer
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
27
499
499
499
0
9
6
10
11
12d
13d
6 + 7
6 + 7
6 + 7
6 + 7
71, 93c
15, 78c
15, 70c
0, 63c
Rh-cut
5
Rh-cut
a
Reactions carried out with H2 b(40 bar) at room temperature for 20 h
(see ESI; single measurements). Determined by chiral GC and GC-MS.
c
Yield for 1-phenylethanol and methyl acetylalaninate, respectively.
Reaction stopped at 40 min.
d
the ketone, hydrogenation of the olefin did not suffer from a yield of olefin reduction product), which further supports the
decrease in product formation. This difference in the influence observed protein-induced chemoselectivity. The group of Lu
of the protein could originate from a limited approach of has previously reported chemoselectivity when Mn–protein artifi-
acetophenone to the metallic center brought forward by the cial enzymes prevented consecutive overoxidation of thioanisole,
proteomic surroundings, or by dative interactions towards the as opposed to the unsupported catalyst.24 Our current study
metal center, e.g. from N and O donors reducing the oxophili- shows a change in reactivity of different chemical functional-
city of the catalyst; in these scenarios promoting the formation ities in different substrates.
of the alkane from the olefin but hampering the formation of
the alcohol from the ketone.
In conclusion, we have demonstrated for the first time catalytic
activity for a soluble artificial metallo-enzyme based on the ASD
Rhodium catalysts are known to show higher catalytic rates inhibition of lipases. The lipase hybrids reported here catalyze
in the hydrogenation of olefins than in the hydrogenation of the hydrogenation of the olefin methyl 2-acetamidoacrylate in
ketones.22 Hence, the difference in yield between 1-phenyl- excellent yields and ambient temperature but show a protein-
ethanol and methyl acetalaninate by Rh-cut does not show by induced discrimination in the hydrogenation of the ketone
itself a selectivity gain derived from the hybridization, but the acetophenone. The more sterically demanding active site of CalB
comparison of the yields achieved in both transformations as anchoring site resulted in exclusive hydrogenation of the olefin
between the unsupported catalyst 5 and the Ru-cut hybrid does by the corresponding hybrid. This complete chemoselectivity was
show a larger influence of the protein over the ketone hydro-
genation. In none of the reactions any enantioselectivity in
product formation was observed.
The latter observations encouraged us to investigate the
anchoring of the rhodium catalyst in a different protein
environment. Candida antarctica lipase B (CalB), a lipase with
similar exposure of the active site to solvent but at a deeper
location,23 was chosen given our experience on its successful
inhibition with metal-functionalized phosphonate esters.9
A
similar methodology as for the previous inhibition was followed
resulting in complete inhibition of this enzyme by a ten-fold
excess of Rh-pNP towards the 1 : 1 metalloprotein Rh-calb.
Treatment of pNPB with this hybrid showed no residual hydro-
lytic activity of CalB according to the release profile of pNP (see
the ESI†). Successful use of a single inhibitor with different
Fig. 2 Reaction outcome of the competitive hydrogenation of acetophe-
none vs. methyl 2-acetamidoacrylate (right) and the corresponding chiral
GC chromatograms (center) comparing catalysts 5 [Rh(cod)(SIMes)Cl] and
Rh-cut (left).
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¨
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¨
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While excellent chemoselective hydrogenation catalysts, prefer-
ring either ketone or olefin reduction, have been developed for
application in organic synthesis,18 our current results represent, to
the best of our knowledge, the first example of chemoselectivity in
reactions catalyzed by artificial metalloenzymes, thereby extending
the selectivity repertoire of this specific class of catalysts. These
findings may lead to the development of more advanced catalytic
tools for the selective conversion of a target substrate in a complex
mixture of substrates, which is of interest to the fields of systems
catalysis and synthetic biology.
This work was supported financially by the Dutch National
Research School Combination-Catalysis (NRSCC). The authors
thank Dr Arjan Barendregt from the Biomolecular Mass Spectro-
metry and Proteomics group at Utrecht University for his help in
the MS analysis of the hybrids.
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