Ring-Closing and Cross-Metathesis with Artificial Metalloenzymes Created by Covalent Active Site-Directed Hybridization of a Lipase

Article abstract of DOI:10.1002/chem.201502381

A series of Grubbs-type catalysts that contain lipase-inhibiting phosphoester functionalities have been synthesized and reacted with the lipase cutinase, which leads to artificial metalloenzymes for olefin metathesis. The resulting hybrids comprise the organometallic fragment that is covalently bound to the active amino acid residue of the enzyme host in an orthogonal orientation. Differences in reactivity as well as accessibility of the active site by the functionalized inhibitor became evident through variation of the anchoring motif and substituents on the N-heterocyclic carbene ligand. Such observations led to the design of a hybrid that is active in the ring-closing metathesis and the cross-metathesis of N,N-diallyl-p-toluenesulfonamide and allylbenzene, respectively, the latter being the first example of its kind in the field of artificial metalloenzymes.

Full text of DOI:10.1002/chem.201502381

DOI: 10.1002/chem.201502381
Full Paper
&
Metathesis
Ring-Closing and Cross-Metathesis with Artificial Metalloenzymes
Created by Covalent Active Site-Directed Hybridization of
a Lipase
Manuel Basauri-Molina, Dide G. A. Verhoeven, Arnoldus J. van Schaik, Henk Kleijn, and
Robertus J. M. Klein Gebbink*[a]
Abstract: A series of Grubbs-type catalysts that contain
lipase-inhibiting phosphoester functionalities have been syn-
thesized and reacted with the lipase cutinase, which leads to
artificial metalloenzymes for olefin metathesis. The resulting
hybrids comprise the organometallic fragment that is cova-
lently bound to the active amino acid residue of the enzyme
host in an orthogonal orientation. Differences in reactivity as
well as accessibility of the active site by the functionalized
inhibitor became evident through variation of the anchoring
motif and substituents on the N-heterocyclic carbene ligand.
Such observations led to the design of a hybrid that is
active in the ring-closing metathesis and the cross-meta-
thesis of N,N-diallyl-p-toluenesulfonamide and allylbenzene,
respectively, the latter being the first example of its kind in
the field of artificial metalloenzymes.
Introduction
serine hydrolases and forms a covalent, irreversible bond with
the serine residue of the catalytic triad. This method links the
metallic center to the former active amino acid residue of the
enzyme, which is the location with greater potential for gener-
ation of a second coordination sphere. Moreover, the activity
of phosphonate esters is not limited to a single lipase; hence,
the choice of different lipases is an alternative strategy to the
use of mutagenic techniques for the screening of protein
scaffolds. Furthermore, catalyst leaching is prevented by the
covalent binding constitution.
The development of semi-synthetic metal–protein hybrids
through the combination of transition-metal catalysts and pro-
tein scaffolds represents a bioinspired approach to catalyst
design. Such hydrides may not only display enhanced catalytic
activities, but also show enhanced catalytic selectivities as
a result of a second coordination sphere that is created around
the metallic center by positioning it within the protein host.[1]
A handful of hybridization strategies have been developed to-
wards the formation of these conjugates, which include: supra-
molecular anchoring of metallated biotin motifs in streptavi-
din,[2] alkylation of cysteine and lysine residues with metallated
electrophilic ligands,[3] cofactor reconstitution,[4] embedding of
nanoparticles in proteins,[5] protein biosynthesis with artificial
amino acids,[6] and active site functionalization of enzymes.[7–8]
With an interest in the development of hybridization strat-
egies and the structural design of metalloproteins, our group
has reported a method for the active-site-directed covalent
anchoring of organometallic catalysts in lipases.[9–10] A phos-
phonate ester that is functionalized with an organometallic
fragment acts as a suicide inhibitor of the enzymatic family of
Following this method, we reported the successful enhance-
ment of the chemoselectivity that is exerted by a Rh(NHC)
hydrogenation catalyst (NHC=N-heterocyclic carbene) through
its covalent anchoring in the active site of the lipases cutinase
and Candida antarctica lipase B (CalB) by a synthetic phospho-
nate–NHC cofactor.[11] Therein, the location of the active site in
CalB was deeper than that of cutinase, which resulted in
a larger extent of sterical influence of the enzyme host over
the metallocatalyst. In continuation of this research, we envi-
sioned the coordination of the designed phosphonate–NHC
cofactor with other metal centers to explore different catalytic
reactions with the corresponding semi-synthetic enzyme
hybrids, which is reported herein.
In the field of artificial metalloenzymes, there is a specific
interest in nonbiological, catalytic applications because these
extend the reactivity repertoire of enzymes, for example in
CÀC coupling reactions, which includes allylic alkylation[12] and
Diels Alder cyclizations.[13] Grubbs-type olefin metathesis
catalysts recently proved to be compatible with proteins as
substrates (in vitro).[14] This prompted the incorporation of
Grubbs-type complexes, based on Ru(NHC)s, in protein
structures to assess the potential promotion of selectivity that
[a] Dr. M. Basauri-Molina, D. G. A. Verhoeven, A. J. van Schaik, H. Kleijn,
Prof. Dr. R. J. M. Klein Gebbink
Organic Chemistry and Catalysis
Institute for Nanomaterials Science, Faculty of Science
Utrecht University, Universiteitsweg 99
Utrecht 3584CG (The Netherlands)
E-mail: r.j.m.kleingebbink@uu.nl
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502381.
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is conferred by the aforementioned generation of a sterically
demanding environment.[15]
Along this line, a number of artificial metalloenzymes for
olefin metathesis have been created.[8,16–18] Ward and Hilvert
reported the alkylation of a cysteine residue of a small heat-
shock-protein variant with a Grubbs catalyst, which contained
an electrophilic haloacetamide group; the resultant covalent
hybrid can perform the ring-closing metathesis (RCM) reaction
of N,N-tosyl diallylamine (TDA).[16] Ward and co-workers per-
formed the same reaction with different metalloenzymes,
which were formed by the site-specific supramolecular anchor-
ing of a biotinylated Grubbs catalyst in streptavidin.[18a] They
further optimized the structure of the hybrids to create an or-
thogonal biotinylation of the Grubbs catalyst, and they extend-
ed the RCM scope to synthesize a coumarin derivative.[18b] Fol-
lowing the alkylation of a cysteine residue, Schwaneberg and
Okuda incorporated the catalyst in a variant of the b-barrel
protein FhuA and performed ring-opening metathesis
polymerization (ROMP) of an oxanorbornene derivative.[17a]
Differences in catalytic activities and a slight influence over E/Z
product selectivity were found when using cofactors of differ-
ent length.[17b] Hirota and co-workers used haloacetamide-func-
tionalized Grubbs catalysts and positioned these in the active
site of the protease a-chymotrypsin to carry out the RCM reac-
tion of TDA and other polar and apolar diallylamines.[8] These
efforts highlight the current interest in the development of
semi-synthetic metalloenzymes for olefin metathesis.
Herein, we have synthesized and studied a series of new
Ru(NHC)–protein hybrid complexes, which have been
produced by the active-site-directed method by using Grubbs
catalysts with orthogonally positioned phosphonate-containing
lipase inhibitors (Figure 1). The bulkiness and tether length of
the tailored NHC ligands influenced both the hybridization
feasibility and the catalytic activity. These semi-synthetic
metalloenzymes for olefin metathesis proved active in the
ring-closing metathesis of TDA as well as the cross-metathesis
of allylbenzene, which is a reaction that has not yet been
explored with artificial enzymes.
Figure 1. Structural variations of Grubbs-like lipase inhibitors lead to
differences in the hybridization and catalytic activity of the targeted active-
site-directed covalent artificial metalloenzymes for olefin metathesis.
Results and Discussion
the imidazolinium salt 7 with the non-nucleophilic base
KHMDS to avoid nucleophilic attack on the phosphorous
center; the free carbene was then directly metallated with
Grubbs–Hoveyda I precursor by using a low concentration of
reagents at room temperature over a long reaction time of 2
days. Under more harsh conditions, it was noticed that intra-
or intermolecular interactions occurred. The purified product 9
still showed a limited lifetime of 5 days under inert conditions
in the presence of residual solvent; therefore, it was used for
the next reaction directly after purification (see below).
The short-tethered SIMes-containing (SIMes=1,3-dimesityl-4,5-
dihydroimidazolinium) preligand 7[11] was used as the building
block in the construction of the lipase inhibitor 1 (Scheme 1).
The Hoveyda chelate (o-propoxyphenyl-methylylidene)[19] was
chosen as the benzylidene ligand because of its wide use in
aqueous olefin metathesis. In parallel, we designed and
synthesized a ligand with a slightly more protecting behavior
towards the ruthenium center. To this end, we substituted the
trimethylphenyl N-substituents for the bulkier diisopropyl-
phenyl groups to give preligand 8 and, consequently, inhibitor
2 (Scheme 1).
Preligand 7 was synthesized through the Suzuki cross-
coupling of hydroboronated ethyl N,N-dimethyl allyl phospho-
namidate 4 and 1-mesityl-3-(4-bromo-2,5-dimethylphenyl)-4,5-
dihydroimidazolinium chloride 5. Metallation to give the
ruthenium complex 9 was achieved by initial deprotonation of
Hartwig has reported that Grubbs catalysts with isopropyl
NHC substituents showed increased resistance to aqueous con-
ditions in the presence of an enzyme compared with the corre-
sponding catalyst with mesityl substituents.[14d] Accordingly,
complex 10 was designed, for which the ligand precursor,
1-(2,6-diisopropylphenyl)-3-(4-bromo-2,6-diisopropylphenyl)-
4,5-dihydroimidazolinium salt
6 was synthesized by the
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lowed by heating at reflux with triethyl orthoformate led to
cyclization to give the desired ligand precursor 6. Afterwards,
the synthesis of preligand 8 was conducted in a similar
manner as that of 7 (see above). Likewise, the metallation pro-
cedure that we had developed for the formation of ruthenium
complex 9 was also successful here to give the complex 10
(Scheme 1), which showed improved stability with no changes
in either the color or the 1H NMR spectrum observed upon
storage. The final step for the synthesis of inhibitors 1 and 2
involved the elimination of dimethylamine by using hydrogen
chloride and then substitution of the resulting halide with
p-nitrophenolate (pNP).
Interestingly, the metallation of the carbene towards both
complexes 9 and 10 resulted in a mixture of isomers, in which
the Hoveyda ligand lies in a plane with the benzylidene in the
direction of either the substituted or the non-substituted mesi-
1
tyl group. According to the H NMR spectrum of the former
moiety, the ratio of these isomers was 72:28 and 74:26 for
complexes 9 and 10, respectively. However, for both inhibitors
1 and 2, only one isomer was observed (see the Supporting
Information for detailed spectra of organometallic species).
Cutinase was chosen as the host enzyme because of its reac-
tivity with hydrophobic substrates, its relatively exposed active
site,[20] and its capability to induce steric hindrance in the artifi-
cial metalloenzymes.[11] Firstly, a reaction to modify cutinase
with inhibitors 1 and 2 was carried out with an excess of com-
plex 1 in accordance with our previously reported biphasic
conditions for a Rh(NHC) inhibitor[11] (Tris-HCl buffer pH 8.5
with CH2Cl2, 5% v/v as co-solvent), which resulted in the suc-
cessful formation of hybrid Ru1–cut. The excess of complex
1 was eliminated by separation of water-insoluble species and
dialysis of the hybrid; the covalent constitution of the hybrid
allowed for the latter procedure without disintegration. ESI-MS
of a dialyzed and denatured sample of Ru1–cut showed the
desired conjugate with incorporation of the Grubbs complex
in a 1:1 stoichiometry (Figure 2, top). This result gives evidence
for the irreversible nature of the hybridization at the former
active site.
Scheme 1. Synthesis of Grubbs–phosphonate-based lipase inhibitors 1 and
2 (9-BBN=9-borabicyclo[3.3.1]nonane, dppf=bis(diphenylphosphino)-
ferrocene).
bromination of 2,6-diisopropylaniline followed by its coupling
with chloro oxallyl anilide 12 (Scheme 2). The resulting diamide
13 was reduced by using a combination of sodium borohy-
dride and trifluoroborane, and the subsequent protonation fol-
On the other hand, when cutinase was treated with inhibitor
2, the formation of the desired hybrid was not achieved even
after several attempts that used different reaction times and
co-solvents. We attributed the latter observation to the bulkier
character of the N-substituents of the inhibitor, in which the
isopropyl moieties possess a larger steric demand than the
methyl groups.[21] The difference between the inhibition of
cutinase with complexes 1 and 2 suggests a fine sensitivity of
cutinase towards these potential inhibitors. Interestingly, when
the reaction to incorporate complex 2 was carried out over
several days at room temperature with DMSO as the co-
solvent, we only observed incorporation of the ligand within
the enzyme without the ruthenium center (Figure 2, bottom,
hybrid L2–cut). This may originate from a slow decomposition
of the Ru–NHC complex in the reaction medium, which allows
cutinase to react with the remaining ligand. This indicated that
the unsuccessful formation of Ru2–cut was due to the
hindrance of the complete inhibitor structure and not only the
bulk of the bis(isopropyl)phenyl groups of the NHC ligand. The
Scheme 2. Synthesis of brominated imidazolinium chloride 6.
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Table 1. Catalytic evaluation of hybrids Ru1–cut and Ru3–cut for the
ring-closing metathesis of N,N-diallyl p-toluenesulfonamide.
Entry
Catalyst
Media[a]
Yield [%]
1
2
3
4
5
6
Ru1–cut
Ru1–cut
Ru1–cut
Grubbs–Hoveyda II
Ru3–cut
Buffer pH 8.5[b,c]
HCl 0.01m
–
–
1
90
–
Buffer pH 5
Buffer pH 5+cutinase
Buffer pH 8.5[c]
Buffer pH 5
Ru3–cut
84
[a] Tris-HCl buffer was used for pH 8.5 and sodium acetate buffer for
pH 5. [b] For 20 and 40 h. [c] With and without MgCl2.
Grubbs–Hoveyda II catalyst was also tested in the presence of
unmodified cutinase to determine if there was an undesired in-
compatibility between these components; however, the reac-
tion took place successfully under these conditions (entry 4).
Interestingly, the high activity of the commercial Grubbs
catalyst in the presence of native cutinase compared with the
notably lower activity of the Ru1–cut hybrid (Table 1, entries 3
and 4) points to little or no risk of leaching of the organome-
tallic fragment from the enzyme. Additionally, treatment of the
catalysis reaction mixture, which contains Ru1–cut, with an
excess of organic solvent to extract the product did not yield
the desired dihydropyrrole product. This result indicates that
the organometallic fragment is also not released after denatu-
ration. These observations stress the importance of the prior
formation and isolation of the hybrid for the evaluation of its
catalytic activity to avoid interference of unsupported catalytic
material in the medium.
Figure 2. ESI-MS spectra of Ru1–cut (top) and L2–cut (bottom), the latter as
a result of the reaction of cutinase with complex 2 for prolonged times.
latter insight led us to design a new Grubbs inhibitor (see
below).
For the evaluation of the catalytic activity of the new hybrid
Ru1-cut catalyst in an RCM reaction, we considered TDA as
a suitable substrate because of its recurrent use in olefin meta-
thesis with artificial enzymes.[8,16,18] Initially, a reaction was car-
ried out with Ru1–cut (5 mol%) in Tris-HCl buffer at pH 8.5,
CH2Cl2 (5% v/v), an excess of MgCl2 as a source of chloride,[22]
and under constant stirring to promote substrate–enzyme in-
teraction, but this resulted in no conversion at room tempera-
ture after 40 h (Table 1, entry 1). Previous reports mention
a negative effect of basic pH on the catalytic performance of
semi-synthetic hybrids.[16,17b,18a] In addition, a histidine side
chain on the enzyme may coordinate to the ruthenium center
and inhibit its activity.[23] Therefore, we dialyzed the hybrid cat-
alyst Ru1–cut and tested it in aqueous hydrochloric acid at
pH 2, but catalysis was still unsuccessful (entry 2); instead, pre-
cipitation of the protein content was observed, which was at-
tributed to denaturation of the cutinase at this very low pH.
Under less acidic conditions (pH 5), native cutinase has demon-
strated both stability and activity, albeit with lower rates.[24] In
view of all this, we prepared and tested the hybrid catalyst
Ru1–cut at pH 5 in acetate buffer; after 2 days, the hybridiza-
tion reaction led to the successful formation of the hybrid
Ru1–cut, which was stored at pH 5. In spite of these changes,
the RCM reaction gave the desired 1-tosyl-2,5-dihydropyrrole
In spite of the well-known solvent accessibility of cutinase’s
active site,[25] our results pointed to the probable blocking of
the metallic center after its embedment in the enzyme host. To
provide insight into this, we constructed computational
models of the hybrid.[26] By the docking of a Grubbs–Hovey-
da II structure that contained the phosphorylated propenyl tail
onto cutinase (Figure 3), it was found that the Grubbs catalyst
is not entirely embedded in the protein, but that the rutheni-
um center is surrounded by amino acid residues that are
known to partially block the entrance of substrates to the cuti-
nase active site (helical flap comprising of residues 81–85 and
a binding loop involving residues 178-186[27]). Moreover, the
benzylidene carbon, the labile site for the substrate, is in close
proximity to the protein scaffold, which surrounds it from
opposite sides (5.2 Š to Leu81 and 5.4 Š to Leu182). Therefore,
steric hindrance from the protein scaffold is a probable cause
of the lack of metathetical reactivity of Ru1–cut.
We then designed the new Grubbs-based inhibitor 3, which
contains the larger hexylene tether (Scheme 3). Accordingly,
phosphonate precursor 15 was synthesized by alkylation of di-
ethyl phosphite and sequential transformation into a chloro-
phosphate and the phosphonamidate 16 in situ. Cross-
coupling with imidazolinium salt 5 and further metallation
in
a
low yield of only 1% (entry 3). The commercial
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complexes 1 and 2 only one isomer was observed (see above).
Inhibition of cutinase was carried out at pH 5 for 30 h by using
an excess of complex 3; analysis by ESI-MS after dialysis
showed the desired hybrid Ru3–cut (see the Supporting
Information).
The new hybrid Ru3–cut gratifyingly afforded
a high
conversion towards the dihydropyrrole product in the RCM re-
action of TDA at pH 5 (Table 1, entry 6). At basic conditions of
pH 8.5 both with or without magnesium chloride, we
observed no conversion (entry 5). From these observations, it
can be deduced that the new ligand, in combination with the
acidic conditions and magnesium chloride, gives rise to a cata-
lytically active olefin metathesis hybrid. Magnesium chloride,
as mentioned above, acts as a chloride source, which is impor-
tant because it has been observed that one chloride ligand
bound to the ruthenium center can promote the metathetical
[18a]
reaction[22] (MgCl2
or KCl[8] have been used with other re-
ported artificial enzymes for olefin metathesis).
Elongation of the cofactor tether successfully allowed for en-
hanced catalytic activity in the hybrid owing to the reduction
of the steric hindrance of protein residues towards substrate
approach (see Figure 1). The increment of the aliphatic tether
could alternatively confer a degree of flexibility to the inhibitor,
which allows for a different directionality of the organometallic
fragment with respect to the enzyme pocket. Notably, an influ-
ence of the protein scaffold in Ru3–cut over the catalytic Ru
center is still observed, which
Figure 3. Docking modeling of Ru1–cut:[26] rear view (top left), side view
(top right), cutinase without the complex (bottom) with the complex in
green and the blocking residues in red.
leads to a difference in the cata-
lytic yield when compared to
the commercial catalyst (Table 1,
entries 4 and 6).
Finally, the activity of the two
metalloenzymes was tested in
the formal cross-metathesis of
allylbenzene (Scheme 4). Under
the optimized conditions men-
tioned above (acetate buffer,
pH 5, 5 mol% of catalyst, RT,
20 h), the desired product 1,4-di-
phenylbut-2-ene was formed in
a very low yield of 2% by Ru1-
cut but in quantitative yield by
Ru3-cut. Product E/Z ratios of
5:3 for Ru1-cut and 4:7 for
Ru3-cut were found, which are
comparable to the values that
were afforded by the unsupport-
ed Grubbs–Hoveyda II catalyst
Scheme 3. Synthesis of lipase inhibitor 3 and hybrid Ru3–cut.
(E/Z=4:9).
steps were equivalent to those of the previous inhibitors. A
mixture of isomers was again observed after the metallation to
give 18 with a ratio of 56:44, which is suggestive of a more
homogeneous mixture compared with the ratio of isomers
that were observed for complexes 9 and 10. Substitution of
the dimethylamine group gave the inhibitor 3, which also
showed a mixture of isomers with a ratio of 8:2, whereas for
Scheme 4. Catalytic evaluation of hybrids Ru1–cut and Ru3–cut for the
cross-metathesis of allylbenzene.
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was obtained from Novonordisk. Et2O, hexane, and toluene were
dried by using an MBraun MB SPS-800 solvent purification system;
dichloromethane and tetrahydrofuran (THF) were dried by distilla-
tion from CaCl2 and sodium/benzophenone, respectively, and
stored over 4 Š molecular sieves. Tris(hydroxymethyl)aminome-
thane buffer (Tris-HCl) and sodium acetate buffer were prepared in
degassed Milli-Q water and stored in Schlenk flasks. 1H, 13C, and
Conclusion
The development of a new hybridization technique represents
a fundamental advance in the search for selective and/or ver-
satile semi-synthetic enzymes. In this work, we have made use
of a number of favorable criteria that, to the best of our knowl-
edge, were separately documented in previous studies of artifi-
cial metalloenzymes for olefin metathesis. Active-site-directed
hybridization, covalent metal–enzyme bonding, orthogonal ori-
entation, and cofactor length tuning have all been integrated
into the semi-synthetic hybrids that are described herein.
Following the covalent inhibition of cutinase with Ru(NHC)–
phosphonate inhibitors, the formed hybrids could be purified
by dialysis, and they keep their covalent character even after
denaturation, as observed by mass spectrometry. This hybrid-
ization technique also produces an important interaction with
the incorporated Grubbs-type catalyst in the pocket around
the original active site of the enzyme.
31P NMR spectra were recorded at 298 K by using
a Varian
AS400 MHz NMR spectrometer at 400, 100, and 162 MHz, respec-
tively. Chemical shifts are reported in ppm and referenced against
the residual solvent signal. GC measurements were performed by
using a PerkinElmer Autosystem XL gas chromatograph equipped
with a PerkinElmer Elite-17 column. GC-MS analyses were per-
formed with
a PerkinElmer gas chromatograph Clarus 680
equipped with a PE Elite 5 MS column and coupled to a PerkinElm-
er mass spectrometer Clarus SQ8T with EI ionization. Electrospray
Ionization (ESI-TOF) mass spectra of chemical products were re-
corded with a Waters LCT Premier XE KE317 Micromass Technolo-
gies spectrometer; mass analyses of protein and hybrid products
were calculated as [M]n+ =(M+n)/n). Ultrafiltration dialysis of pro-
teomic samples was performed with Vivaspin 6 tubes,
10,000 MWCO (PEG membrane). All reactions were carried out by
using standard Schlenk techniques under inert conditions with an
atmosphere of N2.
Successful formation of Ru1–cut compared with the un-
successful formation of the analogue Ru2–cut proves that the
original hybrid design involved a high degree of sensitivity to-
wards steric effects at the enzyme’s active site. The use of the
larger but less rigid inhibitor 3 to overcome the poor catalytic
activity of Ru1–cut was successful by showing a significant
gain in catalytic activity, which suggests a sterical demand in
the binding pocket of cutinase not only in terms of volume
but also in terms of the shape and orientation of the incorpo-
rated organometallic fragment. In our experience with cuti-
nase, we had not before observed rejection of a phosphonate
inhibitor due to chemical hindrance or a variation in hybrid ac-
tivity as a function of the cofactor bulkiness. This sets a clear
example for the need of rationalized cofactor design, and it
states a reference for the sterical limit that cutinase can accom-
modate in its pocket. As reported recently for rhodium–protein
hybrids,[11] the resulting protein scaffold of cutinase has
promoted catalytic selectivity, and this could be enhanced by
using a different lipase. Nonetheless, changes in the activity as
a function of the cofactor is a promising tool for the search of
new artificial enzymes by using cutinase because of the
abundance of this relatively small enzyme.
Synthesis of imidazolinium preligands
Compounds 4, 5, and 7 were synthesized in accordance with our
reported procedures.[11]
4-bromo-2,6-diisopropylaniline 11: The synthesis was adapted
from a literature procedure by Chow et al.[28] A solution of Br2
(0.21 mL, 4.1 mmol) in CH2Cl2/MeOH (10 mL, 1:1 v/v) was added to
a stirred solution of 2,6-diisopropylaniline (0.74 mL, 3.9 mmol) in
CH2Cl2/MeOH (20 mL, 1:1 v/v) at room temperature by an addition
funnel over 1.5 h. The orange-red solution was stirred for 1 day.
Solvents were evaporated, and the resultant red solid was recrys-
tallized from hexane and further purified by column chromatogra-
1
phy (CH2Cl2) to give a light orange-red solid (0.47 g, 99%). H NMR
(400 MHz, CDCl3): d=10.09 (bs, NH2, 2H), 7.35 (s, ArH, 2H), 3.69 (m,
J=26 Hz, iPrCH, 2H), 1.28 ppm (d, J=6.4 Hz, iPrCH3, 12H); 13C NMR
(100 MHz, CDCl3): d=145.0, 128.0, 123.9, 123.8, 29.0, 24.2; HRMS
(EI): m/z calcd for [M]+: 255.0623; found: 255.2681.
2-((2,6-diisopropylphenyl)amino)-2-oxoacetyl chloride 12: Oxalyl
chloride (38.4 mL, 0.44 mol) was placed into a three-necked round
bottom flask, which was equipped with a condenser and an addi-
tion funnel, under Schlenk conditions, and it was cooled to 08C. A
solution of 2,6-diisopropylaniline (7.68 mL, 40.7 mmol) in dry
CH2Cl2 (60 mL) was placed in the addition funnel and added slowly
to the oxalyl chloride solution under a gentle stream of nitrogen
through the reflux condenser. Dry CH2Cl2 (20 mL) was used to rinse
the funnel, and the nitrogen flow was stopped. After the reaction
mixture was stirred overnight, the solvents were evaporated. Dry
Et2O (25 mL) was added, and the solids were removed by filtration.
The solvent was removed under vacuum, and hexane (60 mL) was
added to the residue. The mixture was vigorously stirred before fil-
tration under a nitrogen atmosphere, and the solids were washed
with dry CH2Cl2. Evaporation of the solvent gave the product 12
The difference in reactivity of the hybrids against an unsup-
ported catalyst in the presence of the enzyme is a simple
proof of the difference between metal–protein interactions
inside and outside of a lipase even with an enzymatic cavity
that is accessible by the solvent, as in cutinase. The trend in re-
activity between the metallohybrids in RCM is also reflected in
CM: in both reactions the short-tethered hybrid Ru1–cut is
outperformed by the larger hybrid Ru3–cut. This work
represents the first example of the versatility of artificial
metalloenzymes for mono- and bimolecular olefin metathesis.
1
Experimental Section
(4.94 g, 46%). H NMR (400 MHz, CDCl3): d=7.96 (bs, NH, 1H), 7.36
(t, J=7.6 Hz, ArH, 1H), 7.22 (d, J=7.6 Hz, ArH, 2H), 3.07 (b, 1H),
2.94 (b, iPrCH, 2H), 1.24 (d, J=7.2 Hz), 1.21 ppm (d, J=6.8 Hz,
iPrCH3, 12H); 13C NMR (100 MHz, CDCl3): d=168.9, 153.6, 145.7,
129.4, 128.7, 123.9, 29.0, 23.6 ppm.
Materials and methods
Chemical precursors were purchased from commercial sources and
used without further treatment, unless stated otherwise. Cutinase
Chem. Eur. J. 2015, 21, 15676 – 15685
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Full Paper
N1-(4-bromo-2,6-diisopropylphenyl)-N2-(2,6-diisopropylphenyl)-
oxalamide 13: solution of 4-bromo-2,6-dimethylaniline 11
compound 6 (0.82 g, 80%). 1H NMR (400 MHz, CDCl3): d=8.84 (s,
NCN, 1H), 7.47 (t, J=15.2 Hz, 1H), 7.38 (s, BrArH, 2H), 7.27 (d, J=
7.6 Hz, ArH, 2H), 4.79 (bs, NCH2CH2N, 4H), 2.99 (dsep, J=32.8 Hz,
iPr, 4H), 1.38 (d, J=6.8 Hz, iPrMe, 6H), 1.38 (d, J=6.8 Hz, iPrMe,
6H), 1.26 (d, J=6.8 Hz, iPrMe, 6H), 1.26 ppm (d, J=6.8 Hz, iPrMe,
6H); 13C NMR (100 MHz, CDCl3): d=158.8, 148.4, 146.0, 131.7,
128.5, 125.0, 55.3, 55.1, 53.4, 29.4, 29.3, 25.4, 25.2, 23.7, 23.6 ppm;
HRMS (ESI): m/z calcd for [MÀClÀ]+: 469.2213; found: 469.2233.
A
(5.15 g, 19.3 mmol) and Et3N (2.67 mL, 19.3 mmol) in CH2Cl2
(30 mL) was added dropwise to a solution of compound 12 (4.9 g,
19.3 mmol) in dry CH2Cl2 (100 mL) at 08C. At the end of the addi-
tion, another 0.5 equivalents of Et3N (1.3 mL, 9.7 mmol) was added.
The resulting mixture was stirred at 08C for 10 min, then at room
temperature overnight. The reaction mixture was filtered, and the
collected solid was washed with Et2O and dried under vacuum.
The remaining solids were dissolved in CH2Cl2 (40 mL), washed
with water, dried over MgSO4, and concentrated under vacuum.
The residue was precipitated from Et2O, and the solids were dried
under vacuum to give compound 13 as a white solid (5.04 g,
53%). 1H NMR (400 MHz, CDCl3): d=8.8 (bs, 1H, NH), 7.35 (d, J=
8.0 Hz, ArH, 1H), 7.32 (s, BrArH, 2H), 7.21 (d, J=7.6 Hz, ArH, 2H),
3.01 (dsep, J=34.4 Hz, iPrH, 4H), 1.21 (d, J=3.2 Hz, iPrCH3, 12H),
1.19 ppm (d, J=3.2 Hz, iPrCH3, 12H); 13C NMR (100 MHz, CDCl3):
d=159.7, 159.4, 148.2, 145.7, 129.6, 129.0, 128.9, 127.1, 123.7,
123.1, 29.2, 29.0, 23.5, 23.3 ppm; HRMS (ESI): m/z calcd for [M+H]+:
487.1960; found: 489.1948.
N1-(2,6-Diisopropylphenyl)-N2-(4-(3-((dimethylamino)(ethoxy)-
phosphoryl)propyl)-2,6-diisopropylphenyl)-4,5-dihydro-1H-
imidazol-3-ium chloride 8: In a glovebox, compound 4 (0.24 g,
1.36 mmol) was dissolved in dry, degassed THF (10 mL). 9-BBN
(0.33 g, 1.36 mmol) was dissolved in dry, degassed THF (15 mL) and
slowly added to the solution of 4. The resulting mixture was stirred
for 72 h, after which the solvents were removed under vacuum.
The product was used directly in the next reaction.
Compound 6 (0.627 g, 1.24 mmol) was dissolved in degassed DMF
(10 mL), [PdCl2(dppf)] catalyst (0.091 g, 0.11 mmol) was dissolved in
degassed DMF (20 mL), and K3PO4·H2O (0.29 g, 1.365 mmol) was
dissolved in degassed H2O (10 mL). The solution of compound 4
and 9-BBN (formed in situ, 1.365 mmol) was added to a Schlenk
flask and diluted in degassed DMF (20 mL). While stirring, the solu-
tion that contained compound 6 and the [PdCl2(dppf)] suspension
were added sequentially. Degassed DMF/water (9:1) was added to
obtain a total volume of 100 mL, and then the K3PO4 mixture was
added. The resultant black mixture was stirred at 1008C for 16 h
and then cooled to room temperature. The mixture was filtered
over Celite and washed with DMF (2”35 mL). The solvents were
removed under high vacuum, the resultant solid was dissolved in
CH2Cl2, and the remaining solids were removed by filtration. Owing
to the amphiphilic character of the product, this was extracted into
an aqueous solution with H2O (4”150 mL). The aqueous phases
were dried under vacuum, and the resultant dark oil was dissolved
in CH2Cl2 (15 mL). A spoon of activated carbon (Norit) was added;
the mixture was stirred for 1 h then filtered over Celite, washing
with CH2Cl2 (40 mL), and dried in vacuum to give the product 8
(0.6 g, 80%). 1H NMR (400 MHz, CD2Cl2): d=8.17 (bs, 1H, NCHN),
7.46 (t, J=15.6 Hz, 1H), 7.27 (d, J=7.6, ArH, 2H), 7.06 (s, BrArH,
2H), 4.84 (bs, NCH2CH2N, 2H), 4.13 (m, CH2CH2CH2P, 2H), 3.99 (m,
CH2CH2CH2P, 2H), 3.84 (m, CH2CH2CH2P, 2H), 3.03 (dsep, J=32.8 Hz,
iPr, 4H), 2.67 (bs, NCH3CH3, 3H), 2.64 (bs, NCH3CH3, 3H), 1.39 (d, J=
6.8 Hz, iPrMe, 6H), 1.28 (d, J=7.2 Hz, OCH2CH3, 3H), 1.23 ppm (d,
J=6.8 Hz, iPrMe, 6H); 13C NMR (100 MHz, CD2Cl2): d=158.1, 146.2,
145.2, 131.6, 129.2, 127.2, 125.0, 125.0, 59.2, 59.2, 55.3, 55.2, 53.4,
36.7, 36.5, 36.1, 36.1, 29.2, 29.1, 25.4, 25.4, 16.3, 16.2 ppm; 31P NMR
(81 MHz, CD2Cl2): d=36.09 ppm; HRMS (ESI): m/z calcd for
[MÀClÀ]+: 586.4032; found: 586.4029.
Diethyl hex-5-en-1-ylphosphonate 15: Diethyl phosphite
(1.85 mL, 14.37 mmol, 1.28 equiv) was added dropwise by syringe
to a suspension of sodium hydride (60% disp. in mineral oil,
0.66 g, 15.15 mmol, 1.35 equiv) in dry THF (6 mL). After the initial
gas evolution had ceased, the reaction mixture was stirred for a fur-
ther 30 min at room temperature followed by heating at reflux for
2.5 h. The reaction mixture was cooled to 08C, and 6-bromo-1-
hexene (1.5 mL, 11.22 mmol) was added dropwise by syringe.
Upon complete addition, the ice bath was removed and the reac-
tion mixture was stirred for 20 h at room temperature. H2O (50 mL)
was added, and the mixture was extracted with CH2Cl2 (3”
125 mL). The combined organic phases were washed once with
H2O and concentrated under vacuum. The residue was purified by
column chromatography (Et2O/MeOH, 9:1) to yield compound 15
as a pale yellow oil (1.71 g, 7.79 mmol, 69%). 1H NMR (400 MHz,
CDCl3): d=5.70–5.83 (m, 1H), 4.90–5.03 (m, 2H), 3.99–4.15 (m, 4H),
N1-(4-Bromo-2,6-diisopropylphenyl)-N2-(2,6-diisopropylphenyl)-
ethane-1,2-diaminium 14: H3B·SMe2 complex (4.9 mL, 51.7 mmol)
was added slowly to a suspension of the diamide 13 (5.04 g,
10.3 mmol) in toluene (20 mL) at room temperature. The resulting
mixture was stirred overnight at 958C under a nitrogen atmos-
phere. After the reaction mixture was cooled to room temperature,
HCl(aq) (1m) was added until the stirred solution was acidified
(pH 3), and the resultant mixture was stirred overnight. The precipi-
tate was collected by filtration, washed with Et2O, and dried under
vacuum. The solid was suspended in CH2Cl2 (30 mL) and the mix-
ture was cooled to 08C. Et3N (2.9 mL) was added slowly, and the
mixture was stirred at room temperature for 2 h. The solvent was
removed under vacuum to give a residue, which was purified by
silica gel column chromatography (hexanes/ethyl acetate, 8:2) to
give compound 14 (80 mg, 2%, because of the low yield, the fol-
lowing product, 6, was preferably synthesized directly from com-
1
pound 13, see below). H NMR (400 MHz, CDCl3): d=7.13 (s, BrArH,
2H), 7.06 (d, J=7.6 Hz, ArH, 2H), 6.82 (t, J=15.6 Hz, ArH, 1H), 3.73
(bs, NH, 2H), 2.96 (q, J=18.0 Hz, NCH2CH2N, 2H), 2.89 (dsep, J=
27.2 Hz, iPrH, 4H), 2.80 (q, J=22 Hz, NCH2CH2N, 2H), 1.27 ppm (d,
J=6.8 Hz, iPrCH3, 24H); 13C NMR (100 MHz, CDCl3): d=139.3, 134.6,
132.4, 129.0, 128.2, 125.8, 125.3, 122.8, 118.5, 111.1, 52.4, 28.0,
22.2 ppm; HRMS (ESI): m/z calcd for [M+H]+: 459.2375; found:
459.2405.
N1-(4-Bromo-2,6-diisopropylphenyl)-N2-(2,6-diisopropylphenyl)-
4,5-dihydro-1H-imidazol-3-ium chloride 6 (full synthesis from 13):
BF3·OEt2 (2.62 g, 2.28 mL, 0.0185 mol) was added to a suspension
of compound 13 (1.00 g, 2.05 mmol) and NaBH4 (0.47 g, 0.012 mol)
in THF (25 mL), and the mixture was heated at reflux for 18 h. The
reaction mixture was quenched with MeOH (2 mL) and HCl (1 mL,
10m), and the solvent was removed under vacuum. THF (10 mL)
and HCl (1 mL, 4m) were added, and the resulting mixture was
stirred overnight, after which the solvent was evaporated to give
an off white solid. Et2O (100 mL) and aqueous NaOH (150 mL, 1m)
were added, and the biphasic solution was stirred for 10 min to
dissolve the solid. The organic phase was separated and the aque-
ous phase was extracted with Et2O (2”90 mL). The combined or-
ganic phases were dried over MgSO4, and the resultant filtrate was
concentrated under vacuum, and the residue was dissolved in trie-
thylorthoformate (10 mL). HCl (0.2 mL, 10m) was added, and the
mixture was heated to 1208C for 18 h. The mixture was concen-
trated under vacuum, after which Et2O (70 mL) was added, and the
precipitate was collected by filtration, washing with CH2Cl2, to give
Chem. Eur. J. 2015, 21, 15676 – 15685
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2.01–2.09 (m, 2H), 1.53–1.79 (m, 4H), 1.40–1.51 (m, 2H), 1.30 ppm
(t, J=7 Hz, 6H); 13C NMR (100 MHz, CDCl3): d=138.2, 114.8, 61.4,
33.2, 26.2, 24.8, 21.9, 16.4 ppm; 31P NMR (81 MHz, CDCl3): d=
32.32 ppm.
tion was filtered by cannula. All volatiles were evaporated, and the
product was purified by column chromatography (degassed ace-
tone) collecting the light green band to give complex 9 as a green
oil (58 mg, 74 mmol, 38%). Rf =0.7 (degassed acetone); 1H NMR
(400 MHz, CD2Cl2): d=16.51, 16.45, (s, Ru=CH, 1H), 7.54, 7.08, 7.07,
6.97, 6.95, 6.93, 6.84, 6.82, (ArH, 8H), 4.83 (d, J=4 Hz, CH(CH3)2,
1H), 4.52 (m, CH2CH3, 2H), 4.15 (s, N(CH2)2N, 4H), 2.74 (m, ArCH2,
2H), 2.69, 2.67, 2.12 (s, ArCH3, 15H), 2.45, 2.43 (s, N(CH3)2, 6H), 2.41
(m, PCH2, 2H), 1.46 (d, J=4 Hz, CH(CH3)2, 6H), 1.28 (m, ArCH2CH2,
2H), 1.22 ppm (m, CH2CH3, 3H); 13C NMR (100 MHz, CD2Cl2): d=
295.2, 210.7, 206.3, 154.8, 151.9, 145.0, 142.6, 138.8, 131.8, 129.5,
129.3, 129.2, 128.7, 122.3, 122.1, 113.0, 112.9, 75.1, 73.6, 70.6, 58.9,
51.4, 36.4, 36.3, 35.9, 30.5, 28.9, 25.6, 24.2, 20.8, 20.7, 19.1,
16.0 ppm; 31P NMR (162 MHz, CD2Cl2): d=35.83 ppm; HRMS (ESI):
m/z calcd for [MÀClÀ]+: 754.2478; found: 754.2560.
Ethyl P-(hex-5-en-1-yl)-N,N-dimethylphosphonamidate 16: Oxalyl
chloride (1.97 mL, 23.29 mmol, 3 equiv) was added dropwise by sy-
ringe to a stirred solution of compound 15 (1.71 g, 7.79 mmol) in
CH2Cl2 (20 mL). The reaction mixture was stirred for 17 h at room
temperature, and then the volatiles were removed under vacuum.
This crude material was dissolved in CH2Cl2 (10 mL) and added
dropwise by syringe to a stirred solution of dimethylamine (33% in
absolute ethanol, 27.7 mL, 155.2 mmol, 20 equiv) at 08C. Upon
complete addition, the mixture was stirred for 5 min, the ice bath
was removed, and it was stirred for a further 20 h at room temper-
ature. The reaction mixture was concentrated under vacuum, and
the residue was purified by column chromatography (CH2Cl2, Rf =
0.6, TLC, developed in iodine chamber) to yield compound 16 as
a yellow oil (1.68 g, 7.67 mmol, 99%). 1H NMR (400 MHz, CDCl3):
d=5.70–5.82 (m, 1H), 4.89–5.01 (m, 2H), 3.95–4.06 (m, 1H), 3.79–
3.90 (m, 1H), 2.65 (d, J=9 Hz, 6H), 2.05 (q, J=7.0 Hz, 2H), 1.38–
1.76 (m, 6H), 1.26 ppm (t, J=7.0 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d=138.3, 114.7, 59.0, 36.1, 33.2, 29.9, 25.8, 24.5, 21.6,
16.3 ppm; 31P NMR (81 MHz, CDCl3): d=36.90 ppm.
Inhibitor 1: As a representative synthesis, a solution of hydrogen
chloride in Et2O (1m, 0.65 mL, 0.65 mmol) was added by syringe to
a stirred solution of complex 9 (50 mg, 0.066 mmol) in CH2Cl2
(5 mL) under Schlenk techniques, and the solution was stirred at
room temperature for 3 h. The volatiles were removed under
vacuum, CH2Cl2 (5 mL) was added, and a prepared solution of p-ni-
trophenol (9.2 mg, 0.066 mmol) and Et3N (0.020 mL, 0.14 mmol) in
CH2Cl2 (1 mL) was slowly added at room temperature, and the mix-
ture was stirred for 2 h. The volatiles were removed under vacuum,
and the product was purified by column chromatography (de-
gassed acetone) collecting the front green band to give complex
N2-(4-(6-((dimethylamino)(ethoxy)phosphoryl)hexyl)-2,6-dime-
thylphenyl)-N1-mesityl-4,5-dihydro-1H-imidazol-3-ium chloride
17: 9-BBN (0.46 g, 1.86 mmol, 0.6 equiv) was weighed in the glove-
box and added to a stirred solution of ethyl P-(hex-5-en-1-yl)-N,N-
dimethylphosphonamidate (0.68 g, 3.10 mmol, 1.1 equiv) in dry
THF (60 mL). The reaction mixture was stirred for 20 h at room
temperature and then concentrated under vacuum. A mixture of
[PdCl2(dppf)] catalyst (0.16 g, 0.20 mmol, 7 mol%) and compound
5 (1.15 g, 2.82 mmol) was added followed by degassed DMF
(150 mL). After this mixture was stirred for 5 min, K3PO4·H2O
(0.73 g, 3.44 mmol, 1.11 equiv) was added, and the reaction mix-
ture was stirred for a further 10 min. Degassed H2O (8 mL) was
added, and the reaction mixture was stirred for 20 h at 1008C (con-
version was monitored by TLC: CH2Cl2/CH3OH, 9:1). The reaction
mixture was concentrated under vacuum at 658C, and the residue
was azeotroped once with heptane and purified by column chro-
matography (CH2Cl2/CH3OH, 9:1, Rf =0.45) to yield compound 17
1
1 as a bright green, dense oil (70%). HNMR (400 MHz, CD2Cl2): d=
16.54 (s, Ru=CH, 1H), 8.10 (d, J=8 Hz, OArNO2, 2H), 6.94 (d, J=
8 Hz, OArNO2, 2H), 7.40, 7.38, 7.07, 6.90, 6.87, 6.82, 6.80 (m, ArH,
8H), 4.82, 4.81 (sep, CH(CH3)2, 1H), 4.55, 4.51 (m, OCH2CH3, 2H),
4.16 (bs, NCH2CH2N, 4H), 3.98 (m, ArCH2, 2H), 2.49–2.40 (m, CH2P,
2H), 2.13, 2.12, 1.47 (s, ArCH3, 12H), 1.45 (d, J=8 Hz, CH(CH3)2, 6H),
1.33, 1.26, 1.23 ppm (m, ArCH2CH2, 2H); 13C NMR (100 MHz, CD2Cl2):
d=302.19, 210.57, 206.68, 155.56, 154.93, 151.95, 144.56, 141.65,
138.91, 129.47, 129.26, 128.65, 125.96, 125.59, 122.28, 122.09,
121.04, 121.04, 121.00, 115.56, 112.91, 75.05, 73.62, 70.69, 63.20,
50.39, 30.57, 26.19, 24.79, 20.93, 19.13 ppm; 31P NMR (162 MHz,
CD2Cl2): d=29.90 ppm. HRMS (ESI): m/z calcd for [M]+: 883.1858;
found: 883.1739.
Complex 10: In accordance with the synthesis of complex 9, 8
(78 mg, 0.129 mmol), KHMDS (0.26 mL, 0.5m solution in toluene),
Hoveyda–Grubbs I (59.6 mg, 0.099 mmol) were used, and column
chromatography (degassed acetone then MeOH) gave complex 10
as a bright green, dense oil (45 mg, 51%). 1H NMR (400 MHz,
CD2Cl2): d=16.41, 16.36 (s, Ru=CH, 1H), 7.53–7.50, 7.38, 7.19, 7.04–
6.86 (ArH, 9H), 4.90 (m, OCH(CH3)2, 1H), 4.16–3.98 (bs, NCH2CH2N,
4H), 3.56-3.44 (m, CH2CH3, 2H), 3.42–2.81 (m, ArCH2CH2CH2, 6H),
2.68 (m, OCH(CH3)2, 6H), 2.12, 1.25 (s, N(CH3)2, 6H), 1.23, 1.22 (d,
J=4 Hz, ArCHCH3, 24H), 1.14 ppm (m, CH2CH3, 6H); 13C NMR
(100 MHz, CD2Cl2): d=289.30, 213.76, 213.09, 163.45, 152.13,
149.14, 147.67, 147.66, 147.42, 144.03, 142.41, 142.30, 129.13,
124.19, 122.19, 121.95, 112.84, 74.93, 65.593, 58.927, 58.861, 48.892,
36.789, 36.631, 35.923, 35.883, 30.537, 28.664, 26.250, 23.897,
23.046, 21.455, 16.179, 16.114 ppm; 31P NMR (162 MHz, CD2Cl2): d=
37.97 ppm; HRMS (ESI): m/z calcd for [M+H]+: 888.3341; found:
888.3325; m/z calcd for [MÀClÀ]+: 852.3583; found: 852.3556.
1
as a yellow oil (0.55 g, 1.0 mmol, 36%). H NMR (400 MHz, CDCl3):
3
d=8.84 (s, 1H), 6.93 (d, J=5.5 Hz, 4H), 4.64 (br s, 4H), 3.90–4.02
(m, 1H), 3.75–3.88 (m, 1H), 2.63 (d, 3J=9.0 Hz, 6H), 2.51 (t, 3J=
7.5 Hz, 2H), 2.39 (d, 3J=5.0 Hz, 12H), 2.27 (s, 3H) 1.41–1.88 (m,
7H), 1.20–1.41 ppm (m, 6H); 13C NMR (100 MHz, CDCl3) d=159.1,
145.4, 140.7, 135.0, 130.1, 129.4, 59.0, 53.4, 52.2, 36.1, 35.4, 30.8,
28.7, 25.9, 24.6, 22.0, 21.1, 18.2, 16.3 ppm; 31P NMR (162 MHz,
CDCl3): d=36.94 ppm.
Metallation and synthesis of inhibitors
Complex 9: As a representative synthesis, in the glovebox, potassi-
um hexamethyldisilazide (KHMDS, 0.5m solution in toluene, 0.5 mL,
0.238 mmol, 1.1 equiv) was slowly added to a stirred suspension of
preligand 7 (110 mg, 0.217 mmol, 1 equiv) in toluene (25 mL) at
room temperature, and the mixture was left stirring for 16 h. A so-
lution of Hoveyda–Grubbs I complex (117 mg, 0.195 mmol,
0.9 equiv) in toluene (5 mL) was added to the resultant orange so-
lution. The brown reaction mixture was taken out of the glovebox
and heated to 658C for 2 h, whilst stirring; it was then allowed to
cool to room temperature and stirred for a further 48 h under inert
conditions to give a dark green-brown colored solution. The
volume was reduced to about 10 mL under vacuum, and the solu-
Inhibitor 2: In accordance with the synthesis of inhibitor 1, 10
(15.9 mg, 0.018 mmol), HCl (0.358 mmol, 0.358 mL), p-nitrophenol
(2.5 mg, 0.018 mmol), and triethylamine (0.18 mmol, 0.025 mL)
were used. Column chromatography (degassed acetone) gave in-
hibitor 2 as a green, dense oil (10 mg, 57%). 1H NMR (400 MHz,
CD2Cl2) d=16.43 (s, Ru=CH, 1H), 8.21 (d, J=8 Hz, OArHNO2, 2H),
8.12 (d, J=8 Hz, OArHNO2, 2H), 7.41-7.37, 7.17, 7.00–6.85, 6.81 (m,
Chem. Eur. J. 2015, 21, 15676 – 15685
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Full Paper
ArH, 9H), 4.87 (m, OCH(CH3)2, 1H), 4.30–4.18 (m, CH2CH3,
NCH2CH2N, 6H), 3.56 (m, ArCH(CH3)2, 4H) 3.44, 2.87 (m, ArCH2, 2H),
2.62, 2.15, 2.13, 1.97 (m, ArCH2CH2, 2H), 1.26 (bs, ArCH(CH3)2, 24H)
1.22 (d, J=4 Hz, OCH(CH3)2, 6H), 1.14 ppm (m, CH2CH3, 3H);
13C NMR (100 MHz, CD2Cl2): d=285.22, 212.97, 162.08, 152.15,
149.40, 148.99, 144.01, 142.00, 129.56, 129.27, 125.96, 125.58,
124.33, 124.11, 122.17, 121.92, 121.05, 121.00, 120.92, 115.52,
112.88, 74.93, 63.24, 52.83, 29.63, 28.96, 28.80, 28.58, 26.51, 26.12,
23.15, 22.91, 21.46, 16.18, 16.12 ppm; 31P NMR (162 MHz, CD2Cl2):
d=30.10 ppm; HRMS (ESI): m/z calcd for [MÀClÀ]+: 946.3265;
found: 946.3100.
Catalytic reactions with metalloprotein hybrids
In a typical experiment, N,N-diallyl-p-toluenesulfonamide (TDA) or
allylbenzene and bibenzyl (substrates and internal standard, re-
spectively, were mixed in a stock solution of 10 mm of each com-
ponent in degassed CH2Cl2) were added to a Schlenk flask (1 mL,
1 mmol, 1 equiv) that was charged with a stirring bar and MgCl2
(1 mg, 10.5 mmol, 10.5 equiv; optionally). In the reactions with allyl-
benzene, tetrabutylammonium chloride (TBACl, 2.5 mg, 9 mmol)
was added too. The solvent was removed under vacuum, and the
flask was refilled with N2 gas. The catalytic hybrid (solution of
50 mm in buffer) was added to the Schlenk flask (1 mL, 0.05 mmol,
0.05 equiv) followed by the slow addition of degassed CH2Cl2
(0.05 mL) by syringe while stirring (final total volume=1.05 mL,
which made CH2Cl2 5% v/v and TBACl ꢀ5% w/w; final concentra-
tions: TDA or allylbenzene=0.95 mm, bibenzyl=0.95 mm, catalytic
hybrid=47.6 mm, buffer=47.6 mm, MgCl2 =10 mm). The solution
was stirred vigorously for 20 h at room temperature. The organic
phase was extracted with CH2Cl2 (3”6 mL), concentrated under
vacuum, and dissolved in CH2Cl2 (0.05 mL) for GC analysis.
Complex 18: In accordance with the synthesis of complex 9, 17
(70 mg, 0.127 mmol) and KHMDS (0.25 mL, 0.5m solution in tolu-
ene) were stirred for 5 h (this first step of the reaction requires less
stirring time due to the better solubility of compound 17 in tolu-
ene, in comparison with compounds 7 and 8), Hoveyda–Grubbs I
(72.0 mg, 0.12 mmol) was added, and column chromatography (de-
gassed acetone) gave the complex 18 as a bright green oil (39 mg,
39%). 1H NMR (400 MHz, CD2Cl2): d=16.52, 16.46 (Ru=CH, 1H),
7.54, 7.07, 6.97, 6.95, 6.90, 6.82 (ArH, 8H), 4.88 (m, CH2CH3, 2H),
4.15 (s, NCH2CH2N, 4H), 3.95, 3.85 (m, CH(CH3)2, 1H), 2.66, 2.64 (s,
ArCH3, 15H), 2.60 (m, ArCH2, 2H), 2.44, 2.40 (s, N(CH3)2, 6H), 1.84–
1.64 (tether ÀCH2À, 10H) 1.46 (d, J=8 Hz, CH(CH3)2, 6H), 1.21 ppm
(m, CH2CH3, 3H); 13C NMR (100 MHz, CD2Cl2): d=295.15, 211.06,
210.63, 152.02, 151.92, 145.22, 145.06, 143.79, 138.80, 129.52,
129.31, 128.57, 122.46, 122.22, 122.07, 113.00, 112.87, 75.04, 58.76,
52.84, 35.84, 35.53, 30.58, 29.03, 26.27, 25.82, 24.53, 22.17, 20.89,
16.08 ppm; 31P NMR (162 MHz, CD2Cl2): d=36.37 ppm.
Acknowledgements
The authors thank the Dutch National Research School
Combination-Catalysis (NRSCC) for the financial support.
Keywords: enzyme catalysis
metathesis · ruthenium
· lipase · metalloenzymes ·
Inhibitor 3: In accordance with the synthesis of inhibitor 1, com-
plex 18 (11.8 mg, 0.0142 mmol), HCl (0.14 mmol, 0.142 mL), p-nitro-
phenol (2 mg, 0.0142 mmol), and triethylamine (0.18 mmol,
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1
0.025 mL) gave complex 3 as a green oil (97%). H NMR (400 MHz,
CD2Cl2): d=16.55, 16.47 (Ru=CH, 1H), 8.20, 8.09, 7.51, 7.37, 7.06,
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925.2327; found: 925.2250.
Hybridization reactions
In a typical procedure, a solution of inhibitor 1, 2, or 3 (10 equiv
with respect to cutinase) in degassed CH2Cl2 (0.5 mL) was slowly
added by syringe to a stirred solution of cutinase (50 mm in de-
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round bottom flask and placed on a rotary evaporator with a fast
spin at 258C to remove CH2Cl2. The solids were filtered off three
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ESI-MS analyses, an aliquot of 0.5 mL was further dialyzed and
treated with formic acid for its denaturation before injection.
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Received: June 19, 2015
Published online on September 8, 2015
Chem. Eur. J. 2015, 21, 15676 – 15685
www.chemeurj.org
15685
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim