Synthesis of hybrid transition-metalloproteins via thiol-selective covalent anchoring of Rh-phosphine and Ru-phenanthroline complexes

Article abstract of DOI:10.1039/c0dt00239a

The preparation of hybrid transition metalloproteins by thiol-selective incorporation of organometallic rhodium- and ruthenium complexes is described. Phosphine ligands and two rhodium-diphosphine complexes bearing a carboxylic acid group were coupled to the cysteine of PYP R52G, yielding a metalloenzyme active in the rhodium catalyzed hydrogenation of dimethyl itaconate. The successful coupling was shown by ³¹P NMR spectroscopy and ESI mass spectroscopy. In addition wild-type PYP (PYP WT), PYP R52G and ALBP were successfully modified with a (η⁶-arene) ruthenium(ii) phenanthroline complex via a maleimide linker.

Full text of DOI:10.1039/c0dt00239a

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PAPER
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Synthesis of hybrid transition-metalloproteins via thiol-selective covalent
anchoring of Rh-phosphine and Ru-phenanthroline complexes†
a
b
b
b
b
Ren e´ den Heeten, Bianca K. Mu n˜ oz, Gina Popa, Wouter Laan* and Paul C. J. Kamer*
Received 31st March 2010, Accepted 10th June 2010
DOI: 10.1039/c0dt00239a
The preparation of hybrid transition metalloproteins by thiol-selective incorporation of organometallic
rhodium- and ruthenium complexes is described. Phosphine ligands and two rhodium-diphosphine
complexes bearing a carboxylic acid group were coupled to the cysteine of PYP R52G, yielding a
metalloenzyme active in the rhodium catalyzed hydrogenation of dimethyl itaconate. The successful
3
1
coupling was shown by P NMR spectroscopy and ESI mass spectroscopy. In addition wild-type PYP
6
(
PYP WT), PYP R52G and ALBP were successfully modified with a (h -arene) ruthenium(II)
phenanthroline complex via a maleimide linker.
Introduction
is required. For covalent attachment, usually a suitably located
highly reactive amino-acid functional group, like the nucleophilic
thiol side-chain of a unique cysteine, is employed for anchoring of
Enantioselective catalysis is one of the most efficient strategies for
the preparation of enantiopure compounds. Catalytic reactions
can be achieved by either chemocatalysis or biocatalysis. In the
field of chemocatalysis, homogeneous catalysis with transition
metals has received significant attention during the past decades
and has proven useful and versatile in numerous enantioselective
transformations. Both the nature of the metal center and the
coordinating ligand determine the properties of the catalyst. The
key to the success of organometallic catalysts lies in the relative
ease of catalyst modification by changing the ligand environment.
In biocatalysis, the use of enzymes for organic transformations has
5
the catalyst.
Phosphine ligands are an attractive class of ligands since the
corresponding transition metal complexes are able to catalyze
efficiently a variety of reactions such as olefin hydrogenation,
hydroformylation and allylic substitution, reactions that are (as
yet) not catalyzed by enzymes. In recent years several reports
have been published on the successful non-covalent anchoring of
1
2
7-9
10
phosphine ligands inside proteins and antibodies and the use
of the resulting artificial metalloenzymes in asymmetric catalysis.
The incorporation of phosphine ligands into the macromolecular
host through a covalent linkage has been studied to a lesser
extent, most likely due to the fact that most common methods
for site-specific bioconjugation are unsuitable for phosphines. In
3
received increasing attention. The excellent chemo-, regio- and
enantioselectivity and activity displayed by enzymes, in combina-
tion with the introduction of directed evolution methodologies
4
for performance optimization lie at the basis of this interest.
2
002, Reetz and coworkers reported the covalent anchoring of a
Furthermore, enzymes offer an environment-friendly alternative
to toxic chemical reagents and the field of green chemistry is,
at present, enjoying a high level of attention from the scientific
community.
phosphine ligand within the active site of a lipase, but the obtained
hybrid turned out to be hydrolytically unstable.
11
Clearly, there is a need for reliable synthetic methodology for the
covalent modification of proteins with a wide range of transition
metal complexes. We recently demonstrated the first covalent,
site-selective conjugation of free phosphines to a protein host
With the aim to combine the best of both homogeneous catalysis
and biocatalysis, the development of artificial metalloenzymes has
5
received much interest in the last decade. The principle of the
12
providing a long term stable linkage. Photoactive Yellow Protein
incorporation of a modified coenzyme analogue or a transition
(PYP) was site-selectively functionalised with phosphine-ligands
6
metal complex into a protein, was already developed by Kaiser
and phosphine-palladium complexes, affording artificial metallo-
enzymes active in allylic substitution reactions. Here we show
that the established conjugation method, employing phosphine-
carboxylic acids, can also be applied to the site-selective coupling
of rhodium-diphosphine complexes to the cysteine of PYP R52G,
thus providing a platform for the synthesis of artificial transition-
metalloenzymes for diverse catalytic transformations.
7
and Whitesides in the late 1970’s. In artificial transition metal-
loenzymes the catalytic activity stems mainly from the transition
metal part, while the selectivity of the catalytic transformation is
induced by the chiral environment of the protein.
To create structurally well-defined systems, site-selective func-
tionalization of the host-protein with the organometallic catalyst
The strongly chelating properties of phenanthrolines for a large
number of metals make it an attractive ligand for the synthesis
of hybrid metalloproteins and metalloenzymes. Phenanthroline-
modified proteins have been applied as synthetic metalloenzymes
a
Van’t Hoff Institute for Molecular Sciences, University of Amsterdam,
Nieuwe Achtergracht 166, 1018 WV, Amsterdam, The Netherlands
b
EaStCHEM, Department of Chemistry, University of St. Andrews, St.
Andrews, Fife, KY16 9ST, United Kingdom. E-mail: pcjk@st-andrews.
ac.uk, wwl1@st-andrews.ac.uk; Fax: +44-1334-463808
13
14
for DNA cleavage, asymmetric ester and amide hydrolysis, and
15
Diels–Alder reactions.
†
Electronic supplementary information (ESI) available: Details of protein
production, MS spectra of conjugates and catalysis procedures. See DOI:
0.1039/c0dt00239a
With applications in asymmetric transfer-hydrogenation and
Diels–Alder reactions in mind, we also present here the
1
This journal is © The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 8477–8483 | 8477

Fig. 1 Cartoon representation of (A) the crystal structure of the Photoactive Yellow Protein (PDB ID 2PHY) with Cys69 and Arg52 highlighted, and
B) ALBP (PDB ID 1LIB), with Cys117 highlighted.
(
preparation and characterization of novel metalloproteins ob-
tained via the thiol-selective anchoring of a (h -arene) ruthe-
zolides of phosphino-carboxylic acids 1–7 were synthesised by
reacting them with an excess of CDI in DMF (Scheme 1).
6
12
nium(II) phenanthroline complex to PYP, PYP R52G and ALBP.
Phosphine ligands 1–7 (Scheme 1) are either commercial available
or are readily obtained via short and efficient synthetic routes.
Results and Discussion
Among the different classes of proteins, transporters and enzymes
are most frequently employed for the synthesis of artificial
metalloenzymes. Our study has focused on the use of PYP as
scaffold for the preparation of artificial metalloenzymes. PYP is a
water-soluble, yellow photoreceptor protein, originally isolated
16
from the bacterium Halorhodospira halophila. By definition,
photoreceptor proteins contain a light-absorbing chromophore,
17
for PYP this is p-hydroxycinnamic acid. In 1995, a crystal
18
structure of the protein in the ground state was published (Fig. 1),
revealing two hydrophobic cores: the smaller of these is located
at the N-terminus, while the larger contains the chromophore-
binding pocket. In this pocket, the chromophore is covalently
bound through a thiol ester linkage to the single cysteine of the
19,20
protein, Cys69.
In the mutant PYP R52G, the arginine at position 52 (Arg52) is
replaced by a glycine. This is expected to result in a more accessible
cavity, since Arg52 is believed to shield the chromophore from the
solvent (Fig. 1). The highly nucleophilic thiol group of Cys69 in
combination with the hydrophobic binding-pocket makes PYP an
attractive scaffold for the covalent anchoring of transition metal
complexes.
Scheme 1 Synthesis of phosphine-PYP conjugates via imidazolide forma-
tion of phosphine-carboxylic acids. (a) DMF, overnight, RT. (b) 50 mM
Tris-HCl/H O, pH 8.0, overnight, RT.
2
The coupling of benzoic acid 3 with PYP R52G is described
here in detail as an illustrative example. Compound 3 was activated
with CDI in DMF and subsequently added to one equivalent of
protein in buffered aqueous solution. The mixture was incubated
overnight and the protein was purified by centrifugal ultrafiltration
using a 10 kDa filter. LC-MS analysis of the purified protein
demonstrated the clear presence of a PYP-adduct with a mass
of 16,050 Da which perfectly matches the calculated mass of the
conjugate of ligand 3 to R52G (Fig. 2). The successful coupling
In PYP the chromophore is covalently bound through a thiol
ester to Cys69. The apo-protein can be reconstituted in vitro with
activated forms of the p-hydroxycinnamic acid chromophore; the
21
21
22
use of the thiophenyl ester, the anhydride and the imidazolide
of p-hydroxycinnamic acid all lead to formation of the desired
thioester linkage with the protein. Therefore we explored the use
of carboxylic acid modified phosphine ligands for the site-selective
covalent coupling of the phosphine moiety to the protein.
Because the only by-products of the activation of a carboxylic
acid with N,N¢-carbonyldiimidazole (CDI) and subsequent cou-
3
1
of benzoic acid 3 to R52G was also confirmed with P NMR
spectroscopy. The conjugate displayed a signal at d = -4.8 ppm
which is typical of triphenylphosphine derivatives (Fig. 3). Using
the same approach, ligands 1–7 have been site-selective covalently
12
pling reaction are imidazole and CO
2
, which are easily removed
anchored to PYP R52G. In all cases, the ionization efficiency
during ESI-MS analysis of each hybrid was found to be about 80–
100-fold less than that of the parent-protein, therefore revealing
from the protein after coupling, we chose to use CDI-activated
phosphine-ligands for the protein-functionalization. The imida-
8
478 | Dalton Trans., 2010, 39, 8477–8483
This journal is © The Royal Society of Chemistry 2010

achiral biotin-functionalised rhodium-diphosphine moiety within
7
8
the protein avidin. In later studies, the groups of Chan and in
9
particular Ward further developed this approach. In 2005, De
Vries and coworkers showed that papain, covalently modified at
Cys25 with a monodentate phosphite ligand and complexed with
[
Rh(cod)
2
]BF , is an active catalyst in the hydrogenation of methyl
4
24
a-acetamidoacrylate, but no enantioselectivity was observed.
Inspired by these studies, we explored the challenging goal of
building a covalently enzyme-bound rhodium-phosphine com-
plex for hydrogenation reactions (Scheme 2). Reaction of the
imidazolide of ligand 4 with [Rh(cod)(MeCN)
2
]BF
4
afforded the
corresponding chelating complex displaying two sets of double
3
1
doublets in P NMR spectroscopy at d = 40 and 45 ppm. This
rhodium complex was successfully coupled to PYP R52G as the
Fig. 2 ESI-MS analysis of PYP R52G modified with 3.
31
resulting conjugate showed a broad signal at d = 36 ppm in
P
NMR spectroscopy (Figure S1†). It appears that in addition to
3
1
LC-MS analysis, P NMR spectroscopy is a useful technique for
characterizing phosphorus-based artificial metalloenzymes.
31
Fig. 3
P NMR analysis of PYP R52G modified with 3.
4
Scheme 2 Synthesis of PYP R52G-[(4)Rh(cod)]BF .
that all ligands coupled with more than 90% efficiency. The same
was observed for the hybrids containing metal-complexes (see
below).
The rhodium-catalyzed hydroformylation of alkenes using
artificial metalloenzymes has been studied to a much lesser extent
than the hydrogenation reaction despite the fact that hydro-
formylation is an interesting transformation since it cannot be
achieved by natural enzymes. Using rhodium loaded human serum
albumin (HSA), Marchetti, Paganelli and coworkers reported
the hydroformylation of styrene and 1-octene under aqueous
Analysis of the conjugates by trypsin digestion followed by
MS analysis, failed to provide evidence for protein modification,
probably due to the lability of the thioester under the trypsin di-
gestion conditions. Instead, treatments with dithiothreitol (DTT)
and alkaline conditions were performed on the new phosphine-
containing proteins and the results indicated clearly that PYP
R52G is modified with the phosphine ligands exclusively at
25
biphasic conditions. For the hydroformylation of styrene good
regioselectivity and high turnover numbers were obtained, but
unfortunately, no mention of the enantioselectivity was made.
Notably, the localization of the rhodium catalyst was not defined
since no site-selective anchoring method was applied.
With the exception of this example, the Rh-catalyzed hydro-
formylation of alkenes using artificial metalloenzymes is still un-
explored. We were interested in the synthesis of PYP-based hybrid
catalysts for application in hydroformylation reactions through
our developed cysteine-selective conjugation method. Reaction of
2
12
Cys69.
Another protein scaffold we are exploring is the adipocyte lipid
binding protein (ALBP). ALBP is a small 131 residue protein
with a simple architecture that mainly consists of two orthogonal
planes of b-sheet secondary structure (Fig. 1). A variety of fatty
3
˚
acids bind in a large (600 A ) cavity formed between the two
sheets. The suitability of this protein for our studies is exemplified
by its previous successful application in the construction of
artificial metalloenzymes, whereby organo(metallic) catalysts were
the crude imidazolide of ligand 4 with [Rh(acac)(CO) ] afforded
14,23
31
introduced in the cavity by chemical modification of a cysteine.
a complex which showed two sets of double doublets in P NMR
Unfortunately, the phosphine-conjugation method developed for
PYP lacked the desired chemoselectivity when applied to ALBP.
The modification of lysines occurred in conjunction with thiol-
modification, thus affording proteins containing multiple ligands
spectroscopy at d = 18 and 19 ppm. Using the same procedure as
for the conjugation of the free phosphines, this rhodium complex
could also be selectively coupled to the protein, as revealed by LC-
MS (Fig. 4). A species with a mass corresponding to the protein
containing the ligand and rhodium(acac) was observed.
(
results not shown). A detailed account of this will be presented
elsewhere (manuscript in preparation).
Ruthenium conjugates
Rhodium conjugates
Artificial metalloenzymes have been previously used in transfer
hydrogenation of acetophenone and derivatives by Ward and
Next we focused on the preparation of artificial metalloenzymes,
site-selectively modified with phosphine-rhodium complexes, for
application in hydrogenation and hydroformylation reactions.
From the start of the development of artificial metalloenzymes,
the rhodium-catalyzed hydrogenation of alkenes has been inten-
sively investigated. In 1978, Wilson and Whitesides reported the
construction of a hydrogenation catalyst based on embedding an
27
coworkers. In these studies the (strept)avidin and its mutants
6
were conjugated with (h -arene) ruthenium(II) complexes modified
with ethylenediamine biotinylated ligands (Noyori’s catalyst) and
applied successfully as transfer hydrogenases. Transfer hydro-
6
genation reactions under aqueous conditions using cationic (h -
arene) ruthenium phenanthroline molecular systems have been
This journal is © The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 8477–8483 | 8479

Table 1 Conjugation of 9 to PYP and ALBP
DMWexp
(Dcal 528.5)
Entry
Protein
Conjugate
MWcalcd
MWobs
1
2
3
4
5
6
PYPWT
PYPWT
PYPR52G
PYPR52G
ALBP
—
15861.8
16390.3
15761.4
16289.9
16604.8
17133.3
15855.7
16382.2
15758.5
16286.3
16599.8
17126.2
—
526.5
—
Ru(phen)
—
Ru(phen)
—
527.8
ALBP
Ru(phen)
526.4
Fig. 4 ESI-MS analysis of PYP R52G modified with 4-Rh(acac).
28
studied previously by S u¨ ss-Fink and co-workers. Very recently
Salmain et al. reported the application of papain modified with a
6
phenantroline containing (h -arene) ruthenium(II) complex as an
15
artificial Diels–Alderase. Based on these precedents we decided
6
to synthesise novel (h -arene) ruthenium(II) metalloproteins via
thiol-selective conjugation of a maleimido-phenanthroline ruthe-
nium complex.
The 5-maleimido-1,10-phenanthroline ligand can be easily
synthesised from commercial reagents and obtained in good
yields. The corresponding dicationic p-cymene Ru(II) complex
Fig. 5 ESI-MS analysis of PYP WT modified with Ru complex 9.
29
PYP (WT and mutant) under the analysis conditions. In contrast,
ALBP does not undergo any oxidation during the analysis.
Table 1 shows the main signals observed in the LC-MS. The
difference of the main signal and the signal of the apo-proteins
corresponds to the mass of the conjugation of 9. The new
ruthenoproteins were also characterised by MS of the peptide
fragments obtained by trypsin digestion to confirm cysteine-
selective bioconjugation.
9
was synthesised according to a procedure reported by S u¨ ss-Fink
for similar phenanthrolines.
28
The chemical modification of PYP WT, PYP R52G and ALBP
with 9 was performed by stirring the protein and the ruthenium
complex overnight in 20 mM Tris-HCl buffer at pH 7.0 (Scheme 3).
For PYP WT-9, this analysis showed the signal corresponding
to the unmodified cysteine containing peptide (1528.6 Da), which
was also present in the protein solution. The signal corresponding
to the modified peptide (2056.9 Da = 1528.6 + 528.3 Da) showed
the expected pattern attributed to the ruthenium isotopes (Figure
S4†), in agreement with the calculated mass spectrum of the
modified peptide fragment containing the ruthenium complex
(Figure S5†). Moreover, no other modified peptides were found.
Scheme 3 Synthesis of Ru-phenanthroline-PYP and ALBP conjugates
via thioether bond formation. (a) 20 mM Tris-HCl/H O, pH 7.0,
2
overnight, RT.
This confirms the introduction of the chemical modification on
the cysteine containing peptide fragment. Similar analysis of PYP
R52G-9 and ALBP-9 (Figure S6†) also confirmed thiol-selective
anchoring in these proteins.
The reaction of the ruthenium complex with PYP, PYP R52G
and ALBP afforded in all cases the desired mono-modified protein
and the amount of double coupling was negligible as revealed
by LC-MS analysis (Fig. 5 and Figures S2 and S3†). Moreover,
analysis of the relative ionisation efficiencies showed that the
conversion was more than 90% for each protein. The LC-MS
in Fig. 5 shows the signal corresponding to PYP modified with
We further characterised PYP WT-9 and ALBP-9 by fluo-
rescence spectroscopy. ALBP modified with 5-maleimido-1,10-
phenanthroline (ALBP-Phen) exhibits an emission maximum at
420 nm, which is absent in ALBP and is attributed to the
phenanthroline ligand. The phenanthroline emission is also absent
in ALBP-9, showing that the binding of ruthenium leads to sig-
nificant quenching of the phenanthroline fluorescence (Fig. 6A).
In addition, in ALBP-Phen and particularly in ALBP-9 the
tryptophan fluorescence, centered around 330 nm, is reduced,
likely due to quenching by the phenanthroline. These results
are in good agreement with the results obtained by Distefano
et al., who observed similar fluorescence characteristics of ALBP
2
+
the dicationic fragment (p-cymene)Ru(OH
2
)(Phen-maleimide)
(
16382.2 Da). The signal corresponding to the apo-protein appears
at 15855.7 Da as well as a signal at 15887.6 Da, which we assign
to the doubly oxidised protein. Similar results are obtained with
PYP R52G. The doubly oxidised proteins probably arise from the
oxidation of the methionine terminal group and/or the cysteine of
14a
modified with iodoacetamide-1,10-phenanthroline and copper
8
480 | Dalton Trans., 2010, 39, 8477–8483
This journal is © The Royal Society of Chemistry 2010

acquisition. The CD spectra corresponding to PYP WT modified
with the ruthenium complex showed that the protein seems to keep
◦
◦
its folded structure even at 50 C. However, above 50 C the CD
spectra showed almost no signal suggesting the disappearance of
the chiral environment. At the end of the experiment the protein
solution contained a white precipitate indicating denaturation and
further precipitation of the ruthenoprotein. Based on these results
◦
we decided to study the stability of the protein at 40 C in time.
The CD spectra obtained after three hours showed no significant
changes in the spectra suggesting that the protein was sufficiently
stable under these conditions. At the end of the experiment no
protein precipitate was observed. Similar results were obtained
with PYP R52G modified with the ruthenium complex. A similar
characterization of ALBP-9 is currently ongoing.
Catalysis
Following the successful application of the PYP-based palladium
12
complexes in allylic amination reactions, we performed pre-
liminary catalysis studies with the here reported rhodium- and
ruthenium-hybrid metalloproteins (see Supporting Information).
The R52G-[(4)Rh(cod)]BF conjugate is an active catalyst for
4
the hydrogenation of dimethyl itaconate (Scheme 4) although
organic cosolvents were required to obtain reproducible conver-
sions. We assume that under fully aqueous conditions the metal
center in the R52G adducts are not accessible for substrates. The
addition of organic solvents denatures the protein which results
in a more accessible metal center. This also disrupts the tertiary
structure of the protein which can explain the lack of induction of
enantioselectivity.
Fig.
6
(A) Fluorescence emission spectra of ALBP (grey line),
ALBP-Phen (dashed line) and ALBP-9 (black line) (B) Fluorescence
emission spectra of PYP WT (grey line), PYP-Phen (dashed line) and
PYP WT-9 (black line).
and strongly suggest that the ruthenium is bound to the phenan-
throline in ALBP-9. The relatively higher ratio of phenanthroline
and tryptophan fluorescence observed in our system compared
to that of Distefano may arise from more efficient quenching of
the tryptophan fluorescence and/or an increase in phenanthroline
fluorescence due to a more rigid anchoring of the maleimido-
phenanthroline.
Scheme 4 Hydrogenation of dimethyl itaconate.
The first experiments with the here reported PYP–Rh(acac) and
PYP–Ru conjugates in hydroformylation and transfer hydrogena-
tion reactions, respectively, were unsuccessful. The relatively high
temperature, pressure of syngas or the low pH required for these
reactions appear to be too harsh for the PYP-adducts. The ALBP
conjugate has not been tested yet.
PYP WT modified with phenanthroline (PYP-Phen) does not
show any discernable phenanthroline fluorescence, nor does PYP
WT-9. Absence of phenanthroline fluorescence in phenanthroline-
14b
modified proteins has been reported previously.
PYP-Phen
Conclusions
and PYP-9 do show reduced tryptophan fluorescence compared
to PYP WT, but the phenanthroline induced quenching is less
pronounced than that observed for ALBP (Fig. 6B).
We have developed a general procedure for the site-selective
covalent modification of PYP with phosphine ligands and their
transition metal complexes. The thiol group of Cys69 in mu-
tant R52G is highly reactive towards activated carboxylates. In
this manner, carboxylate-containing phosphine ligands and their
corresponding transition metal complexes were coupled to PYP
R52G. The new artificial metalloproteins were characterised by
S u¨ ss-Fink and coworkers have shown that using ruthenium(II)
phenanthroline complexes as catalysts for transfer-hydrogenation,
low pH and high temperatures favoured the formation of the
28
alcohols. Therefore we explored the temperature and pH stability
of PYP WT-9 and PYP R52G-9. In order to avoid denaturation
of the new PYP metalloproteins the pH was adjusted to 6.5,
in which both metalloproteins were found to be stable. The
stability of the ruthenoproteins towards the temperature was
studied using CD spectroscopy measurements in the near UV
3
1
LC-MS analysis and P NMR spectroscopy, and one of the
metalloproteins was found to be active in the rhodium-catalyzed
hydrogenation in the presence of organic cosolvents.
Novel ruthenium metalloproteins were synthesised via thiol-
6
(
2
250–310 nm) (Figures S7–9†). The temperature was varied from
0 to 80 C, with increments of 10 C and leaving the sample during
selective coupling of a (h -arene) ruthenium(II) complex to PYP
◦
◦
and ALBP; the PYP conjugate was inactive in the initial applica-
an established time to equilibrate the temperature before each
tion in transfer-hydrogenation.
This journal is © The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 8477–8483 | 8481

(dd, J = 5.3 Hz, J = 8.3 Hz, 2H, CH
=
phen), 8.13 (s, 2H, CH=
Experimental
phen), 8.80 (dd, J = 1.2, J = 8.3, 2H, CH= phen), 9.95 (dd, J =
General Procedures
=
13
1
1
.2, J = 5.3, 2H, CH phen). C{ H} NMR (d, D
2
O, ppm):17.6
), 83.1 (C ), 86.5
), 126.6 (CH= phen), 127.7
(
(
(
CH
C
3
), 20.9 (CH(CH
3
)
2
), 30.4 (CH(CH
3
)
2
6
H
4
Chemicals were purchased from Aldrich Chemical Co. and Fluka
and were used as received. 5-maleimido-1,10-phenanthroline
6
H
4
), 101.1 (C ), 103.7 (C
6
H
4
6
H
4
CH= phen), 130.9 (C= phen), 140.3 (CH= phen), 146.0 (C=
2
6
was prepared according to a literature procedure. N,N-
Dimethylformamide was dried over molecular sieves, de-
gassed and stored under argon. THF was distilled from
sodium/benzophenone, Silica gel 60 purchased from Fluka was
used for column chromatography. All air- and water-sensitive
reactions were carried out under dry, air free conditions using
dry degassed solvents and standard Schlenk techniques under an
atmosphere of purified argon. The conjugation of CDI-activated
phosphine-ligands to PYP and PYP R52G was done following
1
9
1
phen), 155.5 (CH= phen). F{ H}NMR (d, D
O, ppm) -79.38
+
2
(
CF SO ). ESI-MS m/z (relative intensity): 528.7 (M ).
3
3
General procedure for the conjugation of 9 to PYP and ALBP
In a Schlenk tube, 0.87 mmol of protein (1 eq.) in 10 mL 20 mM
Tris-HCl, pH 7.0 was degassed by purging with argon for 4 h.
While stirring, a solution of the maleimide ruthenium cationic
complex (9) was added slowly (4.35 mmol, 5 eq., 87 mL of a 50 mM
solution in distilled water for PYP, 10 eq. for ALBP). The clear
12
the procedure recently reported by our group. NMR spectra
were recorded at room temperature on a Bruker Avance II 400
reaction-mixture was stirred overnight at room-temperature for
3
1
spectrometer. P NMR spectra of phosphine-modified proteins
1-2 mM samples, 26.000 scans, 10% D O as reference) were
◦
1
4
6 h. The reaction-mixture was centrifuged at 49.000 g at 4 C for
5 min to remove unreacted ligand. To remove imidazole and any
(
2
measured on a Bruker Avance 500. Positive chemical shifts (d)
remaining dissolved metal-complex, the yellow supernatant was
are given (in ppm) for high-frequency shifts relative to a TMS
transferred to a centrifugal concentrator (Millipore, MWCO =
1
13
31
13
reference ( H and C) or an 85% H
3
PO
P spectra were measured with H decoupling. Coupling constants
J) are reported in Hz. Multiplicities are indicated by: s (singlet), d
doublet), dd (doublet of doublets), sept (septet) and m (multiplet).
4
reference ( P). C and
1
2
0.000 Da) and concentrated to 500 mL followed by dilution with
3
1
1
0 mM Tris-HCl, pH 7.0. This was repeated four times, after
(
(
◦
which the modified protein was stored at 4 C. (Final protein
concentration is 960 mM). LC-MS analysis showed the formation
of the desired conjugates (see supporting information†).
The abbreviation Ar is used to denote aromatic.
Synthesis of chloro-p-cymeme-(5-maleimido-1,10-phenanthroline)
ruthenium(II) chloride [RuCl(phen-maleimide)(p-cymene)]Cl (8)
Fluorescence spectroscopy
◦
Fluorescence measurements were performed at 25 C using a
To a solution of [RuCl
DCM (20 mL) was added phenanthroline-maleimide (56.4 mg,
.32 mmol) and the solution was stirred during 3 h at room
2
(p-cymene)]
2
(95.8 mg, 0.156 mmol) in
Cary Eclips fluorescence spectrophotometer equipped with a
Cary temperature controller. Fluorescence emission spectra were
recorded from 300–500 nm after excitation at 275 nm (5 nm slits
for both monochrometers, 100 nm min scan speed) using protein
solutions of 10 mM in 20 mM Tris-HCl, pH 7.0.
0
temperature. The solvent was removed under vacuum and the
complex was obtained quantitatively and used without further
-1
1
purification in the next step. H NMR (d, CDCl
3
, ppm): 0.96 (d,
), 2.8 (sept, J = 6.9 Hz,
), 6.27 (d, J = 6.2 Hz, 1H,
), 6.21 (d, J = 6.2 Hz, 1H,
), 6.97 (s, 1H, CH maleimide), 6.98 (s, 1H, CH maleimide),
J = 6.9 Hz, 6H, iso-CH
3
), 2.17 (s, 3H, CH
3
CD spectroscopy at variable temperature
1
H, CH), 6.21 (d, J = 6 Hz, 1H, C
6
H
4
C
6
H
H
4
), 6.34 (d, J = 6 Hz, 1H, C
6
H
4
Spectra were recorded on a Jasco, J-810 spectropolarimeter. Data
in the near-UV (250–310 nm) were collected using a 0.5 mm path
cuvette. The scan speed was 20 nm min , with a bandwidth of
1 nm. Samples were prepared at 60 mM protein concentration in
20 mM Tris-HCl, pH 7.0. Each spectrum was measured six times,
and the data were averaged to reduce the noise.
C
6
4
-
1
7
.84 (s, 1H, CH= phen), 7.98 (m, 2H, CH= phen), 8.13 (d, J =
8
.3, 1H, CH= phen), 8.46 (d, J = 8.3, 1H, CH= phen), 10.27 (d,
J = 4, 2H, CH= phen).
Synthesis of aquo-p-cymene-(5-maleimido-1,10-phenanthroline)
ruthenium(II) ditriflate, [Ru(OH
2
)(phen-maleimide)-
General procedure for asymmetric hydrogenation experiments
(
p-cymene)](OSO
2
CF
3
)
2
(9)
The hydrogenation experiments were carried out in a stainless steel
autoclave (total volume is 150 mL) charged with an insert suitable
for 8 or 14 glass reaction vessels with Teflon mini stirring bars for
conducting parallel reactions. In a typical hydrogenation run, a
glass vial was charged with degassed buffer solution containing the
artificial metalloenzyme complex (56 mM) and substrate. Before
starting the catalytic reactions, the charged autoclave was purged
The
dichloro-p-cymene-(1,10)-phenanthroline-5-maleimide)
ruthenium(II) complex (8) (152 mg, 0.32 mmol) was redisolved
in distilled DCM (20 mL). The Schlenk vessel was covered with
aluminium foil to avoid decomposition. Then, solid Ag(OTf)
(
180 mg, 0.70 mmol) was added to the solution and the reaction
was stirred during 2 h at room temperature. The suspension
AgCl) was filtered over celite under argon and the clear filtrate
(
three times with 5 bar of dihydrogen and then pressurized to
◦
was collected in a schlenk vessel. The solvent was removed under
5 bar H
2
. The reaction mixtures were stirred at 25 C for 20 h.
vacuum and the complex was obtained quantitatively as an
Next, the autoclave was depressurized and each reaction mixture
was extracted with EtOAc (3 ¥ 5 mL) and the combined organic
1
orange solid. H NMR (d, D
2
O, ppm): 0.82 (d, J = 6.9 Hz, 6H,
), 2.45 (sept, J = 6.9 Hz, 1H, CH), 6.11
), 6.34 (d, J = 6.5 Hz, 2H, C ), 8.08
iso-CH
3
), 2.14 (s, 3H, CH
3
layers were dried over Na
2
SO and concentrated under reduced
4
(
d, J = 6.5 Hz, 2H, C
6
H
4
6
H
4
pressure. The residue was redissolved in ethyl acetate (100 mL) and
8
482 | Dalton Trans., 2010, 39, 8477–8483
This journal is © The Royal Society of Chemistry 2010

transferred into a micro-GC vial. The conversion was determined
by GC measurement and the enantiomeric excess was measured
by chiral GC using the following column and conditions: Supelco
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◦
◦
-1
b-DEX 225 column (T = 70 C for 50 min, then DT = 25 C min ,
(S) = 51.7 min, t (R) = 52.3 min, t (substrate) = 53.5 min).
t
R
R
R
1
1
1
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Acknowledgements
We thank Prof. Dr D. J. Cole-Hamilton for fruitful discussion.
The plasmids and E. coli strain for expression of PYP and PYP
R52G were kindly provided by Dr Michael van der Horst and
Prof. Dr K. J. Hellingwerf, University of Amsterdam. A plasmid
for expression of ALBP was kindly provided by Prof. D. Bernlohr
and Dr B. Thompson, University of Minnesota. Financial support
from the European Union is kindly acknowledged: NEST Adven-
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