Piano-stool d6-rhodium(III) complexes of chelating pyridine-based ligands and their papain bioconjugates for the catalysis of transfer hydrogenation of aryl ketones in aqueous medium

Article abstract of DOI:10.1016/j.molcatb.2015.10.007

Two half-sandwich d⁶-rhodium(III) complexes of the general formula [(η⁵-Cp?)Rh(N^N)Cl]Cl where N^N is a phenanthroline or a bispyridine methane derivative carrying a thiol-targeting maleimide or chloroacetamide function were synthesized and characterized. Both complexes were able to catalyse the transfer hydrogenation of 2,2,2-trifluoroacetophenone in aqueous medium using formate or phosphite as hydrogen donor. Covalent anchoring of these complexes to the cysteine endoproteinase papain yielded hybrid metalloproteins with transfer hydrogenase properties. Under optimized conditions of pH, hydrogen donor concentration and catalyst load, conversion of substrate was nearly quantitative within 24 h at 40 °C and the (S)-enantiomer was obtained preferably albeit with a modest enantiomeric excess of 7-10%. Covalent docking simulations complemented the experimental findings suggesting a molecular rationale for the observed low enantioselectivity. The harmonious use of experimental and theoretical approaches represents an unprecedented starting point for driving the rational design of artificial metalloenzymes built up from papain with higher catalytic efficiency.

Full text of DOI:10.1016/j.molcatb.2015.10.007

Journal of Molecular Catalysis B: Enzymatic 122 (2015) 314–322
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Piano-stool d6-rhodium(III) complexes of chelating pyridine-based
ligands and their papain bioconjugates for the catalysis of transfer
hydrogenation of aryl ketones in aqueous medium
Nathalie Maderna,b, Nicolas Queyriauxa,b, Alice Chevalleya,b, Mahsa Ghasemia,b
,
Orazio Nicolottic,d, Ilaria Ciofinie, Giuseppe Felice Mangiatordic,∗, Michèle Salmaina,b,∗∗
a Sorbonne Universités, UPMC Univ Paris 6, UMR 8232, IPCM, F-75005 Paris, France
b CNRS, UMR 8232, IPCM, F-75005 Paris, France
c Dipartimento di Farmacia—Scienze del Farmaco, Via Orabona, 4, Università di Bari ”Aldo Moro”, Bari, Italy
d Centro Ricerche TIRES, University of Bari “Aldo Moro”, Via Amendola 173, I-70126 Bari, Italy
e PSL Research University, Chimie ParisTech—CNRS, Institut de Recherche de Chimie Paris, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
6
5
ˆ
ˆ
Article history:
Received 23 July 2015
Received in revised form 2 October 2015
Accepted 10 October 2015
Available online 22 October 2015
Two half-sandwich d -rhodium(III) complexes of the general formula [(ꢀ -Cp*)Rh(NN)Cl]Cl where NN is
a phenanthroline or a bispyridine methane derivative carrying a thiol-targeting maleimide or chloroac-
etamide function were synthesized and characterized. Both complexes were able to catalyse the transfer
hydrogenation of 2,2,2-trifluoroacetophenone in aqueous medium using formate or phosphite as hydro-
gen donor. Covalent anchoring of these complexes to the cysteine endoproteinase papain yielded hybrid
metalloproteins with transfer hydrogenase properties. Under optimized conditions of pH, hydrogen
donor concentration and catalyst load, conversion of substrate was nearly quantitative within 24 h at
40 ◦C and the (S)-enantiomer was obtained preferably albeit with a modest enantiomeric excess of 7–10%.
Covalent docking simulations complemented the experimental findings suggesting a molecular rationale
for the observed low enantioselectivity. The harmonious use of experimental and theoretical approaches
represents an unprecedented starting point for driving the rational design of artificial metalloenzymes
built up from papain with higher catalytic efficiency.
Keywords:
Rhodium
Hydrogenation
Artificial metalloenzyme
Metallocenter assembly
Covalent docking
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
lent anchoring approach still remains very attractive because of
its simplicity, especially when the protein host contains a reac-
Artificial metalloenzymes are biohybrid species relying on the
tight association of a metal-containing species and a biomacro-
molecule (peptide, protein, DNA). Their development is motivated
by the complementary properties of metal catalysts and enzymes
and the ability of these hybrid catalysts to operate in environmen-
tally friendly conditions [1,2]. Incorporation of nonnative metal
cofactors into proteins has been rationalized into three main strate-
gies: covalent, supramolecular and dative anchoring [3,4]. Each
of these strategies has its pros and cons and the supramolecular
assembling approach has provided the most efficient catalysts in
terms on activity and selectivity so far [5]. Nonetheless, the cova-
tive residue that can be specifically targeted and whose location
is appropriate [6]. These features are met in the case of papain (E.C.
3.4.22.2), a cysteine endoproteinase isolated from Carica papaya
[7]. It is found in papaya latex along with three other homologous
enzymes, namely chymopapain, glycyl endopeptidase (GEP) and
caricain [8]. Papain consists of a single polypeptidic chain of 212
aminoacids. Its active site forms a 15 × 25 Å groove between its two
domains [9]. Two aminoacids are essential for the activity of papain,
namely Cys25 and His159. The thiolate group of Cys25 is known to
be highly nucleophilic because of its location and its chemical mod-
ification invariably leads to loss of hydrolytic activity, providing
a convenient means to monitor the reaction between papain and
thiol-targeted reagents [10]. These properties provided a starting
point for Kaiser’s early concept of chemical mutation of proteins
[11–15] and later on for the design of artificial metalloenzymes by
covalent anchoring of metal complexes [16–24].
∗
Corresponding author.
Corresponding author at: Sorbonne Universités, UPMC Univ Paris 6, UMR 8232,
∗∗
IPCM, F-75005 Paris, France.
E-mail addresses: giuseppe.mangiatordi@uniba.it (G.F. Mangiatordi),
michele.salmain@upmc.fr (M. Salmain).
http://dx.doi.org/10.1016/j.molcatb.2015.10.007
1381-1177/© 2015 Elsevier B.V. All rights reserved.

N. Madern et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 314–322
315
To confer papain with transfer hydrogenase activity, we had
previously designed and synthesized monocationic (arene)RuII
and (Cp*)RhIII complexes of 2,2ꢀ-dipyridylamine carrying a thiol-
targeted maleimide function appended to the central nitrogen atom
via an butyl or pentyl spacer [16]. Covalent anchoring of these com-
plexes to papain was achieved and the metallopapains were shown
to display transfer hydrogenase activity on a model substrate. The
RhIII adducts were by far the most active as quantitative conversion
was reached within 60 h at 40 ◦C and neutral pH in the presence
of 1 mol% metallopapain. Disappointingly, the enantiomeric excess
reached 15% at best in favour of the (R)-enantiomer [22]. Notably,
asymmetric transfer hydrogenation (ATH) is a powerful means to
access secondary chiral alcohols and amines from ketones and
imines, respectively [25,26]. Numerous piano-stool d6 rutheniumII,
rhodiumIII and iridiumIII complexes including chiral diamine lig-
ands catalyse the ATH of ketones in neat water using formate as
hydrogen donor [27–41]. Some half-sandwich polypyridine-type
rutheniumII, rhodiumIII and iridiumIII complexes display the same
reactivity [42–51]. In both cases, the reaction mechanism involves
the transient formation of a metal-hydride species resulting from
a ␤-elimination process [34,52]. Artificial transfer hydrogenases
resulting from the supramolecular anchoring of biotinylated piano-
stool diamine complexes to (strept) avidin have been shown to
catalyse the ATH of ketones and imines with high conversion and
enantioselectivity [53–57]. Also, achiral half-sandwich RhIII com-
plexes derived from fatty acids catalysed the ATH of the benchmark
substrate 2,2,2-trifluoroacetophenone (TFACP) with up to 32% ee in
the presence of bovine ␤-lactoglobulin [58,59].
FTIR-4100 spectrometer (Jasco). Uv–visible spectra were recorded
on a Cary 50 spectrometer (Varian). Circular dichroism spectra
were recorded at 20 0.1 ◦C on a J-815CD spectrometer (Jasco)
applying the following parameters: scan speed 50 nm/min, band
width 1 nm, data pitch 0.5 nm, 3 scans. HPLC was carried out
on a System Gold (Beckman Coulter). Reaction mixtures were
analysed on a 150 × 2 mm reverse-phase column (Jupiter Pro-
teo, Phenomenex) under isocratic conditions (H2O/MeCN 60:40
at 0.2 ml/min) or on a 200 × 4 mm chiral column (Nucleodex
␤-CD, Macherey-Nagel) under isocratic conditions (H2O/MeCN
60:40 at 0.4 ml/min). LC-ESI–MS spectra were recorded on a 6210
LC–TOF mass spectrometer (Agilent) equipped with a protein
trap (Zorbax 300SB-C8, 5 um, 5 × 0.3 mm) and RP-HPLC column
(Vydac C4 5 um, 300 Å, 50 × 0.5 mm). A linear gradient from 5
to 95% of MeCN/H2O/TFA 95:5:0.03 in H2O/TFA 100:0.03 was
applied in 15 min. Affinity-purified papain (afPAP) was obtained
from crude papain by affinity chromatography as previously
described [63]. The enzymatic activity of crude papain was mea-
sured on the chromogenic substrates N-alpha-benzyloxycarbonyl-
glycyl-p-nitrophenylester ZGlyONp (Fluka), N-alpha-benzoyl-DL-
arginine 4-nitroanilide BAPNa (Sigma) and l-pyrogLutamyL-l-
phenylalanyl-l-leucine-p-nitroanilide PFLNa (Bachem) in 100 mM
phosphate buffer pH 6.5, 30 mM KCl, 0.1 mM EDTA containing 10%
DMSO (BAPNa and PFLNa) or MeCN (ZGlyONp) (v/v). The pro-
tein concentration was determined by absorbance measurement at
280 nm taking ꢁ= 46,000 M−1 cm−1 for cPAP and 57,600 M−1 cm−1
for afPAP. Alternatively, protein concentration was measured by
the Bradford assay. Thiol concentration was assayed at 324 nm after
reaction with 4,4ꢀ-dithiodipyridine [64].
In this study, we report the synthesis of two new (Cp*)RhIII
complexes containing a polypyridine chelating ligand carrying
a thiol-targeted chloroacetamide or maleimide function without
spacer so as to shorten the distance between the metal centre
and the second coordination sphere and restrict the number of
conformations of the metal complex when bound to the protein.
Their covalent anchoring to papain and the catalytic activity of the
complexes and the metallopapain adducts in the hydrogenation of
TFACP was assessed. In parallel, molecular modelling studies com-
bining quantum mechanics (QM) calculations and covalent docking
simulations allowed us to obtain valuable insights into the posing
of the anchored metal complexes within the protein-binding site. It
is worth noting that molecular docking, a computational technique
commonly employed for drug design, is today considered a valuable
tool to speed up the design process of artificial metalloenzymes,
as recently pointed out by Robles et al. [60]. The theoretical out-
comes, consistent with the observed ee’s as well as with X-ray data
obtained for a similar metal–complex anchored to papain, unveiled
the molecular rationale ruling the observed disappointing enan-
tioselectivity. These outcomes are discussed in the perspective of
providing rational pathways to design more efficient piano-stool
d6-rhodiumIII cofactors.
2.2. Synthesis
2.2.1. 1-(di(pyridin-2-yl) methyl)-1H-pyrrole-2,5-dione 3
A solution of 2 (500 mg, 2.7 mmol, 1 eq) in dry toluene (1 ml) was
added dropwise to a stirred solution of maleic anhydride (265 mg,
2.7 mmol, 1 eq) in dry toluene (5 ml) at room temperature. During
the addition, an exothermic reaction occurred with instantaneous
formation of white precipitates. After the addition was complete,
the resulting suspension was stirred for 1 h and then ZnCl2 (362 mg,
2.7 mmol, 1 eq) was added in one portion. While the resulting
reaction mixture was heated (80 ◦C), a solution of HMDS (834 ␮L,
4 mmol, 1.5 eq) in dry toluene (1 ml) was added slowly over a period
of 10 min, and then the mixture was refluxed for 1 h. The reaction
mixture was cooled to room temperature and poured into satu-
rated NH4Cl. The aqueous phase was extracted with ethyl acetate.
The organic extract was washed with saturated brine and dried over
anhydrous Na2SO4. The solution was concentrated under reduced
pressure to leave the residue, which was purified by silica filtra-
tion (silica gel, petroleum ether/EtOAc 8:2) to afford compound 3
(612 mg, 74%) as a colorless solid. 1H NMR (300 MHz, CDCl3) ∂ in
ppm 8.65–8.55 (m, 2H, H1), 7.68 (td, 2H, J = 7.8, 1.8 Hz, H2 or H3),
7.34–7.20 (m, 4H, H2 or H3 & H4), 6.77 (s, 2H, H8), 6.70 (s, 1H, H6).
13C NMR (75 MHz, CDCl3) ∂ in ppm 170.6 (C7), 158.5 (C5), 149.4
(C1), 136.9 (C4 or C8), 134.5 (C4 or C8), 123.6 (C3 or C2), 123.0 (C2
or C3), 60.6 (C6). MS (CI/NH3): m/z 266.02 [MH+].
2. Experimental
2.1. General
Solvents were dried and distilled by standard procedures
and all reactions and synthetic manipulations were performed
under an inert atmosphere of argon using standard Schlenk and
vacuum-line techniques. 1H and 13C NMR spectra were recorded
on a 300 and 400 MHz FT-spectrometers (Bruker). Crude papain
(cPAP) was purchased from Sigma (reference 76220). Other chem-
icals were purchased from Aldrich or Alfa-Aesar and used as
received. 2-chloro-N-(1,10-phenanthrolin-5-yl) acetamide 1 [61]
and di(2-pyridyl) methylamine 2 [62] were synthesized accord-
ing to literature procedures. ATR-IR spectra were measured on
2.2.2. [(ꢀ5-Cp*)Rh(2-chloro-N-(1,10-phenanthrolin-5-yl)
acetamide)Cl]Cl 1-Rh
[Cp*RhCl(␮-Cl)]2 (0.1 g, 0.162 mmol) was introduced into a
Schlenk tube followed by DCM (10 ml ) and
1 (0.324 mmol,
88 mg). After 3 h, the solvent was removed by evaporation
under reduced pressure and the residue was washed three
times with 10 ml diethyl ether. Compound [(ꢀ5-Cp*)Rh(2-chloro-
N-(1,10-phenanthrolin-5-yl) acetamide)Cl]Cl 1-Rh was obtained
as a red solid (36%) after drying under high vacuum. 1H NMR
(300 MHz, CDCl3) ␦ in ppm 12.20 (s, 1H, NH), 9.99 (d, J = 8.2 Hz,

316
N. Madern et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 314–322
1H, H4), 9.06 (d, J = 54.7 Hz, 1H, H2), 8.98 (d, J = 4.8 Hz, 1H, H9), 8.71
(s, 1H, H6), 8.44 (d, J = 7.8 Hz, 1H, H7), 8.14 (dd, 1H, J = 7.9, 5.5 Hz,
H8), 7.87 (dd, 1H, J = 7.5, 5.5 Hz, H3), 4.91 (d, 1H, J = 14.1 Hz, H13a),
4.84 (d, 1H, J = 14.1 Hz, H13b), 1.74 (s, 15H, 5Me(Cp)). 13C NMR
(75 MHz, DMSO-d6) ␦ in ppm 167.8 (C12), 151.4 (C2), 149.9 (C9),
145.5 (C1a), 142.9 (C10a), 138.5 (C7), 137.7 (C4), 134.6 (C5), 130.7
(C6a), 127.1 (C4a), 126.9 (C3), 126.8 (C8), 118.5 (C6), 97.0 (Cp*),
44.3 (C13), 9.4 (Me). IR ␯ in cm−1: 1705 (amide), 1590 (amide),
2.6. Kinetics of inactivation and assembling of affinity-purified
papain to 1-Rh
A solution of affinity-purified papain (6 ␮M in 20 mM phos-
phate, 0.4 M NaCl pH 7) was treated with 1-Rh (100 ␮M). Aliquots of
the solution were periodically assayed for their enzymatic activity
on PFLNa over a period of 3 h. After 18 h, the solution was concen-
trated by ultrafiltration in a 50 ml stirred cell (Amicon) and excess
complex was separated from the protein by gel filtration (Hiprep
26/10 desalting, GE Healthcare) using 150 mM NaCl as eluent. The
resulting protein solution was diluted with 2 volumes of water and
concentrated by ultrafiltration to yield afPAP-1-Rh.
1023 (Cp*), 727 (aromatic). Uv (H2O) ␭ in nm (ꢁ in M−1 cm−1
)
277 (20,500), 353sh (2900). HR-MS: calcd for C24H25N3OCl2Rh+
544.04242, found 544.04254.
2.2.3. [(ꢀ5-Cp*)Rh(1-(di(pyridin-2-yl)
methyl)-1H-pyrrole-2,5-dione)Cl]Cl 3-Rh
2.7. Transfer hydrogenation of TFACP
To a stirred solution of 3 (300 mg, 1.1 mmol) in dry CH2Cl2 (5 ml )
was added [Cp*Rh(␮-Cl)Cl]2 (283 mg, 0.55 mmol). The mixture
was stirred at room temperature for 16 h. Solvent was evapo-
rated under reduced pressure and diethyl ether was added. The
yellow crystals were filtered and washed with diethyl ether.
Compound [(ꢀ5-Cp*)Rh(1-(di(pyridin-2-yl) methyl)-1H-pyrrole-
2,5-dione)Cl]Cl 3-Rh was obtained as a yellow–orange solid (96%).
1H NMR (300 MHz, DMSO-d6) ∂ in ppm (mixture of chloro and aqua
complexes) 8.93 and 8.71 (d, J = 5.1 or 4.8 Hz, 2H, H1), 8.09 and 7.93
(t or td, J = 7.2 or 8.1 and 1.5 Hz, 2H, H2), 7.64 and 7.38 (d, J = 7.8
or 8.1 Hz, 2H, H4), 7.57 and 7.49 (t, J = 6.3 or 5.7 Hz, 2H, H3) 7.27
and 7.26 (s, 2H, H8), 7.00 and 6.80 (s, 1H, H6), 1.65 (s, 15H, Cp*).
Uv (H2O) ␭ in nm (ꢁ in M−1 cm−1) 253sh (10,200), 351 (2100). MS
(ESI) m/z 538.4 [M+].
Test mixtures (1 ml) containing TFACP (5 or 10 mM), hydro-
gen donor and catalyst in water were incubated at 40 ◦C in a dry
bath. Aliquots (10 ␮l) were retrieved periodically and analysed by
reverse phase or chiral HPLC.
2.8. Modelling studies
In order to evaluate the metal complex posing in the protein-
binding site, covalent docking was carried out on both compounds
1-Rh and 3-Rh. The complexes were docked considering only their
hydride forms, which are the catalytically active species. Being
chiral at the metal centre, both the enantiomers of 1-Rh were con-
sidered (hereafter referred to as 1-Rh(R) and 1-Rh(S)) though 1-Rh
is expected to racemize very quickly because of the lability of the
chloride ligand. As a first step, the Restrained ElectroStatic Poten-
tial (RESP) approach [65] was applied to derive accurate atomic
charges to be employed for docking simulations. Compounds were
optimized at the Density Functional Theory level using the hybrid
B3LYP functional [66]. All atoms were described using a double zeta
valence basis set (6–31+G(d,p)) except for Rhodium, described by
an effective core potential and corresponding valence basis set.
Molecular electrostatic potential (MEP) was then computed on
these structures at B3LYP level of theory using a smaller basis set
(6–31G(d)). Finally, the charge values were fitted in order to repro-
duce the computed MEPs. All these calculations were performed
with the Gaussian 09 [67] package apart from the charges fitting
performed using Antechamber [68], a freely accessible program
that is part of AmberTools [69]. Covalent docking studies were per-
formed on the X-ray crystal structure of papain (PDB code: 9PAP
[70], resolution 1.65 Å). Protein structure was prepared using the
Protein Preparation wizard [71] available from Schrodinger Suite
v2014-4 [72]. The resulting file was used for docking simulations
performed using the Covalent Docking protocol, enclosed in the
Schrodinger Suite [72]. This protocol allows predicting whether
compounds can covalently bind a given receptor and, in case, which
pose is the most suitable. Following such a protocol, Cys25 was
first mutated to alanine, in order to avoid a possible influence of
its side chain conformation on the protein-compound association.
Docking simulations were then performed using Grid-based Lig-
and Docking with Energetics (GLIDE v6.5) software [73] enclosed
in the Schrodinger Suite [72]. Distance constrains were applied
between residue in position 25 and the reactive group of the consid-
ered metal–complex. Furthermore, a preliminary minimization of
the protein residues close to the docked compounds (distance cut-
off = 5 Å) was carried out before selecting suitable docking poses.
Subsequently, the Cys25 side chain was restored and its suit-
able poses explored in the presence of the bound metal-complex.
Finally, the covalent bond was formed and both metal-complex
and Cys25 were minimized to reduce strain. The described cova-
lent docking protocol was performed using the default Force Field
OPLS 2005 [74] except for the atomic charges, which were derived
2.3. Kinetics of inactivation of crude papain by 1-Rh or 3-Rh
A solution of crude papain (2 mg solid/ml in 20 mM phosphate
buffer pH 7.0; [thiol] = 0.11 mM) was treated with excess 1-Rh
(1 mM). Aliquots of the solution were periodically assayed for their
enzymatic activity on BAPNa, PFLNa and ZGlyONp. A solution of
crude papain (10 mg solid/ml in water; 5 ml) was first submitted to
gel filtration to remove low molecular weight thiols contained in
the commercial sample. The resulting protein sample was diluted
to 93 ␮g/ml in PBS ([thiol] = 3 ␮M) and treated with excess 3-Rh
(100 ␮M). Aliquots of the solution were periodically assayed for
their enzymatic activity on ZGlyONp.
2.4. Cation exchange chromatography of cPAP-1-Rh
A solution of crude papain (10 mg solid/ml in 20 mM phos-
phate, 0.4 M NaCl pH 7; [thiol] = 0.56 mM) was treated with 1-Rh
(0.7 mM). After 24 h, the solution was passed on a gel filtration col-
umn (Hiprep D-salt 26/10) using 150 mM acetate pH 5 as eluent.
The protein sample was applied to a Mono S 5/5 cation exchange
column (GE Healthcare) equilibrated with 150 mM acetate pH 5.
Proteins were eluted using a linear gradient of NaCl (31 mM/ml) at
a flow rate of 0.7 ml/min. Fractions of 0.35 ml were collected and
assayed for enzymatic activity on ZGlyONp.
2.5. Assembling of crude papain and 1-Rh or 3-Rh
A solution of crude papain (10 mg solid/ml in 20 mM phos-
phate, 0.4 M NaCl pH 7; [thiol] = 0.56 mM) was treated with 1-Rh
(0.64 mM). After 30 h, NEM (1.28 mM) was added and the mixture
incubated for another 17 h to block residual enzymatic activity.
Unreacted complex and NEM were removed by diafiltration with
water in a stirred cell (Amicon) to yield cPAP-1-Rh. The same pro-
cedure was applied with a slight excess of 3-Rh over thiol (0.83 mM,
1.5 eq.) while omitting the NEM step to yield cPAP-3-Rh.

N. Madern et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 314–322
317
Table 1
following the RESP protocol, as described above. A cubic grid having
Rh/protein ratios of the metallopapains.
an edge of 30 Å centred on the centroid of residues Cys25, Asp158
and Asn64, and all the default setting for covalent docking simula-
tions were used.
Complex
1-Rh
3-Rh
Crude papain
1.5 0.25a
1.2 0.2b
0.9 0.1b
n.d.
Affinity-purified papain
a
Protein concentration determined by the Bradford assay.
Protein concentration determined at 280 nm.
3. Results and discussion
b
3.1. Synthesis
1-Rh was submitted to gel desalting to remove unreacted com-
plex followed by cation exchange chromatography to separate the
individual cysteine endoproteinases. The resulting chromatogram
(Fig. S1) displayed 4 major peaks in agreement with the literature
[79,80]. The first (I) and last peaks (IV) were readily identified as
papain and caricain, respectively while the less well resolved sec-
ond and third peaks (II and III) were identified as chymopapain and
glycyl endopeptidase. Only peak III (v= 21 ml) showed some activ-
ity on ZGlyONp. These experimental data, taken together, suggest
that 1-Rh is unable to inactivate GEP. This behaviour of 1-Rh is con-
sistent with the poor inhibiting properties of both iodoacetate and
iodoacetamide towards GEP [81].
In a similar fashion, gradual loss of activity of a mixture of afPAP
and 1-Rh in excess was observed on PFLNa and the enzyme was
fully inactivated after 18 h. Semi-logarithmic plot of PFLNa hydrol-
ysis rate vs. time was linear (Fig. S2) and gave the pseudo-first
rate constant of inactivation kobs of 0.004 min−1 and the second
order rate constant k2 = kobs/[1-Rh] = 40 M−1 min−1. This value is
ten times smaller than those determined for previously reported
organometallic chloroacetamide derivatives [17,77] and also much
smaller than that of the archetypal irreversible inhibitor TPCK
whose second order rate constant is equal to 1200 M−1 min−1 [17].
Reaction of crude papain with excess 3-Rh was performed with
a protein sample devoid of low molecular weight thiols. Rapid loss
of activity on ZGlyONp was observed (20% activity remaining after
20 min, Fig. 1) but the semi-logarithmic plot of activity vs. time was
nonlinear as depicted in the inset of Fig. 1. This trend reflects the
heterogeneous character of crude papain and the fact that the 4 cys-
teine endoproteinases may be inhibited by 3-Rh at different rates
depending on the relative accessibility of the active site cysteine
residue in each enzyme.
Both 1-Rh and 3-Rh displayed absorption features in the near-
uv range, in particular a band around 350 nm (Figs. S3 and S4).
Accordingly, bioconjugates cPAP-1-Rh, afPAP-1-Rh and cPAP-
3-Rh also displayed absorption around 350 nm owing to the
metal complex as well as a maximum of absorption at 280 nm
typical of proteins (Figs. S3 and S4). The Rh content of the
metallopapains was estimated from the absorbance at 350 nm
(ꢁ350(1-Rh) = 2900 M−1 cm−1; ꢁ350(3-Rh) = 2100 M−1 cm−1) while
the protein concentration was assayed either by the Brad-
ford method or at 280 nm after subtraction of the contribution
of the metal complex (ꢁ280(1-Rh) = 20,500 M−1 cm−1; ꢁ280(3-
Rh) = 3700 M−1 cm−1). The Rh/P ratio (Table 1) for cPAP-1-Rh of
1.5 0.25 was somehow surprising since the thiol/protein ratio is
0.75 for crude papain. Furthermore, since glycyl endopeptidase was
shown not to react with 1-Rh, the Rh/P for cPAP-1-Rh should be
much below 0.75. This might indicate that aminoacids other than
the active site cysteine are involved in the reaction with 1-Rh.
The near-uv CD spectrum of bioconjugate cPAP-1-Rh displayed
a significant increase of ellipticity around 260 nm with respect to
cPAP-NEM prepared in the same conditions (Fig. S5). This increase
may be due to intrinsic (change of environment around the aro-
matic residues of papain) or extrinsic factors.
The chloroacetamide RhIII complex 1-Rh was synthesized in
2 steps from 5-amino-1,10-phenanthroline (Scheme 1). Its amino
group was firstly acetylated with chloroacetyl chloride according
to a published procedure [61] then complexation was achieved
by reaction of [Cp*Rh(␮-Cl)Cl]2 in DCM. 1-Rh was characterized
by 1H and 13C NMR. The 7 protons of the polycyclic system
were non equivalent because of the dissymmetrical nature of the
molecule. Their assignment was done thanks to 2D NMR exper-
iments (COSY, HSQC and HMBC). Interestingly, the two protons
H13 were diastereotopic with a geminal coupling constant of 14 Hz
because of the chirality at the metal center.
The maleimide RhIII complex 3-Rh was synthesized in three
steps for the commercially available oxime (Scheme 2). The oxime
was first converted into di(2-pyridyl) methylamine 2 by reduc-
tion in the presence of ammonia and zinc powder according to a
previously published procedure [62]. Treatment of the amine with
maleic anhydride afforded an intermediate amide. Dehydration to
the maleimide attempted in the classical way with acetic anhydride
gave maleimide 3 with 26% yield. This yield was increased to 80%
by using ZnCl2 and HMDS in toluene at reflux [75]. Finally 3-Rh
was obtained as above and its molecular structure confirmed by 1
H
NMR analysis. Both metal complexes are soluble in polar solvents
including water where they rapidly hydrolyse to the dicationic aqua
complexes.
3.2. Covalent assembling of 1-Rh and 3-Rh to papain
Complexes 1-Rh and 3-Rh were covalently anchored to papain
from 2 different sources, i.e. commercial papain (further called
crude papain or cPAP) which is in fact lyophilized papaya latex con-
taining all 4 cysteine endoproteinases (and an appreciable amount
of low molecular weight thiols) and pure papain (afPAP) obtained
by affinity chromatography of the commercial sample according
to a literature procedure [63]. By analogy with the previously
reported reactions of ␣-chloroacetamides and maleimides with
papain [7,76,77], chemical modification of the active site cysteine
residue of papain and the 3 other cysteine endoproteinases by 1-Rh
and 3-Rh should lead to the loss of their catalytic activity. Therefore,
the hydrolytic activity of a mixture of cPAP and 1-Rh (in excess with
respect to thiol content) was periodically measured on 3 differ-
ent chromogenic substrates, namely BAPNa, PFLNa, and ZGlyONp
(Fig. 1). Papain, caricain and chymopapain have rather broad speci-
ficities (aromatic residue at the P2 position is preferred for papain
[9]) and catalyse the hydrolysis of PFLNa, BAPNa and ZGlyONp. Con-
versely, GEP displays a narrow specificity for substrates including
a glycyl residue at the P1 position and is therefore only active on
ZGlyONp [78].
Time-course plots of residual activity followed a different trend
depending on the substrate. The enzymatic activity of the mixture
of cPAP and 1-Rh on PFLNa decreased rapidly up to complete inac-
tivation in ca. 50 min. The activity on BAPNa decreased more slowly
but complete inactivation was reached within 300 min. For ZGly-
ONp, activity decreased too but reached a plateau at ca. 40% (i.e.
60% inhibition) after ca. 3 h. It clearly highlights some differences
in the reactivity of 1-Rh towards the individual cysteine endo-
proteinases of the crude papain sample. A mixture of cPAP and
Analysis of the metalloprotein resulting from the reaction of
afPAP and 1-Rh by LC-ESI–MS confirmed the formation of the bio-
conjugate as well as the presence of two other papain derivatives
in the sample (Fig. S6). The value of 24,008 Da matched the calcu-

318
N. Madern et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 314–322
13
Cl
Cl
12
O
O
NH
NH
NH2
N
6
5
4
2
7
9
6a
10a
4a
1a
b)
a)
8
3
N
N
N
N
N
Cl-
Rh
1
Cl
Cp*
1-Rh
Scheme 1. (a) chloroacetyl chloride, TEA, DCM, −78 ◦C to RT, 93% ; (b) [Cp*Rh(␮-Cl)Cl]2, DCM, RT, 36%.
8
7
HO
N
O
O
O
O
NH2
N
N
N
4
c)
b)
a)
6
3
2
5
N
N
N
N
N
N
N
1
Rh
2
3
Cl-
Cp*
Cl
3-Rh
Scheme 2. (a) NH4OAc, NH4OH, Zn, H2O/EtOH, ꢂ, 82%; (b) maleic anhydride, then ZnCl2, HMDS, toluene, ꢂ, 74%; c) [Cp*Rh(␮-Cl)Cl]2, DCM, RT, 96.
A)
B)
time (min)
100
80
60
40
20
0
0
5
10 15 20
BAPNa
ZGlyONp
PFLNa
0.0
-0.5
-1.0
-1.5
-2.0
100
80
60
40
20
0
ZGlyONp
10 12 14 16 18 20 22
0
2
4
6
8
0
50 100 150 200 250 300
time (min)
time (min)
Fig. 1. (A) Time-dependent inactivation of crude papain (2 mg solid/ml in phosphate buffer pH 7; [thiol] = 110 ␮M) by 1-Rh (1 mM). Hydrolytic activity periodically measured
on BAPNa (red), PFLNa (blue) and ZGlyONp (green). (B) Time-dependent inactivation of gel filtered crude papain (0.093 mg/ml in PBS pH 7.4; [thiol] = 3 ␮M) by 3-Rh (100 ␮M).
Hydrolytic activity measured on ZGlyONp. Inset: semi-logarithmic plot of% hydrolysis rate vs. time. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
lated mass of the species resulting from bioconjugation of 1-Rh to
papain via alkylation of the thiolate group of Cys25 and replace-
ment of the chloro ligand by a trifluoroacetate ligand and allowed
to rule out direct coordination of the Rh complex to sulphur (calcu-
lated MW = 23,930 Da) that may have occurred owing to rhodium
thiophilicity. No peak corresponding to a double conjugate was
observed on the mass spectrum, indicating that the rhodium con-
Scheme 3. Transfer hydrogenation of TFACP.
tent previously determined by absorption measurements had been
severely overestimated.
and 40 ◦C. Correlatively, the initial rate of conversion expressed as
3.3. Aqueous transfer hydrogenation of TFACP catalysed by the
complexes and the metallopapains
TOF was 3.5 times higher with 1-Rh as compared to 3-Rh (Table 2).
Other aryl ketones were also reduced to the corresponding alcohols
with good conversions in the presence of 1-Rh (Table S1).
Catalytic runs were carried out at the analytical scale on the
activated ketone TFACP using Na formate as hydrogen donor (HD)
(Scheme 3). Time course of the conversion to ␣-(trifluoromethyl)
benzyl alcohol was monitored by HPLC (Fig. 2A). Quantitative con-
version was reached in 1 h with 1-Rh and 25 h with 3-Rh at pH 7.4
Since the (A)TH of ketones catalysed by analogous complexes is
generally pH-dependent [42,46,49,82–84], we thought it relevant
to carry out the catalytic runs at various pH. With 1-Rh, conversion
of TFACP was mostly independent of pH in the range between 3.4
and 8 (Fig. 3) as previously observed for the reduction of NAD+

N. Madern et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 314–322
319
A)
B)
40
35
30
25
20
15
100
80
60
40
20
0
1-Rh (ph 7.4)
10
3-Rh (ph 4.5)
cPAP-1-Rh
afPAP-1-Rh
3-Rh (ph 7.4)
5
0
0
100 200 300 400 500 600
0
200 400 600 800 1000 1200 1400
time (min)
time (min)
Fig. 2. (A) Time course of TH of TFACP in the presence of 1-Rh or 3-Rh. Conditions: [TFACP] = 10 mM, 1 mol% cat., [formate] = 1 M (pH 7.4 or 4.5), 40 ◦C. (B) Time course of TH
of TFACP in the presence of cPAP-1-Rh or afPAP-1-Rh. Conditions: [TFACP] = 5 mM, 1 mol% cat., [formate] = 1 M in citrate-phosphate pH 5.1, 40 ◦C.
Table 2
(Asymmetric) transfer hydrogenation of TFACP catalyzed by 1-Rh, 3-Rh and the
100
metallopapains. Conversion and ee determined by HPLC. Conditions: 1 mol% cat,
1 M formate pH 7.4, 40 ◦C.
80
Catalyst
TON (mol/mol) TOF (h−1
)
Conversion (%) [time, (h)] ee (%)
1-Rh
3-Rh
cPAP-1-Rh
afPAP-1-Rh 48
100
94
30
56
16
1.5
6
100 [1.25]a
95 [25]b
–
–
60
40
20
0
86 [7]b,c
29 [24]b,d
43 [24]b,d
16 [72]b,c
9 (S)e
3 (S)
5 (R)
cPAP-3-Rh
20
–
1-Rh, formate pH 6.1
1-Rh, phosphite pH 7
3-Rh, formate pH 7
a
[TFACP] = 10 mM.
[TFACP] = 5 mM.
Citrate-phosphate pH 4.5.
Citrate-phosphate pH 5.1.
Determined by chiral GC.
b
c
d
e
0
50 100 150 200 250 300
HD/S
1-Rh
Fig. 4. Influence of hydrogen donor (HD) concentration on the conversion of TFACP
catalyzed by 1-Rh and 3-Rh. Conditions: [TFACP] = 10 or 5 mM; 1 mol% cat, 30 min
(1-Rh) or 24 h (3-Rh), 40 ◦C.
3-Rh
100
80
60
40
20
0
cPAP-1-Rh
100
1-Rh
cPAP-1-Rh
80
60
40
20
0
1
2
3
4
5
6
7
8
pH
Fig. 3. Influence of pH on conversion of TFACP catalyzed by 1-Rh (black), 3-Rh (red)
or cPAP-1-Rh (orange). Conditions: [TFACP] = 10 mM, 1 mol% cat, [formate] = 1 M,
30 min (1-Rh), 18 h (3-Rh) or 24 h (cPAP-1-Rh), 40 ◦C. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version
of this article.)
1000
800
600
400
200
0
S/C
Fig. 5. Influence of catalyst load S/C on the conversion of TFACP catalyzed by 1-Rh
or cPAP-1-Rh. Conditions: [formate] = 1 M pH 6.1 (1-Rh) or pH 6.6 with phosphate
buffer (cPAP-1-Rh), 30 min (1-Rh) or 24 h (cPAP-1-Rh), 40 ◦C.
catalysed by [Cp*Rh(bpy)(OH2)]2+ [85]. With 3-Rh, the optimal pH
was found in the acidic range (between 3.4 and 5). Correlatively, the
TOF increased from 16 h−1 at pH 7.4–38 h−1 at pH 4.5 (see Table 2).
The extent of conversion of TFACP by 1-Rh and 3-Rh at constant
catalyst load was also found to increase with the concentration of
formate (Fig. 4). Even with as low as 2.5 eq of formate with respect
to substrate, conversion reached 34% in 30 min in the presence of
1-Rh. Phosphite was found an alternative HD in the reduction of
NAD+ catalysed by [Cp*Rh(bpy)(OH2)]2+ [86]. We found out that
phosphite could act as hydrogen donor in the aqueous TH of TFACP
catalysed by 1-Rh but conversions were lower than with formate
especially as low HD/S (Fig. 4). Conversions in the presence of 1-Rh
also increased with the catalyst load S/C, reaching 60% at 30 min
with S/C = 100 (Fig. 5).
Gratifyingly, metallopapains cPAP-1-Rh, afPAP-1-Rh and
cPAP-3-Rh were able to catalyse the TH of TFACP but at lower
rates and conversions compared to the free complexes (Fig. 2B

320
N. Madern et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 314–322
were able to engage H-bond interactions with the backbones of
Gly66 and Asp158 (Fig. 7C and Fig. 7D), while Tyr67 determined a
distortion of the octahedral metal coordination because of steric
hindrance only in case of 1-Rh(R) enantiomer (Fig. 7B). On the
contrary, H-bond interactions were not observed in the top-scored
pose of 3-Rh having a lower docking score (about 2 kcal/mol vs.
4 kcal/mol in both 1-Rh(R) and 1-Rh(S)). It is worth noting that
the same H-bond interactions herein hypothesized for 1-Rh have
been observed in other X-ray solved covalent inhibitors of papain.
Meaningful examples are given by benzyloxycarbonyl-glycyl-
phenylalanyl-methylenylglycyl derivative (PDB code: 5PAD [76])
and l-leucyl-N-(2-oxopropyl)-l-phenylalaninamide (PDB code:
1KHQ [91]). On the basis of covalent docking simulations, we can
thus derive that the higher enantioselective ability of 1-Rh with
respect to 3-Rh is likely due to the different accessibility of the
metal centre after covalent binding with papain. On one side, we
observed that both 1-Rh enantiomers could establish two H-bond
interactions with the protein, having a stabilizing conformational
effect. On the other, the lack of H-bond interactions allowed the
3-Rh compound a higher conformation freedom, which impacted
its enantioselectivity.
However, it is worth noting that the observed H-bond interac-
tions involved only the amide group of 1-Rh while, on the contrary,
the metallic head was not able to establish any strong interac-
tion with the protein residues, due to the absence of H-bond
donors and/or acceptors substituents. This evidence not only offers
a glimpse of a possible explanation of the observed low enantios-
electivity but could pave the way for the design of new and more
efficient piano-stool d6-rhodiumIII complexes. This point is at the
moment under investigation. Indeed, we are currently screening a
large chemical library of polypyridine rhodiumIII complexes includ-
ing various H-bond acceptors and donors bound to the metallic
head.
Fig. 6. Superimposition between the X-ray structure of papain covalently bound to
an achiral RuII complex (PDB code: 4KP9) and the top-scoring pose of 3-Rh obtained
from covalent docking simulations. For the sake of clarity, only polar hydrogen atoms
are displayed. Metal complexes and important residues are rendered as sticks while
the protein is represented as a surface. Note that carbon atoms of the co-crystallized
metal complex are rendered in yellow while those of the docked compound in
magenta. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
and Table 2). However, the ee’s were disappointingly low, reaching
at best 9% for cPAP-1-Rh. The catalytic activity of metallopa-
pain cPAP-1-Rh followed a dramatically different trend as regards
pH dependence (Fig. 4). The highest conversions were reached
at acidic pH while conversion was the lowest around neutrality.
This increase of conversion at acidic pH was accompanied with
a nearly complete loss of enantioselectivity (ee <3% at pH 3.4).
This behaviour is likely due to a change of conformation of the
metalloenzyme as a function of pH that in turn influences the
accessibility of the metal centre. Indeed, progressive unfolding of
cPAP-1-Rh occurs at 40 ◦C in the presence of 1 M formate at pH 3.4
as shown by the progressive shift of the fluorescence emission from
331 to 344 nm within 19 h (not shown). Eventually, under optimal
conditions of pH and S/C, full conversion of TFACP was reached
within 24 h in the presence of 3.3 mol% cPAP-1-Rh (Fig. 6). A mod-
est enantiomeric excess of 7–10% was obtained in favour of the
(S)-enantiomer.
4. Conclusions
Two half-sandwich polypyridine rhodiumIII complexes includ-
ing a thiol-reactive function for covalent anchoring to papain were
synthesized and characterized by usual spectroscopic means. Both
were shown to inactivate papain in a time-dependent fashion
by reaction with the native thiolate group of the active site cys-
teine, affording the expected biohybrid adducts. These inactivation
experiments highlighted differences of reactivity of 1-Rh and 3-
Rh towards the four homologous cysteine endoproteinases present
in papaya latex and in particular the probable lack of inactivation
of glycyl endopeptidase by 1-Rh. Both complexes were shown to
catalyse the aqueous transfer hydrogenation of an activated aryl
ketone under mild conditions of temperature and pH with the
phenanthroline complex being much more active than the bispyri-
dine one. Covalent insertion of both metallic species within papain
converted it to a transfer hydrogenase. Under optimized condi-
tions of pH and catalyst load, conversion of TFACP was quantitative
within 24 h in the presence of metallopapain cPAP-1-Rh and the
alcohol product was obtained with a modest enantiomeric excess of
7–10% in favour of the (S)-enantiomer. Molecular modelling studies
complemented the experimental data, giving a sound explanation
to the lack of enantioselectivity of the hybrid catalysts. The agree-
ment with experimental findings strongly supported the use of this
theoretical approach combining quantum mechanics and covalent
docking simulations when crystallographic data are not available.
At present, we are screening a large chemical library of polypyri-
dine rhodiumIII complexes with the aim to identify compounds
having low conformational freedom after their covalent insertion
within papain. We do believe that this ongoing attempt might help
to address future design of more selective hybrid catalysis.
3.4. Theoretical insights
As above-mentioned, 1-Rh and 3-Rh were covalently anchored
to the residue Cys25 within the papain active site. Schechter and
Berger established that the binding site of papain contained 7 sub-
sites, each one accommodating an aminoacid of the substrate [87].
These subsites were denominated from S1 to S4 (non-primed bind-
ing sites) and from S’1 to S’3 (primed binding sites). Later on, X-ray
structural data on papain-inhibitor complexes brought to light the
aminoacids involved in each subsite as follows: S1–Gln19, His159,
Gly23; S2–Pro68, Val133, Val157; S3–Tyr61, Tyr67; S’1–Gly20,
Ser21, Cys22; S’2–Gln142; S’3–Trp177.[88,89] These residues may
establish additional non-covalent interactions with compounds
bound to Cys25.
Fig. 6 shows the superimposition of the obtained top-scored
pose of 3-Rh and that taken from an X-ray solved structure of a
similar metal complex bounded to papain having a RuII instead of
RhIII as metal (PDB code: 4KP9, resolution 2.10 Å [90]). Interest-
ingly, the same subsites played similar roles and a good overlap
was observed when superimposing docking and X-ray poses, thus
suggesting that covalent docking is a valuable strategy for the
present investigation. Furthermore, we observed that poses com-
parable to that obtained for 3-Rh were also found for 1-Rh(R) and
1-Rh(S) compounds (see Fig. 7A). Importantly, both enantiomers

N. Madern et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 314–322
321
Fig. 7. Superimposition of the obtained top-scoring poses of 1-Rh(R), 1-Rh(S) and 3-Rh. (A); top scoring pose of 1-Rh(R) (B); 1-Rh(S) (C) and 3-Rh (D). Ligands and important
residues are rendered as sticks while protein is shown as a surface. H-bond interactions are depicted by a dotted line. For the sake of clarity, only the polar hydrogen atoms
are displayed.
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The Agence Nationale de la Recherche (ANR) is gratefully
acknowledged for financial support (project “Artzymes”, grant
number ANR-11-BS07-027-01).This work was supported by the
LabEx MiChem part of French state funds managed by the ANR
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Appendix A. Supplementary data
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