Functionalized cationic (η6-arene)ruthenium(II) complexes for site-specific and covalent anchoring to papain from papaya latex. Synthesis, X-ray structures and reactivity studies

Article abstract of DOI:10.1016/j.tetlet.2008.05.043

The cationic (η⁶-arene)ruthenium complexes 6-9 containing a chloroacetamide or a maleimide functional group on the arene ligand were synthesized and successfully used to introduce ruthenium(II) species to the active site of the cysteine endoproteinase papain in a site-directed and covalent fashion as shown by enzymatic and ESI-MS studies.

Full text of DOI:10.1016/j.tetlet.2008.05.043

Tetrahedron Letters 49 (2008) 4670–4673
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Functionalized cationic (g6-arene)ruthenium(II) complexes for site-specific
and covalent anchoring to papain from papaya latex. Synthesis, X-ray
structures and reactivity studies
Pierre Haquette a, Barisa Talbi a, Sigolène Canaguier a, Samuel Dagorne b, Céline Fosse c,
Annie Martel a, Gérard Jaouen a, Michèle Salmain a,
*
a Ecole Nationale Supérieure de Chimie de Paris, Laboratoire de chimie et biochimie des complexes moléculaires, UMR CNRS 7576, 11 rue Pierre et Marie Curie,
75231 Paris cedex 05, France
b Laboratoire DECOMET, Institut de Chimie, UMR CNRS 7177, Institut Le Bel, 4, rue Blaise Pascal, 67000 Strasbourg, France
c Ecole Nationale Supérieure de Chimie de Paris, Laboratoire de synthèse sélective organique et produits naturels, équipe de spectrométrie de masse, UMR CNRS 7573,
11 rue Pierre et Marie Curie, 75231 Paris cedex 05, France
a r t i c l e i n f o
a b s t r a c t
The cationic (g6-arene)ruthenium complexes 6–9 containing
a chloroacetamide or a maleimide
Article history:
Received 27 March 2008
Revised 18 April 2008
Accepted 7 May 2008
Available online 11 May 2008
functional group on the arene ligand were synthesized and successfully used to introduce ruthenium(II)
species to the active site of the cysteine endoproteinase papain in a site-directed and covalent fashion as
shown by enzymatic and ESI-MS studies.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Ruthenium complexes
Papain
S-Alkylation
We have recently reported the covalent and chemospecific
introduction of several transition organometallic complexes
including various metals to the enzyme papain from Carica papaya.
This was achieved by Michael reaction between the sulfhydryl
group of the sole free cysteine of this enzyme and organometallic
N-substituted maleimides.1 While our main aim was to provide
new heavy metal reagents for protein X-ray crystallography, other
groups have reported the covalent anchoring of transition metal
complexes to the same protein for preparing artificial metallo-
enzymes.2–4 Indeed this concept that relates to the combination
of protein hosts and catalytically active metal centres has recently
witnessed several successes in the area of enantioselective cataly-
sis.5–10 In the particular case of papain, the resulting hybrids were
catalytically active but the measured enantiomeric excesses were
low or even zero.
Nevertheless, we thought it interesting to try to anchor a new
series of complexes with potential catalytic activities to papain.
For this purpose, we selected the family of (g6-arene)ruthenium(II)
complexes as they display versatile catalytic properties and are tol-
erant to water and oxygen.11 Indeed, these complexes with biden-
tate N,N donor ligands catalyze transfer hydrogenation reactions of
ketones and imines.12 Moreover, dicationic (g6-arene)ruthenium
complexes with bidentate ligands are active in Lewis acid-cata-
lyzed Diels–Alder reactions.13–17 Most interestingly, some of these
complexes are even active catalysts in aqueous medium for the
reduction of ketones and imines18–21 and the isomerization of
allylic alcohols.22,23
Therefore we designed and synthesized a series of cationic com-
plexes of the general formula [(g6-arene)Ru(N,N)Cl]+ carrying
either a chloroacetamide or a maleimide moiety on the arene
ligand and studied their reactivity towards papain. These func-
tional groups were selected as they are classical means to specifi-
cally target/ block cysteine residues in protein chemistry. One of
the artificial metalloprotein constructs was characterized by ESI-
MS, which provided good evidence for chemoselective covalent
anchoring of the complexes to papain.
The most general method for preparing ruthenium complexes
of the [RuCl2(g6-arene)]2 type is the reaction of 1,4-cyclohexadiene
derivatives with ruthenium(III) chloride in alcoholic media. The
route used to prepare the complexes described herein, bearing
g6-arene ligands with pendant maleimide or chloracetamide
groups, is depicted in Scheme 1.
Cyclohexadiene derivatives with a terminal amino functionality
C6H7(CH2)nNH2 (n = 1 or 2) 1a and b were prepared by Birch reduc-
tion of commercially available C6H5(CH2)nNH2 compounds. Subse-
quent acylation of 1b with chloroacetyl chloride afforded the
chloroacetamide compound 3 in almost quantitative yield while
* Corresponding author. Tel.: +33 1 44 27 67 32; fax: +33 1 43 26 00 61.
E-mail address: michelesalmain@enscp.fr (M. Salmain).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.043

P. Haquette et al. / Tetrahedron Letters 49 (2008) 4670–4673
4671
Scheme 1. Preparation of (g6-arene)ruthenium(II) complexes 6–9 with a pendant maleimide or chloroacetamide function.
reaction of 1a and 1b with maleic anhydride followed by cycliza-
tion promoted by sodium acetate in acetic anhydride led to the
corresponding maleimide compounds 2a and 2b in moderate yield.
Reduction of ruthenium(III) chloride in refluxing ethanol in the
presence of a stoichiometric amount of 2a and 2b or 3 afforded
orange solids of the dimeric g6-arene complexes 4a, 4b and 5,
respectively, in good yields. These complexes are soluble in
methanol and DMSO but are converted in the latter solvent to
the corresponding [RuCl2(g6-arene)(DMSO)] complex within a
few days. The X-ray structure of the neutral complex RuCl2[g6-
(C6H5(CH2)2NHC(O)CH2Cl)(DMSO)] 10 is depicted in Figure 1.
Complex 10 adopts a typical piano-stool geometry with a pseu-
do-tetrahedral arrangement of the arene, the chloride ligands and
the S atom of the coordinated DMSO ligand around the ruthenium
centre. Bond lengths and bond angles were comparable to the clo-
sely related 1,4,9,10-tetrahydroanthracene ruthenium complex
[(g6-C14H14)RuCl2(DMSO)]24 and the p-cymene ruthenium com-
plex [(g6-C10H14)RuCl2(DMSO)].25
When treated with the N,N donor ligands 2,2’-bispyridyl or
1,10-phenanthroline, the dimeric complexes 4a, b and 5 were con-
verted to the monocationic complexes 6–9 containing N,N-chelate.
Complexes 6–9 are soluble in methanol and water. In the latter sol-
vent, the monocationic chloro complexes are in equilibrium with
the dicationic aqua complexes resulting from the abstraction of
the chloride ligand. The X-ray structure of complex 8 carrying a
pendant chloroacetamide group is depicted in Figure 2.
Figure 1. Molecular structure of complex 10 calculated at 50% probability level.
Selected bond distances (Å) and bond angles (°): Ru(1)–Cl(1), 2.4026(7), Ru(1)–
Cl(3), 2.4125(7), Ru(1)–S(1), 2.3400(6), Ru(1)–C(3), 2.197(3), Ru(1)–C(4), 2.158(3),
Ru(1)–C(5), 2.194(3), Ru(1)–C(6), 2.212(3), Ru(1)–C(7), 2.222(2), Ru(1)–C(8), 2.21-
7(3), Cl(1)–Ru(1)Cl(3), 88.26(3), Cl(1)–Ru(1)–S(1), 86.05(2), Cl(3)–Ru(1)–S(1),
85.96(2).
ꢀ
Compound 8 crystallizes in the triclinic space group P1. The
anthroline and bipyridyl ligands. The distances between Ru and the
6 carbons of the arene ligand range from 2.153 to 2.246 Å, the lon-
gest bond being measured between Ru and the substituted aro-
matic carbon C16. The interplanar angle between the arene and
the bipyridyl planes is 47.94°, which is significantly smaller than
that measured for [Ru(g6-C6H5(CH2)3OH)(bipy)Cl]BF4 (58.5°),26
[Ru(g6-C6H5CH2COOH)(phen)Cl]OTf (58.4°)27 and [Ru(g6-C6H6)-
(phen)Cl]Cl (62.7°).18 The cation displays an eclipsed conformation
for its substituent methylene carbon atom C17 with respect to the
asymmetric unit comprises two molecules of the cationic complex
and two chloride counterions. These counterions are hydrogen-
bonded to the NH amide of neighbouring molecules (dis-
tance = 2.217 Å). Complex 8 adopts a piano-stool, pseudo-tetra-
hedral coordination geometry at the ruthenium atom. The
chloroacetamide chain is moved above the plane of the arene
ligand. The distance between the centroid and the ruthenium atom
is 1.69 Å, very close to related monocationic structures with phen-

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P. Haquette et al. / Tetrahedron Letters 49 (2008) 4670–4673
Table 1
Irreversible inactivation of papain by complexes 6–9
Compound
kobs (minÀ1
)
6a
6b
7a
7b
0.112 0.009
0.090 0.004
0.100 0.004
0.159 0.001
0.036a
NEM
8
9
0.035 0.001
0.049 0.003
0.028b
FcCOCH2Cl
TPCKc
0.121 0.005
Pseudo-first order rate constants kobs measured for [complex] = 100 lM.
a
N-Ethylmaleimide; lit.33
b
Lit.32
c
N-p-Tosyl L-phenylalanine chloromethyl ketone.
the bipy series. Interestingly, all the new maleimide derivatives
inactivated papain much faster than NEM. Such a behaviour was
previously noticed with our former series of organometallic malei-
mides and was attributed to the hydrophobic character of the
complexes.1
Figure 2. Molecular structure of complex 8 calculated at 50% probability level.
Selected bond distances (Å) and bond angles (°): Ru–Cl(1), 2.3978(7), Ru–N(1),
2.091(2), Ru–N(2), 2.083(2), Ru–C(11), 2.210(3), Ru–C(12), 2.153(3), Ru–C(13),
2.192(3), Ru–C(14), 2.196(3), Ru–C(15), 2.213(2), Ru–C(16), 2.246(2), Cl(1)–Ru–N-
(1), 86.67(6), Cl(1)–Ru–N(2), 85.60(6), N(1)–Ru–N(2), 76.84(8).
Both chloroacetamides 8 and 9 were also able to inactivate
papain in a time-dependent fashion, although no reaction had pre-
viously been noticed with cysteine ethyl ester in D2O (see above).
The higher reactivity of papain’s active site cysteine towards elec-
trophiles with respect to cysteine and glutathione has been abun-
dantly discussed.31 It is related to the unusually low pKa of the thiol
of Cys25 (3–4) and the existence of an ion pair with the imidazole
side chain of His159 under physiological conditions. The rates of
inactivation with 8 and 9 were ca. three times slower that those
measured with the maleimide complexes. The phenanthroline
complex 9 reacted slightly faster that the bispyridine derivative 8
as in the maleimide series with the longer spacer arm. The pseu-
do-first order rate constants were in the same order of magnitude
Cl ligand as previously observed in the case of [Ru(g6-C6H5–
(CH2)3OH)(bipy)Cl]BF4.
Reaction of 7b with 1 mole equiv of cysteine ethyl ester was
monitored by 1H NMR in D2O. Immediate disappearance of the sin-
glet at 6.8 ppm assigned to the vinylic protons of the maleimide
double bond was observed on the spectrum of the mixture, indicat-
ing that reaction was fast and quantitative. Conversely, no change
of 1H NMR spectrum of the mixture of 9 and cysteine ethyl ester in
D2O was observed within 2 days, indicating that no reaction
occurred between these compounds. Instead, formation of the
corresponding dicationic aqua complex was shown to occur.
Reactivity studies were then pursued with papain. This protein
is a member of the cysteine endoproteinases family and catalyzes
the hydrolysis of peptide bonds in peptides and proteins.28 Chem-
ical modification of its active site cysteine (Cys25) is known to
induce the loss of its catalytic activity.29 Therefore, if alkylation
of the sulfhydryl group of Cys25 occurs, no more peptidase activity
should be measured for the enzyme treated by the complexes. The
kinetics of inactivation may be monitored indirectly by periodi-
cally assaying the catalytic activity of the enzyme-complex mix-
tures. In a typical experiment, papain (3.5 lM) was incubated
with complexes 6–9 (100 lM, that is, pseudo-first order condi-
tions) in a DMSO/H2O 5:95 mixture. The hydrolytic activity of
the mixtures was periodically assayed on the chromogenic sub-
strate PFLNa.30 Semi-logarithmic plots of initial hydrolysis rates
versus time were linear and gave the pseudo-first order rate
constants of inactivation kobs (Table 1).
It appears that the kinetics of the inactivation process was
weakly dependent on the structure of the complex but markedly
dependent on the nature of the tethered reactive function. In the
malemide series (complexes 6 and 7), the presence of the phenan-
throline ligand had an enhancing effect on the rate of inactivation
of papain when the spacer arm between the maleimide ring and
the complex was long. For the short chain complexes, the effect
was opposite. For the phenanthroline derivatives, shortening of
the spacer arm decreased the rate of inactivation by one third. This
finding is most certainly related to steric hindrance effects. The
presence of the bulky [(g6-arene)Ru(N–N)Cl]+ entity at close prox-
imity of the reactive maleimide group may slow down the rate of
reaction with the thiol of Cys25. No such effect was noticed in
as that reported for ferrocenyl chloromethyl ketone.32 The kobs
/
[complex] values, respectively, equal to 5.8 and 8.2 MÀ1 sÀ1 for 8
and 9 were only two to three times smaller than that measured
for the prototype irreversible inhibitor TPCK which was equal to
20.2 MÀ1 sÀ1 under the same experimental conditions.
To demonstrate that papain inactivation really resulted from
the alkylation of the sulfhydryl group of Cys25, the following
experiment was performed. Freshly affinity-purified papain was al-
lowed to react with an excess of 8 until no more enzymatic activity
was detected for the mixture. After extensive dialysis to remove
excess reagent, the solution was concentrated and analyzed by
ESI-MS. The resulting spectrum (Fig. 3) displayed several peaks
ranging from m/z 997 to 1494.9, corresponding to different charge
states of the protein. The charge state of each peak was first calcu-
lated, assuming that neighbouring peaks differ by one positive
charge. Then the mass was manually determined for each peak
and averaged to yield 23894 2 Da. It was significantly higher
than the theoretical mass of the conjugate resulting from the alkyl-
ation of papain’s sulfhydryl group (23882 Da) but very close within
experimental error to the mass of the adduct where the chloride
ligand is replaced by a formato ligand (23892 Da). This substitu-
tion is likely to occur within the experimental conditions used
for the mass analysis (high concentration of formic acid at high
temperature). Indeed, the ESI-MS analysis of complex 8 performed
in the same conditions showed that this transformation did occur.
In conclusion, several (g6-arene)ruthenium(II) complexes carry-
ing a maleimide or a chloroacetamide function on the arene ligand
were synthesized for the purpose of covalently anchoring procata-
lytic species into the active site of the cysteine endoproteinase
papain. Reactivity studies of the complexes with model cysteine
derivatives in water showed that S-alkylation took place only with
the maleimide derivatives. Nonetheless, both the maleimide and

P. Haquette et al. / Tetrahedron Letters 49 (2008) 4670–4673
4673
Figure 3. ESI-MS of adduct of papain with complex 8.
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Acknowledgements
The French Ministry of Research and the CNRS are gratefully
acknowledged for financial support.
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Supplementary data
Experimental synthetic procedures and characterizations of
ligands and complexes, enzymatic assay protocols; crystal data
and data collection parameters for 8 and 10. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2008.05.043.
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