Receptor-Based Artificial Metalloenzymes on Living Human Cells

Article abstract of DOI:10.1021/jacs.8b04326

Artificial metalloenzymes are known to be promising tools for biocatalysis, but their recent compartmentalization has led to compatibly with cell components thus shedding light on possible therapeutic applications. We prepared and characterized artificial

Full text of DOI:10.1021/jacs.8b04326

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Article
Receptor-based artificial metalloenzymes on living human cells
Wadih Ghattas, Virginie Dubosclard, Arne Wick, Audrey
Bendelac, Régis Guillot, Rémy Ricoux, and Jean-Pierre Mahy
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04326 • Publication Date (Web): 18 Jun 2018
Downloaded from http://pubs.acs.org on June 18, 2018
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Receptor-based artificial metalloenzymes on living human cells
Wadih Ghattas,* Virginie Dubosclard, Arne Wick, Audrey Bendelac, Régis Guillot, Rémy Ricoux,
JeanꢀPierre Mahy*
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Laboratoire de Chimie Bioorganique et Bioinorganique (LCBB)
Institut de Chimie Moléculaire et des Matériaux d’Orsay (ICMMO)
UMR 8182 CNRS
Univ Paris Sud, Université ParisꢀSaclay
9
1405 Orsay cedex, France
ABSTRACT: Artificial metalloenzymes are known to be promising tools for biocatalysis, but their recent compartmentalization
has led to compatibly with cell components thus shedding light on possible therapeutic applications. We prepared and characterized
artificial metalloenzymes based on the A2A adenosine receptor embedded in the cytoplasmic membranes of living human cells. The
wild type receptor was chemically engineered into metalloenzymes by its association with strong antagonists that were covalently
bound to copper(II) catalysts. The resulting cells enantioselectively catalyzed the abiotic DielsꢀAlder cycloaddition reaction of
cyclopentadiene and azachalcone. The prospects of this strategy lie in the organ confined in vivo preparation of receptorꢀbased
artificial metalloenzymes for the catalysis of reactions exogenous to the human metabolism. These could be used for the targeted
synthesis of either drugs or deficient metabolites and for the activation of prodrugs, leading to therapeutic tools with unforeseen
applications.
INTRODUCTION
Human Embryonic Kidney (HEK) cells, hence using human
cell membranes as a compartment to shield metal cofactors
from damage and provide artificial enzymes with promising
therapeutic prospects. To our knowledge, membrane receptors
were never used to form artificial enzymes thus far.
Artificial metalloenzymes are typically chemically prepared
by incorporating synthetic transition metal catalysts into host
10
1,
2
proteins.
These hybrid biocatalysts are imparted with exꢀ
11, 12
Our
traordinary catalytic properties provided by the nonꢀnative
metal cofactor and with a protective and stereoselective enviꢀ
ronment provided by the hosting protein. The development of
these state of the art biocatalysts is still in its infancy and
despite several examples of efficient chemoꢀgenetically optiꢀ
mized artificial metalloenzymes, to our knowledge these are
not being used yet by the chemical industry. Their great poꢀ
tential is however driving their employment in looming theraꢀ
peutic applications. Indeed, artificial metalloenzymes expand
the repertoire of reactions catalyzed by natural enzymes and
could be prepared in selected organs by using metal cofactors
targeting specific host proteins. Subsequently, the formed
artificial metalloenzymes could, for example, activate proꢀ
drugs by the catalysis of abiotic reactions. Ultimately, they
could even be used in treatments to cure metabolic disorders
by catalyzing the synthesis of deficient metabolites. For in
vivo applications, metal cofactors ought to target their specific
host protein in affected organs. Instead, most metal cofactors
strategy targeted the wild type receptor for modifications by
using one of its strong antagonists covalently bound to a copꢀ
per catalyst. We evaluated the potential of the receptor based
artificial metalloenzyme in the catalysis of a prototypeꢀ
reaction i.e. the DielsꢀAlder cycloaddition reaction, which is
3
13, 14
4
rarely catalyzed by natural enzymes
and not by any known
15
human enzyme. The yield and stereoselectivity obtained for
this carbonꢀcarbon bond forming reaction were in agreement
with the catalysis of the reaction by the artificial metalloenꢀ
zyme assembled in the A2A AR embedded in the cytoplasmic
membrane of the living cells. These conclusions were further
corroborated by similar findings we obtained using the puriꢀ
fied receptorꢀbased artificial enzyme.
5
RESULTS AND DISCUSSION
Strategy for targeting the A2A AR by metal cofactors. Exꢀ
tracellular adenosine activates four guanine nucleotideꢀbinding
6
proteinꢀcoupled receptor (GPCR) subtypes: A , A , A and
are damaged by cellular components, leading to their deactiꢀ
vation before formation of artificial metalloenzymes . To
1
2A
2B
16
7
A3 and each of these four human adenosine receptors (ARs)
1
7
plays an essential role in the response to adenosine. More
specifically, the A2A AR is a guanine nucleotideꢀbinding subuꢀ
nit (Gs) coupled receptor that, when activated, results in an
increase of intracellular cyclic adenosine monoꢀphosphate
overcome these limitations scientist have relied on metal coꢀ
factors that remain active in vivo or on compartmentalization
8
in the periplasm in the case of Gramꢀnegative Escherichia coli
9
(
E. coli) bacteria. The therapeutic potential of these apꢀ
(cAMP). This receptor subtype is a prime therapeutic target as
proaches is however limited as human cells do not possess
periplasm and as only a small number of metal cofactors resist
to damage by cellular components. Herein, we report the prepꢀ
aration of an artificial metalloenzyme based on the wildꢀtype
human A2A Adenosine Receptor (AR) at the surface of living
it is implicated in immunoꢀoncological treatments as well as in
1
8
Parkinson’s disease treatments among others. The seek for
therapeutic applications yielded extensive libraries of reversiꢀ
ble A2A ligands with a wide range of affinities. Both structure
activity relationship (SAR) studies as well as the elucidation
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of over thirtyꢀfive 3ꢀD crystal structures of free, ligand bound,
and chimeric A AR have allowed to clarify its binding
frequently used as the active site of artificial metalloenzymes
25, 26, 27, 28, 29, 30, 31
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by us and others,
since it was found to be able
2A
19
mode. Most A2A ligands consist of a core structure with
variable substituents. On the one hand, the core gets bound
inside the adenosine binding pocket and is responsible for the
major contribution to the binding affinity. On the other hand,
the variable substituents extend out of the binding pocket,
pointing toward the extracellular medium, and can be used to
tune the binding affinity and other physical properties relevant
to catalyze cycloadditions efficiently under mild conditions
i.e. in buffered water pH∼7 at 4 ˚C. Hallmark substrates cycloꢀ
pentadiene (6) and 2ꢀazaꢀchalcones (7a-c) reacted at 2.5 mol%
loading of catalyst and afforded up to 50 % yield of isomer
products (8a-c) in 72 h. Under the same conditions, mechanisꢀ
tic probes chalcone (7d) and 4ꢀazaꢀchalcone (7e) that cannot
coordinate copper(II) in a bidentate mode did not react
demonstrating that a reactive electrophilic diene was not obꢀ
tained unless both the pyridine and the carbonyl group of the
substrate were bound to Cu(II) (Scheme 1A). The devised
molecules (12) and (13) were prepared by the reaction of
tosylate ester (6) with two Nꢀmonosubstituted piperazine deꢀ
20
for drug development. Thus, to target the receptor a metal
cofactor had to be attached to a substituent of a strong ligand
of the A2A AR. Binding the metal complex to substituents of
various lengths would allow to explore the effects that the
depth of its insertion in the receptor could induce on the selecꢀ
tivity of the subsequent catalysis. Unlike agonists, antagonists
do not induce a cellular response i.e. increase of intracellular
cAMP and the resulting acceleration of the internalization of
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rivatives (10) and (11) that also contained
a 1,10ꢀ
phenanthroline moiety (Scheme 1B).
21
the receptor. Consequently, receptors containing antagonist
bound metal complexes would remain for a relatively longer
amount of time on the cell surface than their analogues conꢀ
taining agonist bound metal complexes. Preladenant (1) and
SCH 412348 (2) are strong antagonists of the A2A AR (K =
i
1
.1 nM and 0.6 nM, respectively) that were potential drugs
22
developed for the treatment for Parkinson's disease. They
possess related chemical structures as they share the same 2ꢀ
furanylꢀ7Hꢀpyrazolo[4,3ꢀe][1,2,4]triazolo[1,5ꢀc]pyrimidinꢀ5ꢀ
amine adenosineꢀlike core but differ by the terminal Nꢀaryl
substituent on the piperazine ring (Figure 1). It is noticeable
that this substituent weakly affects the binding affinity since
both antagonists exhibit comparable inhibition constants in the
nanomolar range. This was confirmed by massive SAR studꢀ
ies, which proved that this core structure was suitable for
developing A2A antagonists upon substitution with different
residues leading to derivatives such as SCH 58261 (3) and
SCH 4442416 (4) with K = 1.9 nM and 11.1 nM, respectively
i
23
(Figure 1). Furthermore, the core of the antagonist was subꢀ
stituted by bulky groups such as an alexaꢀfluorophore and
resulted in derivative (5) that still showed a high affinity for
2
4
the receptor (Figure 1).
NH
2
NH
2
O
O
N
N
O
N
N
N
N
N
N
O
N
N
N
N
N
N
O
N
N
1
, K
i
= 1.1 nM
i
3, K = 1.9 nM
NH
2
NH
2
F
O
N
N
N
N
N
N
N
Scheme 1. A) DielsꢀAlder cycloaddition leading to four isoꢀ
F
N
N
N
mer products. B) Synthesis of prospected 12 and 13 possessing
N
N
a
4, K
i
= 11.1 nM
a
residual
1,10ꢀphenanthroline
group.
Copꢀ
i
2, K = 0.6 nM
O
per(II)phenanthroline complex or artificial metalloenzyme.
NH
2
NH
2
O
O
Binding of the cofactor into the A2A AR on membrane de-
O
O
SO
3
O
N
N
N
HN
NH
O
bris and on cells. Commercial membrane preparations of A
were used to study the binding of the [ H]CGS21680 radioꢀ
NH
2A
O
3
N
N
N
SO
NH
3
2
ligand (K = 27 nM) in competition with increasing concentraꢀ
d
5
i
, K = 111 nM
2
tions of either 12 or 13. The results indicated that both moleꢀ
cules maintained a high affinity for the receptor as extracted
inhibition constants of 3.8 ± 0.5 nM and 5.2 ± 0.9 nM were
found for 12 and 13 respectively, using the Cheng–Prusoff
Figure 1. Chemical structures and K of various antagonists sharꢀ
i
ing
the
2ꢀfuranylꢀ7Hꢀpyrazolo[4,3ꢀe][1,2,4]triazolo[1,5ꢀ
c]pyrimidinꢀ5ꢀamine core structure.
32
equation (Figure 2A). Functional assays that measure intraꢀ
cellular cAMP concentration were also used to probe the efꢀ
fect of 12 and of 13 on HEKꢀ293 cells that expressed A2A
Inspired by this family of strong antagonists we devised
molecules that comprised their core structure and residual
substituents containing a 1,10ꢀphenanthroline moiety that is
known to be able to bind copper(II), leading to catalysts for
DielsꢀAlder cycloadditions based on the Lewis acidity of the
metal cation. The Cu(II)ꢀ1,10ꢀphenanthroline complex was
(
HEKꢀA ). Incubation of HEKꢀA cells with increasing
2A 2A
concentrations of either 12 or 13 had no effect on the concenꢀ
tration of intracellular cAMP, whereas, in contrast, a concenꢀ
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tration dependent production of cAMP was induced by incubaꢀ
receptor binding (Figure 3). The copper(II) cation was also
found to be coordinated by two chlorine anions and one methꢀ
anol molecule in a typical JahnꢀTeller distorted square pyramꢀ
idal geometry. The 1,10ꢀphenanthroline and chlorine ligands
formed the distorted coordination square plane while the
methanol ligand was found in the elongated axial position. The
(1,10ꢀphenanthroline)NꢀCu(II) (2.041 ± 0.004 Å and 2.033 ±
0.004 Å), and Cu(II)ꢀCl (2.2445 ± 0.014 Å and 2.2583 ± 15 Å)
bond lengths are similar to those observed for the antagonist
free copper(II)(1,10ꢀphenanthroline)dichloride, typically emꢀ
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4
5
6
7
8
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tion with increasing concentrations of agonist 5′ꢀ(Nꢀ
ethylcarboxamido)adenosine (NECA). Furthermore, the proꢀ
duction of cAMP decreased when HEKꢀA2A cells were incuꢀ
bated with a mixture of 1.25 µM NECA i.e. the concentration
that increases intracellular cAMP to 80 %, and increasing
concentrations of either 12 or 13 (Figure 2B). It is noteworthy
that NECA, 12 and 13 had no effect on intracellular cAMP
production by HEKꢀ293 cells that did not express A . These
2A
results indicated that 12 and 13 were antagonists with a strong
34
affinity for A . To form artificial metalloenzymes in the
ployed in preparing artificial DielsꢀAlderases. In the proꢀ
2
A
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cytoplasmic membrane at the surface of living cells, the anꢀ
tagonists 12 and 13 had to be combined with copper(II) and
then incubated with HEKꢀA2A cells. Therefore, copper(II)
complexes were prepared under the conditions that were
thereafter used for catalysis, i.e. by incubation of 40 nM 12 or
posed mechanism for the catalysis of the DielsꢀAlder cycloadꢀ
dition reaction, the 1,10ꢀphenanthroline ligand remains metal
bound while the replacement of the chloride ligands by the
bidentate coordination of substrates 7 on the copper(II) cation
35
leads to reactive electrophilic dienes.
1
3 with 1 equiv. copper(II) nitrate in buffer A (MOPS 75 mM,
pH 7.4, glucose 17 mM, NaCl 50 mM), and were characterꢀ
ized in solution by high resolution electrospray ionization
mass spectrometry (HR ESIꢀMS) before incubation with
HEKꢀA2A cells. The obtained molecular masses and isotopic
patterns (Figure 2 C and D) corresponded to those calculated
2+
ꢀ +
2+
ꢀ +
for [12+Cu +Cl ] and [13+Cu +Cl ] ions respectively,
which demonstrated the formation of 12Cu and 13Cu comꢀ
plexes containing a copper(II) ion coordinated by 1,10ꢀ
33
phenanthroline, and a chlorine anion from the buffer soluꢀ
tion. Complexes 12Cu and 13Cu appeared to be as good anꢀ
tagonists of the A2A AR on cells as 12 and 13, leading to idenꢀ
tical affinity constants, respectively within the experimental
error (Figure 2A and B). These results indicated that copꢀ
per(II) was coordinated in the antagonists away from their
core structure, most probably in the 1,10ꢀphenanthroline group
as expected and in accordance with the HR ESIꢀMS results.
Figure 3. Graphical representation of the 3ꢀD crystal structure of
complex 12Cu. Two coꢀcrystallized methanol molecules were
removed for the sake of more clarity.
Binding stability on cells over time. The binding stability of
the antagonists in the receptor binding pocket was investigated
for 72 h at 4 ˚C using a commercial BOronꢀDIPYrromethene
(BODIPY) fluorophore covalently bound to the AR antagonist
Xanthine Amine Conger (BODIPYꢀXAC ; K = 31 nM). It
d
was previously shown that cytoplasmic membranes were
stained when a similar BODIPYꢀXAC conjugate was incubatꢀ
ed at 22 ˚C with Chinese Hamster Ovary (CHO) cells that
36
express the A adenosine receptor subtype. However, after
1
90 min, the fluorophore was detected inside the cells, which
reflected its internalization. We thus decided to incubate either
HEKꢀ293 or HEKꢀA2A cells at 4 ˚C with 5 nM BODIPYꢀ
XAC. After washing with buffer A, microscopic imaging
revealed that a 2 min incubation was sufficient to stain the
cytoplasmic membranes of HEKꢀA2A cells (Figure 4A), while
HEKꢀ293 cells remained unstained. More importantly, the
stained HEKꢀA2A cells were maintained in buffer A for 72 h at
4
˚C and microscopic imaging revealed that more than 90 % of
BODIPYꢀXAC remained at the surface of the HEKꢀA2A cells
(Figure 4B) while in contrast, at 22 ˚C internalization was
observed after only about 10 min (Figure 4C). These results
demonstrated that when HEKꢀA2A cells were kept at 4 ˚C,
antagonists could be maintained bound in the A2A AR for at
least 72 h. Finally, the incubation at 4˚C of HEKꢀA2A cells
with a mixture of 5 nM BODIPYꢀXAC and increasing concenꢀ
trations of 12Cu gradually prevented the staining of cytoplasꢀ
mic membranes indicating that 12Cu was binding the A2A AR
instead of BODIPYꢀXAC (Figures 4 D1 to D3).
Figure 2. A) inhibition of the binding of radio ligand
3
[
H]CGS21680 by A2A in the presence of increasing concentraꢀ
). B)
Quenching of TRꢀFRET signal of an antibody of cAMP by inꢀ
creasing concentrations of NECA ( ), and increase of the TRꢀ
FRET signal with addition of increasing concentrations of 12 (
), 13 ( ), 12Cu ( ) and 13Cu ( ) in the presence of 1.2 µM
tions of 12 ( ), 13 ( ), 12Cu ( ) and 13Cu (
NECA. HR ESIꢀMS spectra of C) 12Cu, and of D) 13Cu.
Furthermore, single crystals of complex 12Cu could be obꢀ
tained by slow diffusion of ether in a solution of 12Cu in
methanol and were suitable for Xꢀray diffraction analysis. The
solved crystal structure proved that indeed only the nitrogen
atoms of the 1,10ꢀphenanthroline residue were bound to copꢀ
per(II), while the remainder of the antagonist was available for
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tected with the three substrates and could be caused by the
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hindering of the access of the substrate to the copper center by
A2A protein residues. More importantly, because A2A protein
residues orchestrated the positioning of the substrate around
the copper center, they should also be responsible for the
obtained endo/exo ratios and ees. Similarly, 13Cuꢀbased
metalloenzymes on cells were active and provided yields and
selectivities similar to those obtained in the positive control
experiments. Bearing in mind that 13Cu has a comparable
affinity for the receptor as 12Cu, and that in 13Cu the 1,10ꢀ
phenanthroline residue is covalently bound further away from
the core of the antagonist than it is in 12Cu, it can be assumed
that 13Cu was also bound into the receptor at the surface of
cells but its catalytic center was predominantly exposed to the
extracellular buffer medium. Therefore, 13Cu remained active
when combined with HEKꢀA2A cells but did not induce selecꢀ
tivity for any of the four products.
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Figure 4. Fluorescent Spinning disk confocal laser microscopic
FSDCLM) imaging of, in blue 5 µg/mL Hoechst 33342 and in
red 5 nM BODIPYꢀXAC stained living HEKꢀA2A cells A) after
incubation for 2 min at 4 ˚C and washing in buffer A, B) after
further incubation for 72 h at 4 ˚C, C) after further incubation for
10 min at 22 ˚C. FSDCLM imaging of, in blue 5 µg/mL Hoechst
(
3
3342 and in red 5 nM BODIPYꢀXAC stained living HEKꢀA2A
cells in the presence of D1) 25 nM, D2) 2.5 µM, and D3) 25 µM
2Cu and incubation for 2 min at 4 ˚C followed by washing in
buffer A.
Mechanistic probes 7d-e did not react under all assayed
conditions and only 7a-c that could bind Cu(II) in a bidentate
mode, were reactive substrates. This implied that the reaction
was indeed taking place only on metal centers. On the contraꢀ
ry, if any template effect had catalyzed the reaction in the case
of 7a-c it should have also catalyzed the cycloaddition with
1
Catalysis with the artificial enzymes at the surface of cells.
The artificial enzymes resulting from combining either 12Cu
7
d-e. In fact, results showed no background catalysis with 7d-
or 13Cu with A , at the surface of living HEKꢀA cells
2A
2A
e in the presence of cells. This indicates that the main backꢀ
ground catalysis observed in the case of 7a-c was most probaꢀ
bly due to transition metals leaked by dead cells.
suspended in buffer A, were used at 2.5 mol% to study the
catalysis of the DielsꢀAlder cycloaddition of 6 and 7a-e at 4˚C.
In addition to the catalysis with the enzymes, positive and
negative controls were performed i.e. the catalysis using either
38
Finally, 1000 equiv. of XAC (K = 2.75 nM) were added
d
1
2Cu or 13Cu in absence of cells, and the catalysis with
to 12Cu and then the mixture was incubated with HEKꢀA2A
cells and used for catalysis. After 72h, yields of 12 % ± 2 of
8a, 9 % ± 2 of 8b and 5 % ± 2 of 8c without ee, were obtained,
hence similar to the results of the catalysis with metal comꢀ
plexes in the presence of HEKꢀ293 cells. The reason for this is
most likely the fact that XAC occupied the receptor instead of
12Cu, thus preventing the assembling of the artificial metalꢀ
loenzyme and leaving the complex interacting nonꢀspecifically
with cell components, which lead to the inhibition of the caꢀ
talysis.
HEKꢀ293 and HEKꢀA2A cells in the absence of the metal comꢀ
plexes, respectively. Similar experiments were also performed
using either 12Cu or 13Cu combined with HEKꢀ293 that do
not express A . Reactions were stopped after 12, 24, 48 and
2A
7
1 h by extraction with ethyl acetate and samples were anaꢀ
lyzed by chiralꢀHPLC with MS/MS detection (Figure S2,
Table 1).
After 72 h, positive controls showed that both 12Cu and
1
7
3Cu catalyzed the reaction with similar yields. Starting from
a, 8a was obtained with about 50 % yield (20 TON), 84/16
The kinetics of the reaction was followed in the presence of
12Cu as catalyst combined with either HEKꢀ293 or HEKꢀA2A
cells. Using 12Cu combined with HEKꢀ293 cells, the reaction
quickly slowed down and the yield reached 14 % after 24 h
and only 16 % after 72 h. Additionally, there was no signifiꢀ
cant ee in favor of any of products 8a. These results indicate
that the catalysis was inhibited by HEKꢀ293 cells, most probaꢀ
bly via nonꢀspecific binding of the complex. In the presence of
endo/exo ratio and no enantiomeric excess (ee). Compounds
b and 7c were less reactive and yielded 40 % of 8b, 83/17
7
endo/exo ratio and 33 % of 8c with 65/35 endo/exo ratio, reꢀ
spectively and both without ee. Negative controls yielded 9 ±
2
% (3.6 TON) of 8a, 4 % of 8b and < 3 % of 8c. This backꢀ
ground reaction could be assigned to the catalysis of the reacꢀ
tion by template effects that could occur in the different hyꢀ
drophobic cavities at the surface of cells including those in
12Cu inserted into the receptor at the surface of HEKꢀA2A
3
7
membrane proteins. It could also have resulted from the
remainders of dead cells including leaked transition metals
possessing Lewis acid properties. Catalysis with either 12Cu
or 13Cu combined with HEKꢀ293 cells provided similar low
yields with a maximum of 16 % (6.4 TON) of 8a, suggesting
that the catalysis with the metal complexes was mostly inhibꢀ
ited by the cells via nonꢀspecific binding. The catalysis with
cells, the reaction was faster, yielding 27 % after 24 h and 42
% after 72 h, which demonstrates the protective effect of the
protein on the catalyst. An ee was observed from the beginꢀ
ning of the reaction (8 % ee after 12 h) indicating that the
catalysis was taking place within the artificial enzyme and was
not only due to background catalysis. The ee increased with
time to reach 14 % after 72 h, suggesting that the catalysis by
the artificial enzyme was dominant. Furthermore, the initial
rate of the reaction catalysed by the artificial enzyme was
similar to that of the reaction catalysed by the Cu(II) catalyst
in solution in absence of cells. (Figure 5).
1
2Cuꢀbased artificial metalloenzymes at the surface of living
HEKꢀA2A cells yielded up to 42 ± 4 % (17 TON) in the case of
8a and an ee of up to 28 ± 0.5 % in the case of 8c. The enꢀ
do/exo ratio of products 8a-c also was affected by the selectivꢀ
ity of the artificial enzyme as the endo isomer was further
favored. This was remarkable with 8c where endo/exo ratio
reached 80/20. As the complex remained active, it demonstratꢀ
ed the protective effect of the embedment of the catalyst inside
the A2A AR. A decline in yields vs. positive controls was deꢀ
These results further confirm the binding assays, the funcꢀ
tional assays and the imaging data to prove that the artificial
enzyme was assembled at the surface of living cells expressing
the A2A receptor, which simultaneously protected the metal
complex and induced stereoselectivity in the catalysis.
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Journal of the American Chemical Society
Table 1. Results of the catalysis of the Diels-Alder cycloaddition reaction by different catalysts.
1
2
3
4
5
6
7
8
9
c
Catalyst
Yield (%)
8b
endo/exo
ee
8
a
8c
8a
8b
8c
8a
8b
8c
n.d.
n.d.
n.d.
n.d.
0
12
13
< 3
< 3
< 3
< 3
< 3
< 3
n.d.
n.d.
n.d
n.d.
n.d. n.d.
n.d. n.d.
< 3
< 3
n.d.
n.d.
b
b
HEKꢀA2A
9 ± 2
4 ± 2
4 ± 2
84/16 83/17
84/16 83/17
84/16 83/17
84/16 83/17
84/16 83/17
84/16 83/17
82/18 78/22
84/16 83/17
n.d.
0
0
0
0
HEKꢀ293
9 ± 2
n.d.
a
1
2Cu
51 ± 4
50 ± 4
16 ± 2
15 ± 2
42 ± 4
50 ± 4
40 ± 3 33 ± 3
40 ± 3 32 ± 3
65/35
65/35
65/35
65/35
80/20
65/35
0
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
13Cu
0
0
0
a
1
1
2Cu + HEKꢀ293
10 ± 2
9 ± 2
6± 2
6± 2
0
0
0
a
3Cu + HEKꢀ293
0
0
0
a
b
1
2Cu + HEKꢀA
28 ± 3
39 ± 3
22± 2
32± 3
14
0
18
0
28
0
2A
a
b
A
13Cu + HEKꢀA2
a
b
c
Reactions were performed in buffer A at 4 ˚C for 72 h with 1.6 mM 6 and 1.6 µM 7a. 40 nM, 86 nM A , ± 0.5 %, calculated on the
2
A
endo isomers of products 8a-c.
artificial enzyme and regain the function of a DielsꢀAlder
cycloaddition biocatalyst.
Catalysis using the purified artificial enzyme. These findꢀ
ings were corroborated by the results we obtained by preparꢀ
ing and studying the purified form of the 12Cuꢀbased artificial
enzyme. The protein was expressed, purified and used to
build the 12Cuꢀbased artificial metalloenzyme in solution.
Catalysis with the pure protein was performed under the same
conditions as those used with the enzyme at the surface of
living cells except for the buffer (buffer B) that further conꢀ
39
tained 6 % glycerol, 0.05 % nꢀdodecylꢀβꢀ ꢀmaltopyranoside
(DDM) and 0.01 % 3βꢀhydroxyꢀ5ꢀcholestene 3ꢀhemisuccinate
(CHS), which were necessary for maintaining the correct
D
39
folding of the receptor in solution. The obtained results were
indicative of a catalysis performed by an artificial enzyme
resulting from the combination of 12Cu with the purified
receptor and were similar to those obtained with the enzyme at
the surface of living cells, most notably the same enantiomer
was favored. Yield were however lower and the ees were
higher. The outcomes of these experiments demonstrated the
formation of receptor based artificial enzymes both in solution
and at the surface of living cells. The differences in yields and
in ees can be caused by various factors. Firstly, the difference
in buffer constituents and secondly, the absence of background
catalysis with the pure receptor in the absence of 12Cu (Table
2).
Figure 5. Kinetic, yield and the ee of the catalysis of the Dielsꢀ
Alder cycloaddition reaction by HEKꢀA2A cells ( ), HEKꢀ293
cells ( ), 12Cu + HEKꢀ293 cells ( ), 12Cu + HEKꢀA2A
(
)
cells and 12Cu( ). ees were not determined for < 10 % yield.
Sustainability. The suspended HEKꢀA2A cells combined
with 12Cu that were used for catalysis were isolated from the
reaction mixture after 72 h at 4˚C. 85 ± 10 % of the isolated
cells were found to remain alive. They were adherently culꢀ
tured and provided new cell batches that maintained A2A exꢀ
pression. The catalytic system was thus shown to be sustainaꢀ
ble, requiring only the addition of 12Cu or 13Cu to reform the
Table 2. Results of the catalysis of the Diels-Alder cycloaddition reaction by the purified A , cofactor 12Cu, and the puri-
2A
fied artificial metalloenzyme.
c
Catalyst
Yield (%)
8b
33 ± 4 24 ± 3
< 3 < 3
18 ± 2 16 ± 2
endo/exo
8b
ee
8
a
8c
8a
8c
8a
8b
8c
a
1
2Cu
44 ± 4
< 3
84/16
83/17
65/35
n.d.
n.d.
n.d.
b
A2A
n.d.
n.d.
n.d.
n.d.
19
a
n.d.
23
b
n.d.
35
c
a
b
A
12Cu + A2
28 ± 4
82/18
77/23
81/19
Reactions were performed in buffer B at 4 ˚C for 72 h with 1.6 mM 6 and 1.6 µM 7a-c. 40 nM, 86 nM, ± 0.5 %, calculated on the endo
isomers of products 8a-c.
Comparison with related artificial metalloenzymes. The
only other known artificial enzyme that was prepared in vivo
catalyzed metathesis reactions in the periplasm of bacterial
cells. In its purified form, the enzyme yielded 55 TON while
9
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Journal of the American Chemical Society
it was less active in living bacteria where it provided 3.6 TON. metal complex, which thus far can only be used in vitro for
Page 6 of 8
4
7
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Using genetic optimization, the yield was increased up to 6.6
TON in vivo. A direct comparison between the in vivo Dielsꢀ
Alder and the metathesis catalytic systems is biased not only
because of the different nature of the catalyzed reactions, but
also because of the use of different respective host systems.
Nonetheless, it is noticeable that the metathesis reactivity was
drastically decreased by the encapsulation of the enzyme in
bacterial periplasm (3.6 in vivo vs. 55 TON in vitro per enꢀ
zyme) while on the contrary, higher yields were obtained via
the exposure of the DielsꢀAlderase on the surface of human
cells (20 TON in vivo vs. 11 TON in vitro per enzyme). This
effect of localization of artificial enzymes is further corroboꢀ
rated by a recent study in which a rhodium based artificial
polymerase was prepared in the outer membrane of lyophiꢀ
lized bacterial cells and used to catalyze phenylacetylene
transꢀselective polymerization. These exposed enzymes on the
bacterial cells also showed an increase in the TON as opposed
to in vitro catalysis (about 6100 TON on cells vs. 5000 in vitro
enhancing their catalytic properties.
CONCLUSIONS
This work validates the concept of transforming wild type
surface receptors of living cells into artificial enzymes. Our
results show that the chemical modification of the wild type
A2A membrane receptor at the surface of living human cells
allowed its successful transformation into an artificial enzyme
that stereoselectively catalyzed DielsꢀAlder cycloaddition
reactions. This first demonstration of preparing artificial
metalloenzymes based on human cells for the in vivo synthesis
of organic molecules opens new therapeutic horizons as it
broadens the repertoire of reactions catalyzed by the human
metabolism which could enable the targeted synthesis of either
deficient metabolites or drugs starting from biologically inacꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
49, 50
tive substrates.
Furthermore, such artificial metalloenꢀ
zymes provide potential new tools for the selective activation
of prodrugs by catalyzing abiotic reactions in a targeted theraꢀ
40
51
per enzyme).
py approach. Therefore, reactions with artificial enzymes
based on organꢀspecific membrane receptors in combination
with upstreamꢀreactions prior to internalization could be exꢀ
ploited for organ confined catalysis at body temperature.
Clearly several challenges inherent to the development of
novel therapeutic tools, such as inhibition by digestive fluids
or serum must be overcome first, however, our results prove
the potential of artificial metalloenzymes as novel therapeutic
tools. This is especially true since the majority of marketed
drugs comprise ligands of membrane receptors, and since
metal complexes are the most efficient manꢀmade catalysts
that are routinely used to form the catalytic sites of artificial
metalloenzymes.
On the other hand, artificial DielsꢀAlderases that were preꢀ
pared based on Cu(II) complexes have provided comparable
results to those we obtained with the receptor based artificial
enzyme on living human cells (50 % yield of 8a, and 81/19
endo/exo ratio with 35 % ee of 8c). Those that were prepared
employing Cu(II)ꢀ1,10ꢀphenanthroline complexes provided 51
% maximum yield, which was obtained with the complex in
Geobacillus thermocatenulatus lipase (GTL) and was accomꢀ
3
0
52
panied with 0 % ee and 94/6 endo/exo ratio. In terms of ee,
the highest reported value (28 %) was obtained with the comꢀ
plex in Lactococcal multidrug resistance Regulator (LmrR)
and was accompanied with 90/10 endo/exo ratio and 29 %
5
3, 54
29
yield. These artificial DielsꢀAlderases were however outperꢀ
formed by their genetically optimized peers, which have been
ASSOCIATED CONTENT
Supporting Information
shown to provide up to quantitative yields, 99/1 endo/exo ratio
25, 28, 29, 30, 31
and >97 % ee.
Such chemoꢀgenetic optimization
The Supporting Information is available free of charge via the
Internet at http://pubs.acs.org.
are often performed to lead to more efficient artificial enꢀ
41
zymes. Effects of mutations on the A2A receptor have been
extensively studied and helped in developing the membrane
4
2
Experimental data including synthesis and characterization of all
described compounds, animal cell culture and microscopy, exꢀ
pression and purification of AR A2A in bacteria as well as emꢀ
ployed equipment and chiral HPLCꢀMS/MS parameters (PDF).
Crystallographic data of Cu12 (CIF).
receptor thermostablization technique which was applied for
4
3, 44, 45
the crystallization of membrane receptors.
These studies
provide valuable information that could lead more efficient
mutant receptor based enzymes at the surface of cells, but
these would lack therapeutic potential since they are not natuꢀ
rally expressed in living organisms. Additionally, other Cu(II)
complexes were also used to prepare artificial DielsꢀAlderases.
These included 2,2’ꢀbipyridine, 2,2′:6′,2′′ꢀterpyridine, N,Nꢀ
di(methylꢀ2ꢀpyridine)amine and Nꢀmesitylimidazoleꢀ2ꢀylidene
Cu(II) complexes and provided less efficient enzymes than
AUTHOR INFORMATION
Corresponding Author
*
*
wadih.ghattas@uꢀpsud.fr
jeanꢀpierre.mahy@uꢀpsud.fr
1
2, 25, 29, 31, 46, 47
those obtained with Cu(II)ꢀ1,10ꢀphenanthroline.
Author Contributions
Cu(II)ꢀphthalocyanine was also employed bound in various
serum albumins. The mechanism of the catalysis with this
complex remains inconclusive but when bovine serum album
was the host protein, it provided the best conversions with up
to 91 % yield for 7c and an 98 % ee for the corresponding
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
48
Funding Sources
product 8c. Other methods for preparing artificial enzymes
This work was financially supported by the ANR Programme
Retour PostꢀDoctorant : REBAR ANRꢀ12ꢀPDOCꢀ0007ꢀ1.
rely on covalent bonding for anchoring metal complexes in
proteins. These methods target either SH, OH or NH funcꢀ
2
tions of cysteine, serine and lysine amino acid residues in
proteins, respectively, and are thus protein nonꢀspecific. The
preparation of artificial enzymes following such methods is
incompatible with in vivo applications as it does not target an
aimed host protein. This also applies to the covalent anchoring
of chiral inducers in artificial enzymes at the proximity of the
ACKNOWLEDGMENT
The authors thank Prof. Dr. Grisshammer (The National Institutes
of Health, Rockville, MD 20852, USA) for providing the pRG/IIIꢀ
hsꢀMBP plasmid.
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Journal of the American Chemical Society
greve, M. Biophysical Mapping of the Adenosine A 2A Reꢀ
ceptor. J. Med. Chem. 2011, 54 (13), 4312–4323.
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