Human carbonic anhydrase II as host protein for the creation of artificial metalloenzymes: The asymmetric transfer hydrogenation of imines

Article abstract of DOI:10.1039/c3sc51065d

In the presence of human carbonic anhydrase II, aryl-sulfonamide-bearing IrCp* pianostool complexes catalyze the asymmetric transfer hydrogenation of imines. Critical cofactor-protein interactions revealed by the X-ray structure of [(η⁵-Cp*)Ir(pico 4)Cl] 9 WT hCA II were genetically optimized to improve the catalytic performance of the artificial metalloenzyme (68% ee, k_cat/K_M 6.11 × 10 ^-3 min^-1 mM^-1).

Full text of DOI:10.1039/c3sc51065d

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Human carbonic anhydrase II as host protein for the
creation of artificial metalloenzymes: the asymmetric
transfer hydrogenation of imines†
Cite this: DOI: 10.1039/c3sc51065d
a
a
ab
b
Fabien W. Monnard, Elisa S. Nogueira, Tillmann Heinisch, Tilman Schirmer
and Thomas R. Ward*a
Received 22nd April 2013
Accepted 31st May 2013
In the presence of human carbonic anhydrase II, aryl-sulfonamide-bearing IrCp* pianostool complexes
catalyze the asymmetric transfer hydrogenation of imines. Critical cofactor–protein interactions revealed
DOI: 10.1039/c3sc51065d
5
by the X-ray structure of [(h -Cp*)Ir(pico 4)Cl] 9 3 WT hCA II were genetically optimized to improve the
6.11 ꢀ 10ꢁ min mM ).
3
ꢁ1
ꢁ1
www.rsc.org/chemicalscience
catalytic performance of the artificial metalloenzyme (68% ee, kcat/K
M
sulfonamides display high affinity for the catalytic Zn ion which
lies at the bottom of a deep hydrophobic funnel-shaped cavity.
1
Introduction
Articial metalloenzymes result from the incorporation of an We thus speculated that upon tethering an aryl-sulfonamide to a
organometallic moiety within a protein scaffold. In this context, bidentate ligand may allow to anchor an organometallic catalyst
18
three anchoring strategies have been pursued: covalent, supra- within hCA II. This was recently conrmed with an X-ray
1
–17
molecular and dative.
strategies, we hypothesized that human Carbonic Anhydrase II
hCA II hereaer) may provide a suitable scaffold for the crea-
In the context of dative anchoring
structure of an aryl-sulfonamide-bearing Ru pianostool complex
19,20
incorporated within hCA II.
However, this latter proved
(
catalytically inactive for a variety of transformations. Herein we
tion of articial metalloenzymes. Indeed, para-substituted aryl- report on our efforts to create an articial transfer hydrogenase
based on the incorporation of a catalytically active iridium pia-
nostool complex within hCA II, Fig. 1.
2
Results and discussion
2.1 Ligand synthesis
With the aim of identifying the most suitable scaffold for
incorporation within hCA II, we set out to synthesize different
ligands bearing a p-aryl-sulfonamide anchor. As previously
2
+
demonstrated, the {IrCp*} moiety has proven superior to its
2
+
2+
{
RhCp*} and {Ru(arene)} congeners for the reduction of
21,22
imines in water.
The synthesis of bis-pyridine 1, bipyridine
2, amino-pyridine 3 and sulfonamido-pyridine 4 and 5 ligands
Fig. 1 Artificial transfer hydrogenase for imine reduction. An aryl-sulfonamide-
bearing bidentate ligand (green) ensures anchoring of an IrCp*-moiety within and the corresponding complexes 6–10 is presented in Schemes
human carbonic anhydrase II (blue). Variation of the bidentate ligand combined
1
and 2 (see ESI† for full experimental details).
The synthesis of bis-pyridine 1 was reported in 2011. The
with genetic modification of the host protein allows a chemogenetic optimization
19
of the catalyst's performance for the synthesis of salsolidine.
preparation of bipy 2 started with an aldol condensation reac-
tion between 4-(methylthio)benzaldehyde 11 and 2-acetylpyr-
idine 12, to afford the corresponding phenyl-substituted-
bipyridine 13. The sulfonamide anchor was installed in three
steps via oxidation of the thiomethyl function, which was
a
Department of Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel,
23
Switzerland. E-mail: thomas.ward@unibas.ch; Fax: +41 61 267 10 05; Tel: +41 61
24
2
67 10 04
b
Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, converted to the sulfonamide using the method disclosed by
Switzerland. E-mail: tilman.schirmer@unibas.ch; Fax: +41 61 267 21 09; Tel: +41
25
Zhang et al. The sulfonamide-bearing bipyridine ligand 2 was
obtained in 8% overall yield, Scheme 1.
The synthesis of 2-picolylamine-bearing ligand 3 relied on a
palladium-catalyzed cross-coupling strategy as key step.
Commercially available chloropyridine 14 and aryl-sulfonamide
6
1 267 20 89
†
Electronic supplementary information (ESI) available: Modied protocol for the
expression of hCA II in E. Coli. Detailed description of the X-ray structure.
Synthesis and characterization of all new compounds. See DOI:
1
0.1039/c3sc51065d
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Scheme 1 Synthesis of pianostool complexes 6 and 7 bearing bis-pyridine-type
2 2 3
bidentate ligands. Reagents and conditions: (a) [Cp*IrCl ] , H CCN, reflux, 4 h; (b)
˚
NaOH aq./MeOH, RT; (c) ethyl vinyl ether, 4 A molecular sieves, THF, RT; (d)
H
0
2
ꢂ
NO$HCl, H
3
CCN, reflux; (e) KMnO
C, (ii) trimethylsilyl chloride, RT; (g) (i) TBAF,THF, reflux, (ii) hydroxylamine-O-
O, RT.
4 2 2 2
/MnO , CH Cl , RT; (f) (i) DIPA, n-BuLi, THF,
sulfonic acid, H
2
1
5 were coupled under Suzuki conditions. Reduction of the
nitrile with (AlCl )$LiAlH , followed by deprotection with TFA
afforded the corresponding ligand 3 in 40% overall yield.
3
4
26,27
For the synthesis of ligands pico 4–H and pico 5–H, the
oxime 17 was identied as advanced intermediate, easily
accessed on large scale from commercially available 4-bromo-2-
methylpyridine 16. Its synthesis was based on key steps starting
Scheme 2 Synthesis of pianostool complexes 8–10 bearing picolylamine-type
ꢂ
bidentate ligands. Reagents and conditions: (a) Pd(PPh
3
)
4
, Na
2
CO
3
, THF, 80 C; (b)
LiAlH
4
$AlCl
3
, THF, RT; (c) TFA, RT; (d) [Cp*IrCl
2
]
2
, EtOH, reflux; (e) m-CPBA, CH
NO$HCl, NaHCO , MeOH,
, Na CO , H O/
2 2
Cl ,
28,29
from a Boekelheide reaction
and followed by an oxidation to
ꢂ
RT; (f) TFAA, CH
2
Cl
2
, 0 C; (g) MnO
2
, CH
3
Cl, reflux; (h) H
2
3
30
ꢂ
the corresponding aldehyde. Treatment with hydroxylamine RT; (i) TFA, Zn, 0 C; (j) arylsulfonylchloride, DIPEA, RT; (k) Pd(PPh
3
)
4
2
3
2
ꢂ
dioxane, microwave, 150 C.
hydrochloride yielded the oxime 17. Reduction by Zn in tri-
31

uoroacetic acid followed by treatment with an aryl-sulfonyl-
chloride derivative yielded the sulfonamide-pyridines 18 and
32
2.2 Catalysis results
19, respectively. Finally, the benzene sulfonamide anchor was
introduced via Suzuki cross-coupling reaction to afford the In order to evaluate the transfer hydrogenase activity of the
3
3
bidentate ligands pico 4–H and pico 5–H in 5% overall yield.
The aminosulfonamide-bearing ligands 1–5 were reacted selected as prochiral imine. The sulfonamide-bearing catalysts
with [Cp*IrCl to afford the corresponding mononuclear pia- 6–10 (1.8 mol% vs. imine, 5% DMSO in MOPS buffer, pH 7.5,
nostool complexes [(h -Cp*)Ir(bispy 1)Cl] 6, [(h -Cp*)Ir(bipy 2)- 40 C, 20 hours, 150 eq. HCO
pianostool complexes 6–10, the salsolidine precursor was
34
2 2
]
5
+
5
ꢂ
2
Na) were tested both in the
Cl] 7 and [(h -Cp*)Ir(pico 3)Cl] 8, [(h -Cp*)Ir (pico 4)Cl] 9 and absence and in the presence of hCA II (see ESI† for experimental
(h -Cp*)Ir(pico 5)Cl] 10 respectively. These were puried by details). From these data, it appears that both bispy- and bipy-
precipitation in acetonitrile or CH Cl to afford analytically pure bearing complexes [(h -Cp*)Ir(bispy 1)Cl] 6 and [(h -Cp*)-
aryl-sulfonamide-bearing iridium pianostool complexes for Ir(bipy 2)Cl] 7 are very poor transfer hydrogenation catalysts
+
5
+
5
5
[
5
+
5
2
2
+
anchoring within hCA II, Schemes 1 and 2.
(Table 1, entries 1–4). In contrast, the complex bearing the
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ꢂ
Table 1 Selected results for the chemogenetic optimization of artificial transfer good TONs and similar ees at 40 C (Table 1, entries 11 and 12).
a
ꢂ
C
hydrogenases for the production of salsolidine at 40
ꢂ
5
Upon decreasing the temperature to 4 C, the bare catalyst [(h -
Cp*)Ir(pico 4)Cl] 9 showed low ATHase activity (Table 2, entry 5).
Entry
Complex
hCA II
Conv. %
ee %
5
5
In contrast, both [(h -Cp*)Ir(pico 4)Cl] 9 3 WT hCA II and [(h -
Cp*)Ir(pico 4)Cl] 9 3 I91A afforded salsolidine with signi-
1
2
3
4
5
6
7
8
9
6
6
7
7
8
8
8
8
9
9
9
9
10
10
10
10
—
WT
—
WT
—
WT
I91A
K170A
—
WT
I91A
K170A
—
WT
I91A
K170A
3
0
14
0
n.d.
n.d.
n.d.
n.d.
Rac.
5 (S)
1 (S)
3 (S)
1 (S)
32 (S)
ꢂ
cantly improved selectivity at 4 C (68% ee (S), 27% conv. and
4% ee (S), 23% conv. respectively). Upon increasing the catalyst
loading to 9%, the conversions could be upgraded to 82% and
6
Quant.
20
46
34
81
69
67
40
86
51
58
25
7
0% respectively aer 44 hours (Table 2, entries 9 and 10).
2
.3 Kinetic- and thermodynamic characterization
Para-substituted aryl-sulfonamides are widely studied inhibi-
29 (S) tors for hCA II and their affinities vary over several orders of
10
11
12
13
14
15
16
18
30 (S)
2 (R)
30 (S)
5 (R)
14 (S)
magnitude. In order to ensure that, under catalytic conditions
(
i.e. [hCA II] ¼ 0.4 mM; [complex] ¼ 0.35 mM), the bulky pia-
nostool complexes were quantitatively incorporated within hCA
II, their affinity towards hCA II was determined (see ESI† for
It is interesting to note that the sulfonamide-
bearing pianostool complexes [(h -Cp*)Ir(pico 4)Cl] 9 and [(h -
19,37–41
details).
a
ꢂ
The reaction was carried out for 20 h at 40 C with 0.35 mM complex
5
5
(
nal concentration, 1.8 mol% vs. substrate), 0.4 mM hCA II in MOPS
buffer (0.4 M, pH 7.5, 200 mL containing 5% DMSO), 20 mM imine Cp*)Ir(pico 5)Cl] 10 displayed signicantly higher affinity
substrate and 3 M formate. The yield and ee is an average of at least
5
towards hCA II compared to the picolylamine complex [(h -Cp*)-
Ir(pico 3)Cl] 8 (compare 15 nM and 17 nM for complexes 9 and
two catalytic runs d ee: ꢃ1%, d conv.: ꢃ4%.
+
10 respectively to 1280 nM for complex 8, Table 3, entries 3–5).
This suggests that a secondary hydrophobic contact between
the additional aryl group of the ligand and the hydrophobic
funnel-shaped hCA II active site contributes to signicantly
5
+
amino-pyridine ligand [(h -Cp*)Ir(pico 3)Cl] 8 performed well
in the absence of hCA II (>55 turnover numbers, TON). Disap-
pointingly, incorporation within hCA II lead to a signicant
erosion of its catalytic performance (TON 11, ee 5% (S), Table 1,
entries 5 and 6). A recent report by Çetinkaya suggests that the
42
increase the affinity of these complexes.
Having ensured that the pianostool complexes are indeed
quantitatively incorporated within hCA II under catalytic
6
transfer hydrogenase activity of d -pianostool complexes with a
sulfonamide-bearing 2-picolylamine is superior to that of the
corresponding the 2-picolylamine-containing catalyst. We
thus set out to synthesize and evaluate the activity of [(h -Cp*)-
Ir(pico 4)Cl] 9. Although less active than [(h -Cp*)Ir(pico 3)Cl] 8
in the absence of host protein (45 vs. >55 TONs), incorporation
within hCA II yielded salsolidine in 69% yield and 32% ee (S)
5
conditions, the saturation kinetics behavior of [(h -Cp*)Ir(pico
5
5
4
)Cl] 9, [(h -Cp*)Ir(pico 4)Cl] 9 3 WT hCA II and [(h -Cp*)-
35
Ir(pico 4)Cl] 9 3 I91A hCA II was determined, Fig. 2. Upon
5
5
+
Table 2 Selected results for the chemogenetic optimization of artificial transfer
a
ꢂ
hydrogenases for the production of salsolidine at 4
C
(
Table 1, entries 9 and 10). Inspired by Carreira's observation
Entry
Complex
hCA II
Conv. %
ee %
that uorination of the aryl-moiety of amino-sulfonamide
ligands leads to improved transfer hydrogenase activity in
1
2
3
4
5
6
7
8
9
8
8
8
8
9
9
9
9
—
WT
I91A
K170A
—
78
7
9
8
7
27
23
21
82
70
57
10
7
Rac.
9 (S)
2 (R)
3 (R)
5
water, we tested the diuorinated catalyst [(h -Cp*)Ir(pico 5)Cl]
36
1
0. Unfortunately, its catalytic performance remained modest
both in the absence and within hCA II (Table 1, entries 13–16).
These results highlight the critical inuence of the second
coordination sphere interactions on the catalytic performance
of pianostool complexes with a well dened rst coordination
sphere.
Rac.
WT
68 (S)
64 (S)
67 (S)
70 (S)
66 (S)
68 (S)
2 (R)
40 (S)
15 (S)
16 (S)
I91A
K170A
WT
I91A
K170A
—
WT
I91A
K170A
b
9
b
With the aim of identifying critical residues in the most 10
9
b
11
9
promising ATHase based on WT hCA II, the X-ray structure of
5
12
10
10
10
10
[
(h -Cp*)Ir(pico 4)Cl] 9 3 hCA II was determined (vide infra, see
13
14
15
Fig. 3). The structure revealed the proximity of residues I91 and
K170 to the articial cofactor, lining the hemisphere potentially
occupied by the substrate. With the aim of tailoring more space
for the substrate binding pocket, hCA II single mutants I91A
9
6
a
ꢂ
Reactions were carried out for 44 h at 4 C with 0.35 mM complex (nal
concentration), 20 mM substrate, 0.4 mM protein, MOPS buffer (0.4 M,
b
and K170A were produced, puried and tested as host protein pH 7.5, 200 mL, 5% DMSO) and 3 M formate. Reactions were carried
ꢂ
out for 44 h at 4 C with 0.35 mM complex (nal concentration), 4
(
see ESI† for details).
As for WT hCA II, both ATHase mutants [(h -Cp*)Ir(pico 4)-
mM substrate, 0.4 mM protein, MOPS buffer (0.4 M, pH 7.5, 200 mL,
% DMSO) and 3 M formate. The yield and ee is an average of at least
5
5
5
Cl] 9 3 I91A and [(h -Cp*)Ir(pico 4)Cl] 9 3 K170A displayed two catalytic runs d ee: ꢃ2%, d conv.: ꢃ5%.
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Table 3 Summary of affinity constants for sulfonamide-bearing pianostool protein has a major impact on the respective kinetic proles of
complexes 6–10 towards WT hCA II
the three catalysts.
Entry
Complex
d
K /nM
2
.4 X-ray structure of the hCA II metalloenzyme
a
1
2
3
4
5
6
120 ꢃ 20
270 ꢃ 40
1280 ꢃ 200
15 ꢃ 2
5
To gain structural insight on the ATHase [(h -Cp*)Ir(pico 4)Cl] 9
3 hCA II, diffraction data were collected to 1.3 A resolution of a
hCA II crystal aer soaking in a solution of complex 9. Upon
solving the crystal structure, residual density was apparent in
the funnel-shaped active site of hCA II in close proximity to the
a
7
˚
a
8
b
9
a
10
17 ꢃ 8
a
Affinity determined using the rate of hydrolysis of p-
b
nitrophenylacetate. Affinity determined using competition with catalytic zinc (Fig. 3). The pyridine and terminal benzene-
dansylamide (see ESI for experimental details).
sulfonamide moieties of complex 9 were modeled into the
density with the sulfonamide forming a coordinative bond with
the catalytic zinc and the benzene and pyridine ring arranged
varying the imine concentration and determining by HPLC the
virtually coplanarly. A strong peak in both the F –F and the
o
c
initial rate of salsolidine formation, very different kinetic
anomalous difference density map (23s and 8s, respectively)
indicated the presence of the Ir atom in close proximity to the
pyridine. The atom, however, is not fully occupied as found
upon renement, which yielded 30% occupancy. The low
occupancy may be due to partial dissociation of the metal upon
complex binding to the protein. The iridium is coordinated by
the pyridine nitrogen and the nitrogen of the secondary ben-
zenesulfonamide. Consistent with the low occupancy, no elec-
tron density was observed for the Cp* ring, the chlorine, and the
secondary benzenesulfonamide. In the nal model of the fully
coordinated Ir atom the occupancy of these groups was set to
5
proles were obtained, Fig. 2. For the bare catalyst [(h -Cp*)-
Ir(pico 4)Cl] 9, the reaction rate was essentially independent of
the imine concentration at [imine] > 1.17 mM. Unfortunately,
lowering the imine concentration below 1.17 mM (i.e. less than
5
three eq. vs. [(h -Cp*)Ir(pico 4)Cl] 9) renders the pseudo rst
order kinetics approximation invalid and thus precludes the
precise determination of kcat for the reaction in the absence of
hCA II. Two possibilities may be put forward to explain the
5
behaviour of the free cofactor [(h -Cp*)Ir(pico 4)Cl] 9: (i) K
M
<
1.17 mM or (ii) the substrate is not involved in the rate deter-
mining step.
o c
30%. Upon renement no negative density emerged in the F –F
In stark contrast, upon incorporation within hCA II iso-
5
map. The pianostool moiety of the complex 9 is located in an
hydrophobic groove formed by residues F131, V135, L198, P202
and L204.
forms, saturation kinetic proles were observed. For [(h -Cp*)-
Ir(pico 4)Cl] 9 3 WT hCA II, severe substrate inhibition was
5
observed (K 45 mM). A single point mutation with [(h -Cp*)-
i
Ir(pico 4)Cl] 9 3 I91A hCA II removed the substrate inhibition.
ꢁ
3
ꢁ1
ꢁ1
ꢁ3
5
ꢁ1
ꢁ1
(k
cat/K
M
5
: 9.9 ꢀ 10 min mM vs. 6.1 ꢀ 10 min mM
for [(h -Cp*)Ir(pico 4)Cl] 9 3 WT hCA II and [(h -Cp*)Ir(pico 4)-
Cl] 9 3 I91A hCA II, respectively, Fig. 2.) Comparing the satu-
ration kinetics behavior of the articial metalloenzyme with
that of the organometallic catalyst demonstrates that the host
Fig. 3 Close-up view of the active site in the crystal structure of complex 9 3
hCA II (PDB code 3ZP9). Complex 9 is depicted in stick representation. The protein
is represented in surface representation with oxygens and nitrogens in red and
blue color, respectively. Residues of the hydrophobic wall (P131, V135, P202,
L204) and those that were chosen for genetic optimization (I91, K170) are
highlighted through the surface as stick representation. The occupancies of the
+
{
IrCp*Cl} fragment and the secondary benzenesulfonamide in the eclipsed-
5
+
5
+
Fig. 2 Kinetic profiles of [(h -Cp*)Ir(pico 4)Cl] 9 (red), [(h -Cp*)Ir(pico 4)Cl]
9
conformation are 30%. The salsolidine precursor (orange) was qualitatively
45 ꢃ 15 mM, kcat 6.60 ꢀ 10ꢁ
+
2
ꢃ
M
modeled in the active site to yield the observed (S)-enantiomer of the product.
3
WT hCA II (black trace: K
M
5
6.67 ꢃ 2.20 mM, K
i
2
ꢁ1
1
8
.17 ꢀ 10ꢁ min ) and [(h -Cp*)Ir(pico 4)Cl] 9 3 I91A hCA II (blue trace: K
o c
The electron density of the F –F omit map (grey color) and the anomalous
.63 ꢃ 1.30 mM, kcat 5.28 ꢀ 10ꢁ ꢃ 0.23 ꢀ 10ꢁ2 min ).
2
ꢁ1
difference density (green color) are contoured at 3s and 4s, respectively.
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Residual density in the F
proximity to the secondary benzenesulfonamide. Rotation of
o
–F
c
omit map was encountered in
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ꢂ
lytic performance both in terms of activity and selectivity at 4 C
(up to 68% ee). Saturation kinetic analysis of the WT ATHase
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revealed however severe substrate inhibition. Guided by the X-
ray structure of complex [(h -Cp*)Ir(pico 4)Cl] 9 3 WT hCA II,
the catalytic site was expanded by mutation of close-lying
isoleucine 91 into an alanine. This chemogenetically optimized
ATHase [(h -Cp*)Ir(pico 4)Cl] 9 3 I91A hCA II no longer
suffered from substrate inhibition and displayed signicant
rate enhancement over the organometallic catalyst. Current
efforts are aimed at further engineering the catalytic site by
genetic introduction of additional substrate recognition
elements.
5
2
0 D. Can, B. Spingler, P. Schmutz, F. Mendes, P. Raposinho,
C. Fernandes, F. Carta, A. Innocenti, I. Santos,
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51, 3354.
2
2
1 M. D u¨ rrenberger, T. Heinisch, Y. Wilson, T. Rossel,
E. Nogueira, L. Kn ¨o rr, A. Mutschler, K. Kersten,
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Acknowledgements
23 J. G. Cordaro, J. K. McCusker and R. G. Bergman, Chem.
Commun., 2002, 1496.
FWM thanks the Novartis Foundation and the Swiss Nano-
science Institute for nancial support. EN thanks Marie Curie
ITN (Biotrains FP7-ITN-238531). TH thanks the Marie Curie ITN
2
4 A. Shaabani, P. Mirzaei, S. Naderi and D. G. Lee, Tetrahedron,
004, 60, 11415.
2
2
5 I. K. Khanna, Y. Yu, R. M. Huff, R. M. Weier, X. Xu,
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C. M. Koboldt, J. L. Masferrer, W. E. Perkins, K. Seibert,
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(BioChemLig FP7-ITN-238434) TRW thanks C. Fierke for the
hCA II plasmid and Umicore for a loan of Ir.
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