Carbonic anhydrase II as host protein for the creation of a biocompatible artificial metathesase

Article abstract of DOI:10.1039/c5ob00428d

An artificial metathesase results from incorporation of an Hoveyda-Grubbs catalyst bearing an arylsulfonamide anchor within human carbonic anhydrase II. The optimization of the catalytic performance is achieved upon combining both chemical and genetic means. Up to 28 TONs were obtained within four hours under aerobic physiological conditions.

Full text of DOI:10.1039/c5ob00428d

Organic &
Biomolecular Chemistry
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Carbonic anhydrase II as host protein for the
creation of a biocompatible artificial metathesase†
Cite this: Org. Biomol. Chem., 2015,
13, 5652
Jingming Zhao, Anna Kajetanowicz and Thomas R. Ward*
Received 3rd March 2015,
Accepted 13th April 2015
An artificial metathesase results from incorporation of an Hoveyda-Grubbs catalyst bearing an arylsulfon-
amide anchor within human carbonic anhydrase II. The optimization of the catalytic performance is
achieved upon combining both chemical and genetic means. Up to 28 TONs were obtained within four
hours under aerobic physiological conditions.
DOI: 10.1039/c5ob00428d
www.rsc.org/obc
Artificial metalloenzymes result from the incorporation of an thus selected for further optimization. With no MgCl2 added
abiotic cofactor within a host protein.1 With biomedical appli- and at pH 7.0, catalyst 2 and the corresponding metathesase 2
cations in mind, it would be desirable to capitalize on a host ⊂ WT hCA II afforded 23 and 14 turnovers after four hours at
protein which is overexpressed on the surface of cancer cells. 37 °C. Performing catalysis under strict exclusion of oxygen
Accumulation of the abiotic cofactor, which displays high yielded very similar results.
affinity for the latter protein, may allow to site-specifically
Reactions carried out at pH 7.0 and in the presence of
uncage a drug.2 In this context, the ring-closing metathesis 154 mM NaCl (corresponding to physiological conditions)
(RCM) is an attractive reaction as unactivated diolefins can be yielded 32 and 21 TONs for 2 and 2 ⊂ WT hCA II respectively
viewed as bioorthogonal. Furthermore, the intramolecular (Table 1, entries 15 and 16). As can be appreciated, the TON of
nature of the RCM may facilitate the reaction under highly the catalyst is pH dependent, both in the presence and in the
dilute aqueous conditions.3 Herein, we report on our efforts to absence of hCA II. The best performance is obtained at lower
exploit human Carbonic Anhydrase II (hCA II hereafter) for the pH and high salt concentration (Table 1, entries 12 and 13). As
creation of a biocompatible artificial metathesase, Scheme 1. for other artificial metalloenzymes, we do not believe that the
Certain forms of cancer overexpress hCA IX, a membrane pI of the host protein influences significantly the catalyst
bound variant of hCA. These arylsulfonamide binding proteins performance.6
are thus privileged targets for cancer therapy.2,4a
Compared to the other four artificial metathesases reported
Introduction of an arylsulfonamide-anchor on an Hoveyda- to date,3a–e the system presented herein presents the following
Grubbs 2nd generation-type catalyst ensures its localization advantageous features (Table 2): (i) it does not require an inert
within carbonic anhydrase.4 For this purpose, complexes Boc-1, atmosphere; (ii) the substrate concentration is the lowest of all
Boc-2 and Boc-3,5 were deprotected in situ and reacted with systems reported to date; (iii) except for the metathesase based
4-sulfamoylbenzoic acid to afford the corresponding sulfon- on FhuA (which requires SDS, a surfactant), it displays the
amide-bearing metathesis cofactors 1, 2 and 3, Scheme 2 (see highest turnover frequency and (iv) it catalyses RCM at pH 7.0,
ESI† for full Experimental details).
temperature 37 °C and physiological [NaCl] concentrations.
The catalytic performance of the artificial metathesases was These results thus suggest that WT hCA II is a suitable host
evaluated using the ring-closing metathesis of N-tosyl diallyl- for the creation of artificial metathesases operating under
amine in the presence of 1 mol% ruthenium. To ensure an physiological conditions and at low catalyst concentrations
homogeneous mixture, water/DMSO (9/1) was selected.
(i.e. 10 µM).
To gain insight into the localization of rac-2 within WT hCA
Comparison of catalysts 1–3 in the absence of hCA II at pH
6.0 in the presence of 0.1 M MgCl2 reveals that the bulkiest II, both enantiomers were docked using the GOLD program,7
catalyst 2 outperforms catalysts 1 and 3 (Table 1, entries 1–3). Fig. 1. As can be appreciated, the cofactor fits nicely within the
The same trend is observed upon incorporation of the cofac- cone-shaped binding funnel of hCA II, presenting its alkyl-
tors 1–3 into WT hCA II (Table 1, entries 4–6). Catalyst 2 was idene moiety at the surface of the protein (Fig. 1). With the aim
of improving the TON of the artificial metathesase, residues
I91, F131, L198, K170 were subjected to site-directed mutagen-
esis (Table 1, entries 20–27). Lypophylic, polar and potentially
coordinating aminoacid residues were engineered into these
Department of Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel,
Switzerland. E-mail: thomas.ward@unibas.ch
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
positions. A selection of mutants tested is presented in
c5ob00428d
5652 | Org. Biomol. Chem., 2015, 13, 5652–5655
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Scheme 1 Artificial metalloenzyme for ring-closing metathesis. Tethering an arylsulfonamide anchor (green) to an Hoveyda-Grubbs type catalyst
(black) ensures the localization of the metal moiety within human Carbonic Anhydrase II (blue).
Table 1 Selected results for the ring-closing metathesis of N-tosyl
diallylaminea
Entry
Catalyst
hCA II
pH
[MClx] mol/l
TONb
1
1
2
3
1
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
—
—
—
6.0
6.0
6.0
6.0
6.0
6.0
6.0
7.0
7.0
7.0
7.0
5.0
5.0
6.0
8.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
MgCl2 0.1
MgCl2 0.1
MgCl2 0.1
MgCl2 0.1
MgCl2 0.1
MgCl2 0.1
NaCl 0.2
NaCl 0.2
—
20 0.4
48 0.8
25 0.7
13 1.3
45 2.0
16 1.0
40 1.5
28 1.1
23 2.1
14 0.5
20 2.3
85 1.0
78 2.5
23 2.6
21 1.2
32 2.0
21 1.8
32 1.8
29 1.2
18 3.3
16 1.3
18 1.6
22 0.1
28 0.6
15 1.7
14 0.1
15 2.0
2
3
4
WT
5
WT
6
WT
Scheme 2 Synthesis of olefin metathesase cofactors 1–3 bearing an
arylsulfonamide anchor for incorporation within hCA II.
7
WT
8
WT
9
—
10
11c
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
WT
—
—
WT
—
MgCl2 0.5
MgCl2 0.5
—
WT
Table 1, entries 20–27. To our delight, 2 ⊂ hCA II L198H
yielded significantly improved catalytic performance: up to 28
TONs under physiological conditions (Table 1, entry 24). Con-
sidering that both His198 and Phe198 affort similar TONs, we
do not believe that the former residue coordinates to ruthe-
nium. At this point however, it is difficult to rationalize or
predict the effect a point mutations on the outcome of
catalysis.
WT
WT
—
—
NaCl 0.154
NaCl 0.154
NaCl 0.5
NaCl 1.0
—
—
—
—
NaCl 0.154
—
—
—
WT
WT
WT
I91A
F131A
L198F
L198H
L198H
L198A
L198Q
K170A
In order to ensure localisation of the cofactor 2 within hCA
II under catalytic conditions, its affinity was determined using
the dansylamide displacement assay.8 Dansylamide (DNSA)
displays enhanced fluorescence upon incorporation within
hCA II. The non-covalent probe can be displaced by high
affinity arylsulfonamide-bearing hCA II inhibitors, leading to a
decrease in fluorescence.
a Reaction conditions: [substrate] 1 mM, [catalyst] 10 µM, [hCA II] 12
µM, Vtot 200 µL (VDMSO 20 µL), 37 °C for 4 hours. The reactions were
carried out in triplicate. Very similar results were obtained under
rigorous exclusion of oxygen. b Turnover number. c [substrate] 5 mM,
[catalyst] 50 µM, [hCA II] 60 µM.
For DNSA, we obtained a Kd = 4.83 µM and 17.35 µM for
WT hCA II and L198H hCA II respectively. The displacement
assay (see ESI† for details) yielded Kd = 90.40 nM and specific binding on the surface of hCA II, we feel that the
205.10 nM for 2 ⊂ WT hCA II and 2 ⊂ hCA II L198H respecti- exquisite specificity of arylsulfonamides most probably
vely, Fig. 2. Although we cannot exclude additional non- ensures selective binding to the Zn(His)3 moiety. We tenta-
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Table 2 Summary of the catalytic performance of artificial metathesases reported to date
Hilvert3b
Ward3a
Schwaneberg3d,e
Matsuo3c
Ward
[substrate]
Reaction type
Anchoring of
Ru-cofactor
RCM
Covalent
RCM
Supramolecular
ROMP
Covalent
RCM
Covalent
RCM
Dative
Host protein
MjHSPa
4 mol%
45 °C
12 h
Avidin
4.8 mol%
40 °C
16 h
FhuA ΔDCVFtev b
0.08 mol%
25 °C
α-Chymotrypsin
0.63 mol%
25 °C
hCA II
1 mol%
37 °C
4 h
Temp.
Time
68 h
2 h
pH
Reaction
conditions
2
4
7
7
7
10 mM HCl, Water/
t-BuOH 4/1 under air
0.1 M acetate Water/
DMSO 5/1 0.5 M MgCl2,
under air
Water/THF 9/1 SDS
1% under N2
Degassed 100 mM
KCl, under N2
0.1 M phosphate Water/
DMSO 9/1 under air
TON
25
20
955
20
28
a MjHSP: M. jannaschii small heat shock protein. b FhuA ΔDCVFtev: engineered variants of the β-barrel ferric hydroxamate uptake protein
component A.
Conclusions
In summary, we have developed an artificial metathesase
relying on hCA II as host protein. Importantly, the present
system operates under aerobic physiological conditions and at
low catalyst concentrations (i.e. 10 µM). Current efforts are
directed at evaluating hCA IX, a cell-surface hCA variant which
is overexpressed in various forms of cancer.
Fig. 1 Docked structure of (R)-2 ⊂ WT hCA II (a) and (S)-2 ⊂ WT hCA II
(b). The ruthenium cofactor is displayed as stick and hCA II as solvent
Experimental section
Typical procedure for the ring closing metathesis
accessible transparent surface. Residues subjected to mutagenesis are
highlighted in magenta.
The following stock solutions were prepared: catalyst 2 (200
µM) in DMSO, N-tosyldiallylamine (20 mM) in DMSO and hCA
II isoform solution (13.3 µM) in phosphate buffer (0.1 M,
pH 7.0).
To a small glass vial, the catalyst 2 stock solution (10 µL)
was added to the protein stock solution (180 µL). The mixture
was incubated at 37 °C for 20 min. The N-tosyldiallylamine
stock solution (10 µL) was added and the reaction vial was
placed in the incubator (37 °C) for 4 h at 200 rpm.
After completion of the reaction, 2-phenylethanol (100 µL,
used as internal standard, 1 mM in H2O) and MeOH (700 µL)
Fig. 2 Determination of the dissociation constant of 2 ⊂ hCA II using
the dansylamide displacement assay: WT hCA II (a) and L198H hCA II (b)
[hCA II] = 0.25 µM, [Dansylamide] = 20 µM, see ESI† for full Experimental
details. All measurements were performed in duplicate.
were added. The mixture was transferred to an eppendorf tube
and centrifuged at 14 000 rpm for 15 minutes to precipitate
hCA II. The supernatant (500 µL) was transferred in an HPLC
vial and water (500 µL) was added. The sample was then sub-
jected to reversed phase HPLC for TON analysis.
tively attribute the modest quality of the fit to the use of a
rac-2 (and thus the presence of two inhibitors with poten-
tially different affinities). Under catalytic conditions 96%
and 93% of 2 is bound to WT hCA II and hCA II L198H
respectively.
Acknowledgements
Financial support for this project was provided by the seventh
framework programme project METACODE (KBBE, “Code-
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