Carbonic anhydrase activity of dinuclear CuII complexes with patellamide model ligands

Article abstract of DOI:10.1039/c3dt53135j

The dicopper(ii) complexes of six pseudo-octapeptides, synthetic analogues of ascidiacyclamide and the patellamides, found in ascidians of the Pacific and Indian Oceans, are shown to be efficient carbonic anhydrase model complexes with k_cat up to 7.3 × 10³ s^-1 (uncatalyzed: 3.7 × 10^-2 s^-1; enzyme-catalyzed: 2 × 10 ⁵-1.4 × 10⁶ s^-1) and a turnover number (TON) of at least 1700, limited only by the experimental conditions used. So far, no copper-based natural carbonic anhydrases are known, no faster model systems have been described and the biological role of the patellamide macrocycles is so far unknown. The observed CO₂ hydration rates depend on the configuration of the isopropyl side chains of the pseudo-octapeptide scaffold, and the naturally observed R*,S*, R*,S* geometry is shown to lead to more efficient catalysts than the S*,S*,S*,S* isomers. The catalytic efficiency also depends on the heterocyclic donor groups of the pseudo-octapeptides. Interestingly, the dicopper(ii) complex of the ligand with four imidazole groups is a more efficient catalyst than that of the close analogue of ascidiacyclamide with two thiazole and two oxazoline rings. The experimental observations indicate that the nucleophilic attack of a Cu^II- coordinated hydroxide at the CO₂ carbon center is rate determining, i.e. formation of the catalyst-CO₂ adduct and release of carbonate/bicarbonate are relatively fast processes.

Full text of DOI:10.1039/c3dt53135j

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Carbonic anhydrase activity of dinuclear CuII
complexes with patellamide model ligands†
Cite this: Dalton Trans., 2014, 43,
3144
Peter Comba,*a Lawrence R. Gahan,b Graeme R. Hanson,c Marcel Maederd and
Michael Westphala
The dicopper(II) complexes of six pseudo-octapeptides, synthetic analogues of ascidiacyclamide and the
patellamides, found in ascidians of the Pacific and Indian Oceans, are shown to be efficient carbonic
anhydrase model complexes with kcat up to 7.3 × 103 s−1 (uncatalyzed: 3.7 × 10−2 s−1; enzyme-catalyzed:
2 × 105–1.4 × 106 s−1) and a turnover number (TON) of at least 1700, limited only by the experimental
conditions used. So far, no copper-based natural carbonic anhydrases are known, no faster model
systems have been described and the biological role of the patellamide macrocycles is so far unknown.
The observed CO2 hydration rates depend on the configuration of the isopropyl side chains of the
pseudo-octapeptide scaffold, and the naturally observed R*,S*,R*,S* geometry is shown to lead to more
efficient catalysts than the S*,S*,S*,S* isomers. The catalytic efficiency also depends on the heterocyclic
donor groups of the pseudo-octapeptides. Interestingly, the dicopper(II) complex of the ligand with four
imidazole groups is a more efficient catalyst than that of the close analogue of ascidiacyclamide with two
thiazole and two oxazoline rings. The experimental observations indicate that the nucleophilic attack of a
CuII-coordinated hydroxide at the CO2 carbon center is rate determining, i.e. formation of the catalyst-
CO2 adduct and release of carbonate/bicarbonate are relatively fast processes.
Received 6th November 2013,
Accepted 27th November 2013
DOI: 10.1039/c3dt53135j
www.rsc.org/dalton
transition metal coordination chemistry, in particular with
CuII, has been studied in some detail.6–16 However, the biologi-
Introduction
Ascidiacyclamide and the patellamides are pseudo-octapep- cal function of these cyclic pseudo-peptides is still largely
tides containing four N-based heterocycles and four amide unclear. An interesting observation in this context is that many
nitrogen groups as possible donors for metal ion coordination of the ascidians are host to symbionts, and for Lissoclinum
(see Scheme 1). These and other cyclic peptides present in the patella, from which ascidiacyclamide and the patellamides are
ascidians of the Pacific and Indian Oceans have attracted isolated, the symbiont is photosynthetic, i.e. the prokaryote
scientific interest for many years and for various reasons. Prochloron.17,18 The natural role of the cyclic pseudo-octapep-
Their biosynthesis has been reported,1–4 there is a consider- tides possibly involves CuII coordination, and among the
able interest in their pharmaceutical activity,1,5 and their rich suggested biological functions are CuII transport and
storage,17,19 copper detoxification,17 CO2 hydration (carbonic
anhydrase reactivity),12–14,17,20,21 oxygen activation,17,19,22 and
phosphoester hydrolysis (phosphatase reactivity),15 and the
aUniversität Heidelberg, Anorganisch-Chemisches Institut, INF 270,
CuII cyclic peptide complexes might also be cofactors in
D-69120 Heidelberg, Germany. E-mail: peter.comba@aci.uni-heidelberg.de;
enzymes.17 Carbonic anhydrase activity has been suggested on
Fax: +49-6226-546617
bSchool of Chemistry and Molecular Biosciences, The University of Queensland,
Brisbane, Queensland 4072, Australia
the basis of the structural and spectroscopic characterization
of carbonato-bridged dicopper(II) complexes.12–14 Studies
involving EPR spectroscopy in combination with molecular
mechanics (MM) and density functional theory (DFT) based
molecular modeling as a structural probe,14,23–25 together with
cCentre for Advanced Imaging, The University of Queensland, Brisbane,
Queensland 4072, Australia
dDepartment of Chemistry, The University of Newcastle, Callaghan, NSW 2308,
Australia
†Electronic supplementary information (ESI) available: The ESI includes a figure 18OH− and 13CO2 labeling and electrospray mass spectrometry
(ESI-MS),14 have suggested that the carbonate emerges from
which illustrates the dependence of the kcat values from the ligand system and
pKa value of the sacrificial base, a plot of the pH as a function of time for
the H4pat1-based system with the four base/indicator systems used, and the
atmospheric CO2, and catalytic activity was assumed on the
basis of NMR experiments and the gravimetric quantification
entire set of relevant kinetic traces, plots of experimental vs. fitted electronic
of CO32−. However, so far there has been no clear proof for cata-
spectra and concentration profiles not shown in the manuscript. See DOI:
10.1039/c3dt53135j
lytic turnover and the kinetics and catalytic efficiency have not
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Scheme 1 Relevant cyclic pseudo-octapeptides; H4Asc and H4PatA–G are natural products, H4pat1–5 and H4LascA are the synthetic analogues used
in the present study.
been studied; these topics are therefore the focus of the biological systems (photosynthesis, respiration, bone meta-
present report.
bolism) as well as the regulation of pH, e.g. in the blood and
Carbonic anhydrases are ubiquitous and responsible for stomach. Carbonic anhydrases are among the most efficient
the efficient hydration of CO2 at pH > 7 and dehydration of enzymes and accelerate the hydration of CO2 around 108-fold.
HCO3− at pH < 7, allowing the fixation and transport of CO2 in They generally have mononuclear ZnII sites and their
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mechanism has been of interest to biochemists, biologically
oriented coordination and computational chemists.26–35 One
of the exciting features of the dicopper(II)-patellamide chem-
istry discussed here is that these complexes might be the first
structural, spectroscopic and very efficient functional models
for a copper-based carbonic anhydrase. Note however that the
−
in vitro results presented only show that CO2 hydration/HCO3
dehydration might be a biological function, and confirmation
of this suggestion obviously will need biological studies. Most
of the small molecule model complexes reported so far are
mononuclear ZnII based but a few other mimics have been
reported, in particular dicopper(II) complexes.36–39 The fixation
of CO2 also has potential for environmental and technological
applications40,41 and, importantly, the dicopper(II) complexes
of the patellamide-based cyclic peptides discussed here are the
most efficient model systems for carbonic anhydrase known so far.
Scheme 2 Equilibria involved in the hydration of CO2.43
chlorophenol red (pKa 6.21, 6.25), imidazole and bromothymol
blue (pKa 7.14, 7.10), Tris [(tris-hydroxymethyl)-aminomethane]
and metacresol purple (pKa 8.07, 8.30), 1,2-dimethylimidazole
and metacresol purple (pKa 8.22, 8.30).26,42 The various equili-
bria shown in Scheme 2 (eqn (1) and (3)) have been analyzed
in detail previously,43–45 and for the analysis of the CuII–patel-
lamide systems presented here, we have adopted the same
experimental setup and largely the same parameters. Note,
however, that, due to solubility problems, we had to revert to
methanol–water mixtures (10% MeOH).‡ For the evaluation of
the kinetic parameters (see below) we have therefore carefully
tested the fundamental parameters involved in Scheme 2 and
found that a small variation of these parameters did not lead
to significant changes in the kCat values (Table 1 shows the
parameters used for fitting the stopped-flow traces; see ESI†
for the effect of variation of the least well-determined para-
meters KCat and KH). The time-dependent spectra of a solution
containing H4pat1 : Cu : base (1 : 2 : 4) with CO2 in the
optimum pH range (Tris, pKa = 8.07; note that the pH value
varies during the reaction and, therefore, pKa rather than pH
values are discussed in the text and given in Table 1) are
shown in Fig. 1 (similar Figures for the other dicopper(II) com-
plexes of the synthetic analogues (H4pat2–H4pat5, H4LascA) and
at different pH values are given as ESI†).
Results and discussion
The kinetic analysis of the uncatalyzed and catalyzed hydration
of CO2 in general is not trivial because the gas phase and
ionized species have very different solubilities in H2O with
strong dependencies on temperature, pressure and pH. In
addition, all relevant species are colorless and there are a
number of protonation equilibria, also including those of the
catalyst involved, which are difficult to assess quantitatively
(Scheme 2, eqn (1)–(3); note that the kinetics related to
eqn (1a) and (3c) are generally analyzed as pseudo-first-order
processes; Ind: indicator, Cat: catalyst).
k1
ꢀ!
CO ðaqÞ þ ðH OÞ
H CO
3
ð1aÞ
ð1bÞ
ꢀ
2
2
2
k
ꢀ1
k2
CO2ðaqÞ þ OHꢀ
HCO
3
ꢀ
ꢀ!
ꢀ
k
ꢀ2
Ka1
ꢀ
CO32ꢀ þ Hþ
HCO3ꢀ þ Hþ
OHꢀ þ Hþ
HCO
ð1cÞ
ð1dÞ
ð1eÞ
ð2Þ
ꢀ
ꢀ!
3
Ka2
ꢀ!
H CO
ꢀ
2
3
Kw
ꢀ!
H O
2
ꢀ
The reaction is limited by the amount of base used
(8.5 mmol); CO2 was used in excess (17 mmol) and the catalyst
concentration was 5 μmol (0.06 mol%), that of the indicator
12.5 μmol; depending on the base used, the pH variation is
around 2 pH units (see ESI†). The complex formation and the
various CuII complexes present in equilibrium in solution have
KInd
ꢀ
þ
Ind þ Hþ
Cat þ CO2
Cat þ Hþ
IndH
ꢀ!
KCat
ꢀ!
CatCO
CatH
ð3aÞ
ð3bÞ
ð3cÞ
ꢀ
2
KH
ꢀ!
ꢀ
kCat
‡Note that our earlier work focusing on solution structures and equilibria of the
CatCO2 þ ðH2OÞ ꢀ! Cat þ H2CO3
mono- and dinuclear copper(II) complexes of cyclic pseudo-peptide ligands was
The analysis of the CO2 hydration activity of carbonic anhy- performed in pure but not dry methanol, i.e. the only difference in terms of
solvent to the experiments reported here is the amount of water present. Since
drase and model systems is usually based on stopped-flow
kinetics, involving the spectrophotometric measurement of
in the various structural analyses reported before, and to which we refer in the
present kinetic study,13,14 OH2 and OH− are terminal and bridging ligands to the
the time dependence of pH via the color change of a pH indi-
copper(II) centers, we assume that the moderate change in terms of conditions
cator (eqn (2)),26,30 using matched pairs of bases and pH indi-
for our kinetic analysis does not lead to significant differences in terms of the
cators. The four pairs used in our studies are 3,5-lutidine and reported structural models.
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Table 1 Fixed general parameters for the hydration of CO2 at 25 °C (see Scheme 2; the hydration of CO2 (eqn (1a), k1*) is treated as a pseudo-first-
order reaction, [H2O] = 55.6 M)43,57–59 a
k1* (s−1
k−1 (s−1
k2 (M−1 s−1
k−2 (s−1
)
)
3.7(2) × 10−2
24.8(4)
Ka1 (M−1
Ka2 (M−1
K1*
)
)
6.3(4) × 10−11
2.0(1) × 10−5
1.5(1) × 10−3
3.02(1) × 107
KH
18.0(2) × 10−9
104
a
a
Kcat
)
12.1(4) × 103
40(1) × 10−5
)
K2 (M−1
)
a In addition to the usual fixed parameters for fitting kinetic data of CO2 hydration, the pKa value of CuII-coordinated water (see Schemes 2 and 3,
KH; pKa [CuII(H2O)6]2+ = 7.50)48 and the association constant of CO2 with the catalyst (see Scheme 2, log KCat = 4)36,47,60 were estimated from
published data (standard deviations given in brackets).
pseudo-octapeptides are the catalytically active species (see
Scheme 3). The fitted rate constants of the six copper-patella-
mide catalysts used are given in Table 2. Fig. 2 shows a com-
parison of the experimental spectra at the beginning and end
of the reaction with those simulated with the fitted kinetic
parameters, using the dicopper(II) complex of H4pat1 as cata-
lyst and Tris/metacresol purple as base/indicator couple for
detection (similar plots of the other catalysts are given as
ESI†). The good agreement between theory and experiment
supports the quality of the fitting procedure and parameters
given in Table 2. From the time-dependence of the concen-
tration of the relevant species (CO2, HCO3−, pH, base, baseH+)
given in Fig. 3 (similar plots of the other catalysts are given as
ESI†) it follows that under the optimized conditions used there
is
a nearly quantitative yield of the hydration reaction
(>8 mmol), leading to the maximum TON (turnover number)
of close to 1700 under these conditions.
The kinetic parameters in Table 2 indicate that all six cata-
lysts are of similar efficiency with a maximum catalytic rate
between 1.7 × 103 and 7.3 × 103 s−1 with Tris as base (pKa =
8.07). The uncatalyzed hydration of CO2 at 25° is 3.7 ×
10−2 s−1 26,43 i.e., there is an approx. 105-fold acceleration by the
,
dicopper(II)–patellamide catalysts, compared to the enzymatic
rates which are in the region of 2 × 105–1.4 × 106 s−1 26,46
i.e.,
leading to an up to approx. 108-fold acceleration. Rates of
,
other model systems generally are smaller (1 × 102–5 × 103 s−1
,
typically in the pH range of 7–9),29–32,37,39,47 and the systems
discussed here are the most efficient CO2 hydration catalysts
known so far.
The proposed mechanism (see Fig. 4) involves CO2 coordi-
nation to one of the CuII centers, nucleophilic attack of a
hydroxido group, coordinated to the other CuII center, at the
−
CO2 carbon atom, leading to a bridging HCO3 anion, and
−
release of HCO3 to reform the catalytically active hydroxido-
dicopper(II)-patellamide complex [the important forms of the
complexes (unbridged, hydroxido-bridged, carbonato-bridged)
have been thoroughly characterized by electrospray mass-
spectrometry (ESI-MS), UV-vis-NIR, CD, EPR spectroscopy,
X-ray crystallography, 13C and 18O labeling studies and compu-
tational methods)‡ and preliminary conformational analyses
(Monte Carlo and molecular dynamics simulations) support
the release of the bridging carbonate].13,14
Fig. 1 Two-dimensional (absorbance vs. wavelength, recorded in the
first 2 s, top) and three-dimensional (absorbance vs. wavelength vs.
time, bottom) representation of absorbance data during the hydration of
CO2 by the [Cu2II(H2pat1)(OH)]+ complex. Reaction with 8.5 mM Tris [(tris-
hydroxymethyl)-aminomethane] and 17 mM CO2 in a water–methanol
9 : 1 mixture at 25 °C; corresponding figures for the other dicopper(II)
cyclic peptide complexes are given as ESI.†
been studied in detail for the cyclic pseudo-octapeptides used
The maximum rate with Tris as the base (pKa = 8.07,
105-fold acceleration) and relatively small rates at lower pH
here (see Scheme 3).‡ 13,14
From these studies13,14 and the data presented below, it (3,5-lutidine pKa = 6.21, uncatalyzed; imidazole pKa = 7.14,
emerges that the hydroxido-dicopper(II) complexes of the cyclic
103-fold acceleration) indicate that the pKa value of the
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Scheme 3 Complexation and deprotonation reactions of the six patellamide ligands (for detailed structures see Scheme 1). The assumed catalytically active
species is the monohydroxo dicopper(II) complex (water exchange rates of CuIIaq are in the range of 7 × 108–5 × 109 s−1 49 the pKa value [CuII(H2O)6]2+ is 7.50).48
;
Table 2 Hydration rates of CO2, catalyzed by the dicopper(II) complexes of the six analogues of the patellamides given in Scheme 1 (kcat; reactions
with 8.5 mmol base, 17 mmol CO2, 5 μmol catalyst, 12.5 μmol indicator, in a water–methanol 9 : 1 at 25 °C; analysis based on parameters given in
Table 1; average and standard deviation of 5 independent measurements)
kcat [s−1
]
Ligand
pKa 6.21
pKa 7.14
pKa 8.07
pKa 8.22
H4pat1
H4pat2
H4pat3
H4pat4
H4pat5
H4LascA
1.71 × 10−3 9.1 × 10−4
1.54 × 10−3 1.3 × 10−3
1.10 × 10−3 9.4 × 10−4
2.27 × 10−3 1.3 × 10−3
1.94 × 10−3 1.3 × 10−3
1.41 × 10−3 1.1 × 10−3
7.08 × 101 2.3 × 100
6.96 × 101 1.4 × 100
7.32 × 101 6.0 × 100
7.74 × 101 1.6 × 101
7.25 × 101 2.6 × 100
7.49 × 101 4.6 × 100
7.32 × 103 3.7 × 102
1.72 × 103 1.1 × 102
1.86 × 103 8.0 × 101
1.92 × 103 1.1 × 102
2.05 × 103 2.9 × 101
4.68 × 103 2.0 × 102
6.00 × 102 1.8 × 102
1.15 × 102 1.3 × 101
1.18 × 102 3.8 × 101
1.12 × 102 2.2 × 101
1.11 × 102 1.6 × 101
2.59 × 102 2.3 × 101
Fig. 2 Comparison of experimental (solid lines) and simulated (dotted lines;
parameters listed in Tables 1 and 2 were used for the simulation) absorbance
−
II
data of the CO2 hydration reaction of the [Cu2 (H2pat1)(OH)]+ complex using the
Fig. 3 pH and concentration profile of important species (CO2, HCO3
,
base, baseH+) during the hydration of 17 mM CO2 with 8.5 mM Tris by
the [Cu2II(H2pat1)(OH)]+ complex at 25 °C; corresponding figures for the
other cyclic peptide systems are given as ESI.†
Tris/metacresol purple base/indicator system (pKa 8.07/8.30; λmax 578) at the
beginning (black lines) and the end (blue lines) of the reaction; corresponding
figures for the other dicopper(II) cyclic peptide complexes are given as ESI.†
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Fig. 4 Proposed mechanism for CO2 hydration by the [Cu2II(H2patn)(OH)]+ complexes.14
CuII-coordinated OH2 molecule, responsible for a maximum carbonate or bicarbonate to reform the resting state is assumed
concentration of the catalytically active hydroxido complex, is to be a facile process.14 This is supported by the fact that the
7.1 < pKa < 8.1, and this is as expected for CuII aqua ions48 and observed optimum rates of all six catalysts are similar (1.5–7.5 ×
the assumed pKa values of the two coordinated water mole- 103 s−1), and this is due to the similarity of the geometric and
cules in our complexes in H2O–MeCN as solvent (pKa = 6.8 electronic structures of the catalytically active hydroxido com-
and 7.5), based on the analysis of the phosphoester hydrolysis plexes.14 While we do not have quantitative information on the
kinetics.15 We assume that coordination of CO2 (see Fig. 4) is a stability constants of the carbonato-bridged intermediate, it is
fast process since the water exchange rates of CuII are in the interesting to note that for the most efficient of the catalysts
aq
range of 7 × 108–5 × 109 s−1 49 Moreover, the fact that the studied here, the dicopper(II) complex of H4pat1, we have, in
.
hydration rate decreases at higher pH (see Table 2 and ESI†) contrast to the other systems, not been able to trap and spec-
suggests that the dihydroxido complex is a much less efficient troscopically characterize any carbonato-bridged complexes.
catalyst (see discussion of the pKa values above). This is in This might indicate that the configuration of the substituents,
agreement with the expectation that ligand exchange (rate of which for H4pat1 is identical to that of the natural systems, is
CO2 coordination) of the hydroxido complex is less efficient. related to the high efficiency of this system. The only other
The nucleophilic attack by the OH− group is expected to be rate macrocycle with the same configuration, H4LascA, which also
determining, and the subsequent release of the emerging has the same heterocycle combination as observed for
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ascidiacyclamide and the patellamides, has a significantly (however, the relative stabilities of the patellamide–CuII com-
lower efficiency but still a faster catalytic rate than the com- plexes and the relative metal ion concentrations in the ocean
plexes of the four other ligand systems studied here. This compared to that in various species support the assumption
suggests that both the orientation of the side chain and the that these complexes play an important role).19 The fact that
type of heterocycle have an influence on the structure, stability many ascidans live in symbiosis with photosynthetic prokary-
and flexibility of the macrocycle and its complexes, and this is otes shows the need for efficient carbonic anhydrases, and
as expected on extensive computational work of the metal-free the function of the patellamide–CuII complexes might be to
macrocycles (which also define the type of side-chain as provide the Prochloron symbionts with CO2. That the dehy-
decisive)13,50–52 and some initial studies of the dicopper(II) dration of HCO3 which is abundant in sea water, rather than
−
complexes.14
hydration of CO2, might be of importance for the L. patella –
Prochloron couple is supported by the fact that the optimum
pH for the hydration reaction is relatively high (around pH 8),
and the reverse reaction should then occur at around physio-
logical pH.
Conclusion
The structures and solution equilibria of the CuII complexes of
six synthetic pseudo-octapeptides [Scheme 1, H4patn (n = 1–5)
and H4LascA] are known in much detail, and under the con-
ditions of the experiments discussed here (neutral to slightly
basic aqueous solution), the main species present are the
Experimental section
Materials and methods
cyclic-peptide-dicopper(II)-hydroxido complexes (Scheme 3). All materials obtained commercially were of reagent grade and
There are various possible forms of these compounds (i.e. were used without further purification. Indicators, bases, CuII
mono- or dihydroxido, moreover, one of the hydroxides can triflate (Cu(CF3SO3)2) and methanol were purchased from
bridge the two copper centers) and the corresponding struc- Fluka and Aldrich, respectively. Solvents were used in pure
tures have been analyzed in detail by X-ray crystallography analytical grade. All solvents were degassed and kept under
and/or the combination of EPR spectroscopy with compu- argon to prevent any dissolution of CO2 from the air. The CuII
tational structure optimization.13,14 The hydroxido complexes complexes were prepared according to published pro-
are known to hydrate CO2 in a reaction that involves a cedures,13,14,16 and were generated in situ by mixing the solu-
complex-CO2 adduct and the nucleophilic attack of a co- tion of the corresponding cyclic peptide (1 mM, MeOH)
ordinated hydroxide at the CO2 carbon atom, leading to a brid- with the CuII solution (Cu(CF3SO3)2, 25 mM, MeOH) and base
ging carbonate or bicarbonate.14 The results presented here ([N(C4H9)4](OMe), 25 mM, MeOH).
clearly show that this reaction is catalytic and very efficient,
Stopped-flow measurements were performed with a P.D.1
photodiode-array stopped-flow spectrometer from Applied
i.e. the fastest of the systems (H4pat1) hydrates CO2 with kcat
=
7.3 × 103 s−1, and this is five orders of magnitude faster than Photophysics. Experiments were performed in a mixture of
the uncatalyzed reaction and only one to two orders of magnitude water–methanol ratio of 9 : 1. 30 min prior to the catalysis
slower than carbonic anhydrases.
experiments a methanol solution of complex H4patn–CuII–
From a comparison of the rates for the dicopper complexes MeO− 1 : 2 : 4 (c(H4patn) 1 mM) was prepared. In the catalytic
of cyclic pseudo-octapeptides with different heterocyclic donor experiments 220 µL of the complex (c = 10 μM), indicator (c =
groups (imidazole vs. thiazole vs. oxazoline) and from the con- 25 μM) and base (c = 17 mM) containing water–methanol
figuration of the side chains (significant dependence on the (80 : 20) solution were mixed with 220 µL of CO2-saturated
configuration of the isopropyl side chain, small influence only water (c = 34 mM) at room temperature. The CO2 concentration
on the heterocycle) it emerges that (i) the nucleophilic attack was attained by relative gas-flow rates and published saturation
at the CO2 carbon atom is rate determining, (ii) coordination concentrations.53 After mixing, the rapid absorbance change
of CO2 to one of the CuII centers is in fast preequilibrium (i.e. was recorded and the absorption change correlated to the
the electronic perturbation by the heterocyclic N donor is rela- amount of hydrated CO2. Each measurement was performed
tively unimportant), and (iii) inhibition by the carbonate five times to ensure consistency of the measuring conditions
product is only of minor importance and consequently only and settings. The ReactLab™ software was used for the kinetic
slightly influences the catalytic efficiency at the concentrations analysis (Jplus Consulting Ltd).54 This software suite is
formed in these catalytic reactions.
designed to carry out the fitting of reaction models of either
The important result is that our in vitro data suggest that a kinetic or equilibrium multiwavelength data sets. For fitting
possible biological function of the patellamides is carbonic the kinetic parameters the software suite’s “Kinetics” tool was
anhydrase activity of their copper(II) complexes. This is exciting used. All calculations are handled internally and the user pro-
for two reasons, i.e., (i) there is no reliable information on the vides the experimental measurements and a reaction scheme
role of the patellamide macrocycles in the ascidians and (ii) so that is to be fitted to the data. A range of relevant supporting
far no copper-based carbonic anhydrases have been reported. options is available as well as comprehensive graphics tools
However, we point out that there is not even proof that CuII for visualization of the data and results. Data, models and
complexation of the patellamides is relevant for the ascidians results are provided in pre-formatted Excel(R) spreadsheats.55,56
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19 S. H. Wright, A. Raab, J. Feldmann, E. Krupp and
M. Jaspers, in Handbook of Marine Natural Products,
ed. E. Fattorusso, W. H. Gerwick and O. Taglialatela-Scatati,
Springer, Heidelberg, New York, 2012, p. 861.
20 B. P. Kremer, R. Pardy and R. A. Lewin, Phycologia, 1982,
21, 258.
Acknowledgements
Financial support by the German Science Foundation (DFG),
the University of Heidelberg and the University of Queensland
are gratefully acknowledged.
21 M. Kühl and A. W. D. Larkum, in Cellular Origin and Life in
Extreme Habitats, ed. J. Seckbach, Kluwer Acad. Publ.,
Dordrecht, 2002, vol. 3: Symbiosis, Mechanisms and Model
Systems, p. 273.
22 R. Latifi, M. Bagherzadeh, B. F. Milne, M. Jaspars and
S. P. De Visser, J. Inorg. Biochem., 2008, 102, 2171.
References
1 B. F. Milne, P. F. Long, A. Starcevic, D. Hranueli and
M. Jaspars, Org. Biomol. Chem., 2006, 4, 631.
2 E. W. Schmidt, J. T. Nelson, D. A. Rasko, S. Sudek,
J. A. Eisen, M. G. Haygood and J. Ravel, Proc. Natl. Acad. 23 P. V. Bernhardt, P. Comba, T. W. Hambley, S. S. Massoud
Sci. U. S. A., 2005, 102, 7315. and S. Stebler, Inorg. Chem., 1992, 31, 2644.
3 W. E. Houssen, J. Koehnke, D. Zollman, J. Vendome, 24 P. Comba, Y. D. Lampeka, A. Prikhod’ko and
A. Raab, M. C. M. Smith, J. H. Naismith and M. Jaspars,
ChemBioChem, 2012, 13, 2683.
4 J. Koehnke, A. Bent, W. E. Houssen, D. Zollman,
F. Morawitz, S. Shirran, J. Vendome, A. F. Nneoyiegbe,
G. Rajaraman, Inorg. Chem., 2006, 45, 3632.
25 P. Comba, L. R. Gahan, G. Haberhauer, G. A. Hanson,
C. J. Noble, B. Seibold and A. L. van den Brenk, Chem.–Eur.
J., 2008, 14, 4393.
L. Trembleau, C. H. Botting, M. C. M. Smith, M. Jaspars 26 R. G. Kalifah, J. Biol. Chem., 1971, 246, 2561.
and J. H. Naismith, Nat. Struct. Mol. Biol., 2012, 19, 767.
5 B. M. Degnan, C. J. Hawkins, M. F. Lavin, E. J. McCaffrey,
D. L. Parry and D. J. Watters, J. Med. Chem., 1989, 32, 1354.
6 A. L. van den Brenk, D. P. Fairlie, G. R. Hanson,
27 S. Lindskog and J. E. Coleman, Proc. Natl. Acad. Sci.
U. S. A., 1973, 70, 2505.
28 D. N. Silverman and S. Lindskog, Acc. Chem. Res., 1988, 21,
30.
L. R. Gahan, C. J. Hawkins and A. Jones, Inorg. Chem., 29 E. Kimura, T. Shiota, T. Koike, M. Shiro and M. Kodama,
1994, 33, 2280. J. Am. Chem. Soc., 1990, 112, 5805.
7 P. Comba, R. Cusack, D. P. Fairlie, L. R. Gahan, 30 X. P. Zhang, R. van Eldik, T. Koike and E. Kimura, Inorg.
G. R. Hanson, U. Kazmaier and A. Ramlow, Inorg. Chem.,
1998, 37, 6721.
8 P. V. Bernhardt, P. Comba, D. P. Fairlie, L. A. Gahan,
Chem., 1993, 32, 5749.
31 R. Alsfasser, S. Trofimenko, A. Looney, G. Pakin and
H. Vahrenkamp, Inorg. Chem., 1991, 30, 4098.
G. R. Hanson and L. Lötzbeyer, Chem.–Eur. J., 2002, 8, 1527. 32 R. Alsfasser, M. Ruf, S. Trofimento and H. Vahrenkamp,
9 A. L. van den Brenk, J. D. A. Tyndall, R. M. Cusack, Chem. Ber., 1993, 126, 703.
A. Jones, D. P. Fairlie, L. R. Gahan and G. R. Hanson, 33 E. Kimura, Acc. Chem. Res., 2001, 34, 171.
J. Inorg. Biochem., 2004, 98, 1857.
34 J. H. Satcher Jr., S. E. Baker, H. J. Kulik, C. A. Valdez,
10 L. Grondahl, N. Sokolenko, G. Abberate, D. P. Fairlie,
G. R. Hanson and L. R. Gahan, J. Chem. Soc., Dalton Trans.,
1999, 1227.
11 P. Wipf and C. Wang, Org. Lett., 2006, 8, 2381.
12 A. L. van den Brenk, K. A. Byriel, D. P. Fairlie, L. R. Gahan,
G. R. Hanson, C. J. Hawkins, A. Jones, C. H. L. Kennard,
R. L. Krueger, F. C. Lightstone and R. D. Aines, Energy
Procedia, 2011, 4, 2090.
35 W. Sattler and G. Parkin, Chem. Sci., 2012, 3, 2015.
36 C. Bazzicalupi, A. Bencini, A. Bianchi, V. Fedi, V. Fusi,
C. Giorgi, P. Paoletti, L. Tei and B. Valtancoli, J. Chem. Soc.,
Dalton Trans., 1999, 1101.
B. Moubaraki and K. S. Murray, Inorg. Chem., 1994, 33, 37 C. Bazzicalupi, A. Bencini, A. Bencini, A. Bianchi,
3549.
F. Corana, V. Fusi, C. Giorgi, P. Paoli, P. Paoletti,
B. Valtancoli and C. Zanchini, Inorg. Chem., 1996, 35, 5540.
38 A. Escuer, F. A. Mautner, E. Penalba and R. Vicente, Inorg.
Chem., 1998, 37, 4190.
13 P. Comba, N. Dovalil, G. R. Hanson and G. Linti, Inorg.
Chem., 2011, 50, 5165.
14 P. Comba, N. Dovalil, L. R. Gahan, G. Haberhauer,
G. R. Hanson, C. J. Noble, B. Seibold and P. Vadivelu, 39 A. Company, J.-E. Jee, X. Ribas, J. M. Lopez-Valbuena,
Chem.–Eur. J., 2012, 18, 2578.
15 P. Comba, L. R. Gahan, G. R. Hanson and M. Westphal,
Chem. Commun., 2012, 9364.
L. Gomez, M. Corbella, A. Llobet, J. Mahia, J. Benet-
Buchholz, M. Costas and R. v. Eldik, Inorg. Chem., 2007, 46,
9098.
16 P. Comba, N. Dovalil, G. Haberhauer, K. Kowski, 40 E. A. Parson and D. W. Keith, Science, 1998, 282, 1053.
N. Mehrkens and M. Westphal, Z. Anorg. Allg. Chem. 41 H. Arakawa, M. Aresta, J. N. Armor, M. A. Barteau,
(Special Issue Bioinorg. Chem.), 2013, 639, 1395.
17 L. A. Morris and M. Jaspers, in A Cu2+ selective marine
metabolite, RSC Special Publications, 2000, vol. 257, p. 140.
18 C. E. Salomon and D. J. Faulkner, J. Nat. Prod., 2002, 65,
689.
E. J. Beckman, A. T. Bell, J. E. Bercaw, C. Creutz, E. Dinjus,
D. A. Dixon, K. Domen, D. L. DuBois, J. Eckert, E. Fujita,
D. H. Gibson, W. A. Goddard, D. W. Goodman, J. Keller,
G. J. Kubas, H. H. Kung, J. E. Lyons, L. E. Manzer,
T. J. Marks, K. Morokuma, K. M. Nicholas, R. Periana,
This journal is © The Royal Society of Chemistry 2014
Dalton Trans., 2014, 43, 3144–3152 | 3151

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Paper
Dalton Transactions
L. Que, J. Rostrup-Nielson, W. M. H. Sachtler, L. D. Schmidt, 52 G. Haberhauer, A. Pinter, T. Oeser and F. Rominger,
A. Sen, G. A. Somorjai, P. C. Stair, B. R. Stults and
W. Tumas, Chem. Rev., 2001, 101, 953.
42 DATABASE, United States National Library of Medicine and
Chem Dplus Advaced online database, 2013.
43 X. Wang, W. Conway, R. Burns, N. McCann and M. Maeder,
J. Phys. Chem. A, 2010, 114, 1734.
44 X. Wang, W. Conway, D. Fernandes, G. A. Lawrance,
R. Burns, G. Puxty and M. Maeder, J. Phys. Chem. A, 2011,
115, 6405.
Eur. J. Org. Chem., 2007, 1779.
53 D. Linde, CRC Handbook of Chemistry and Physics, CRC
Press, Boca Raton FL, 2007.
54 Jplus, Jplus Consulting Pty Ltd, http://www.jplusconsulting.
com, Palmyra, WA 6957, Australia.
55 M. Maeder, P. King and K. Varmuza, Analysis of Chemical
Processes, Determination of the Reaction Mechanism and
Fitting of Equilibrium and Rate Constants, Chemometrics in
Practical Applications, InTechs, 2012.
45 W. Conway, X. Wang, D. Fernandes, R. Burns, G. A. Lawrance, 56 M. Maeder and Y.-M. Neuhold, Practical Data Analysis in
G. Puxty and M. Maeder, J. Phys. Chem. A, 2011, 115, 14340.
46 J. C. Kernohan, Biochim. Biophys. Acta, 1965, 96, 304.
47 D. A. Palmer and R. van Eldik, Chem. Rev., 1983, 83, 651.
48 G. Schwarzenbach, Angew. Chem., 1958, 70, 451.
49 D. H. Powell, L. Helm and A. E. Merbach, J. Chem. Phys.,
1991, 95, 9258.
Chemistry, Elsevier, Amsterdam, 2007.
57 H. S. Harned and S. R. Scholes, J. Am. Chem. Soc., 1941, 63,
1706.
58 M. Maeda, O. Hisada, Y. Kinjo and K. ITO, Bull. Chem. Soc.
Jpn., 1987, 60, 3233.
59 H. S. Weast and R. J. Davis, Handbook of Chemistry and
Physics, Chemical Rubber Co., Cleveland, 1970–1971,
p. D122.
50 P. Wipf, Alkaloids: Chemical and Biological Perspectives,
Pergamon, New York, 1998.
51 G. Haberhauer, E. Drosdow, T. Oeser and F. Rominger, 60 G. M. Harris and T. P. Dasgupta, J. Indian Chem. Soc., 1977,
Tetrahedron, 2008, 64, 1853.
54, 62.
3152 | Dalton Trans., 2014, 43, 3144–3152
This journal is © The Royal Society of Chemistry 2014