Triazacyclophane (TAC)-scaffolded histidine and aspartic acid residues as mimics of non-heme metalloenzyme active sites

Article abstract of DOI:10.1039/c1ob06806g

We describe the synthesis and coordination behaviour to copper(ii) of two close structural triazacyclophane-based mimics of two often encountered aspartic acid and histidine containing metalloenzyme active sites. Coordination of these mimics to copper(i)

Full text of DOI:10.1039/c1ob06806g

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PAPER
Triazacyclophane (TAC)-scaffolded histidine and aspartic acid residues as
mimics of non-heme metalloenzyme active sites†
a
b
a
c
b
H. Bauke Albada,‡ Fouad Soulimani, Hans J. F. Jacobs, Cees Versluis, Bert M. Weckhuysen and
a
Rob M. J. Liskamp*
Received 19th August 2011, Accepted 9th November 2011
DOI: 10.1039/c1ob06806g
We describe the synthesis and coordination behaviour to copper(II) of two close structural
triazacyclophane-based mimics of two often encountered aspartic acid and histidine containing
metalloenzyme active sites. Coordination of these mimics to copper(I) and their reaction with molecular
oxygen leads to the formation of dimeric bis(m-hydroxo) dicopper(II) complexes.
Introduction
of true biomimetic models has been restricted to large peptidic
constructs and the behaviour of biologically relevant amino acid
5
It has been estimated that metalloenzymes comprise 40% of
all enzymes known to date. They can catalyze reactions like
based coordinating groups in small constructs lagged behind.
To bridge this gap, we recently described the mimicry of
copper-coordinating tris-histidine active sites, as in tyrosinase and
hemocyanin, for instance, by attaching these histidine residues
1
the hydrolysis of (bio)molecules or the oxidation of organic
substrates using activated small molecules like water or oxygen,
2
respectively. In general, their scope of catalyzed reactions is
6
to a synthetic triazacyclophane (TAC)-scaffold. In order to
less limited than that of non-metalloenzymes. For immobiliza-
tion of the catalytically active metal ion(s) in a special site in
the protein, tetrapyrroles like heme and/or amino acid side-
chain functionalities such as histidyl imidazole, aspartyl and/or
glutamyl carboxylate, or cystinyl thiolate groups are frequently
complete this small series of true biomimetic ligands, we have
now added two mimics of metalloenzymes using aspartic acid
(
Asp, D) and histidine (His, H) residues attached to the TAC-
scaffold. With this addition, three TAC-based structural mod-
els of prominent metalloenzyme active sites have now been
constructed.
1
utilized. Whereas immobilization of metal ions by tetrapyrrole
has been successfully mimicked by synthetic porphyrins, mimicry
of the immobilization by amino acid functionalities has been
mainly limited to the use of small synthetic organic molecules
Results and discussion
3
that lack resemblance with natural amino acid counterparts.
Synthesis of the mimics
Nevertheless, many of these small unnatural models have been very
4
useful to study important features of the mimicked enzymes, even
For the preparation of the mimics, a TAC-scaffold was pre-
pared containing Fmoc groups on the benzylic amines and an
Alloc group on the middle aliphatic amine group (see ESI for
synthesis†). Using this scaffold, the two mimics could be prepared
by straightforward solid-phase synthesis procedures (Scheme 1).
First, the Fmoc groups of resin-bound scaffold 1 were removed
and Fmoc-His(Trt)-OH or Fmoc-Asp(OtBu)-OH were coupled
using BOP and DiPEA. After replacement of the Fmoc groups
with acetyl groups—in order to mimic the backbone of the
metalloenzyme and to prevent coordination of terminal amine
groups with metal ions—the middle position was functionalized.
leading to the identification of active site intermediates before their
presence in the enzyme was revealed. However, the application
3
a
Utrecht Institute for Pharmaceutical Sciences, Medicinal Chemistry &
Chemical Biology, Faculty of Science, University of Utrecht, Univer-
siteitsweg 99, Utrecht, 3584 CG, The Netherlands. E-mail: r.m.j.liskamp@
uu.nl; Fax: +31-30-2536655; Tel: +31-30-2537396
b
Inorganic Chemistry and Catalysis group, Department of Chemistry,
Faculty of Science, University of Utrecht, Universiteitsweg 99, Utrecht, 3584
CG, The Netherlands
c
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for
Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences,
Faculty of Science, University of Utrecht, Universiteitsweg 99, Utrecht, 3584
CG, The Netherlands
0
For this, the Alloc group was removed using Pd followed
by attachment of Fmoc-Asp(OtBu)-OH or Fmoc-His(Trt)-OH
using BOP and DiPEA. After N-terminal acetylation, side-chain
deprotection and cleavage of resin-bound constructs resulted
in two mimics: one with two histidine and one aspartic acid
residues (the “2-His-1-Asp” or HDH-mimic (2)) and one with
one histidine and two aspartic acid residues (the “1-His-2-Asp” or
DHD-mimic (3)).
†
Electronic supplementary information (ESI) available: synthesis of
scaffold precursors, assignment of all infrared and Raman signals, diffuse
reflectance UV-vis spectra and models of the complexes. See DOI:
1
0.1039/c1ob06806g
‡
Present address: Chair of Inorganic Chemistry I-Bioinorganic Chem-
istry, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum,
Bochum, Germany.
1
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Scheme 1 Solid-phase synthesis of the tridentate mimics.
Coordination of the mimics to copper(II)
After this, lyophilized samples from the UV-vis studies were
subjected to infrared and Raman spectroscopic analysis in order
to determine changes in functional group vibrations as result
of their coordination to Cu(II). From this it became clear that
coordination indeed occurred by histidyl imidazole ring(s) as well
as the carboxylate group(s) of the aspartic acid residue(s) and that
coordination of amide–bond oxygen atoms to Cu(II) was absent.
Coordination of histidyl imidazole rings was especially clear from
The coordination chemistry of these mimics was assessed by
mixing stoichiometric ratios of these ligands with CuSO in water
4
at low pH. As the pH value of the mixture was raised by the
addition of base, the carboxylate functionality from aspartic acid
residues and the imidazole ring from the histidine residues became
available for coordination to Cu(II), which was evident from the
blue-shift and increase of the absorption maxima (Fig. 1). At pH
-1
an increase in intensity in the bands between 1200 and 1100 cm
7
.2, an absorption maximum was observed at 672 nm for the
in the IR spectra, going from the mimics to the complexes (Fig. 2).
These bands originated from breathing vibrations of the imidazole
rings and donation of electron density of the ring towards the
HDH-mimic (2), whereas for the DHD-mimic (3) a maximum
was found at 688 nm. For the HDH- this value corresponds
roughly to a square-planar geometry with two imidazole rings
and one carboxylate functionality. A similar geometry with
two carboxylate functionalities and one imidazole ring can be
6
Cu(II) ion resulted in a significant increase in intensity. In
-1
addition, bands around 1600 cm in the Raman spectra indicated
t
coordination by the N -nitrogen atom of the ring (see ESI†).
7
envisioned for the DHD-mimic. The remaining position of the
Additional marker bands for this coordinating tautomer, usually
square-plane could be occupied by either the sulfate ion or
a water molecule. Additionally, the isotope pattern found in
mass spectrometric analyses of these two complexes at pH 7.2
corresponds with the presence of predominantly 1 : 1 complexes
-1
observed at 997 cm , were obscured by breathing vibrations of the
aromatic ring of the TAC-scaffold. Additionally, coordination of
the carboxylate groups to Cu(II) seemed to be monodentate due
-1
-1
to a nas at 1593 and 1594 cm , and a n
s
at 1397 and 1398 cm
(
see ESI†).
(
Fig. 2), for carboxylate groups in the HDH and DHD complexes,
-
1
respectively. The resulting D (nas - n ) of 196 cm is a hallmark
s
for monodentate coordination of carboxylate groups to transition
Fig. 1 Absorption bands of d–d transitions at different pH values. Left:
HDH-Cu(II) complex: (a) pH 1.1; (b) pH 3.4; (c) pH 3.8; (d) pH 4.2; (e)
pH 4.4; (f) pH 5.3; (g) pH 6.8; (h) pH 7.2. Right: DHD-Cu(II) complex: (i)
pH 1.2; (j) pH 2.2; (k) pH 3.0; (l) pH 3.7; (m) pH 4.5; (n) pH 5.9; (o) pH
Fig. 2 Infrared spectra of the two ligands (grey traces) and their Cu(II)
6
.5; (p) pH 7.2.
complexes (black traces).
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8
metal ions. Next to these functional group vibrations, Cu–N and
the Cu
2
(OH)
2
-core for both complexes (see ESI†). In addition, Cu–
Cu–O bonds were also observed in the Raman spectra, at 466 and
around 400 cm , respectively (see ESI†).
(m-OH)
2
–Cu diamond core breathing vibrations were observed by
-
1
-1
intensities at 739 cm in the Raman spectra of both mimics (arrow
(
a), Fig. 3). Even though, Cu–N and Cu–O bonds can also be
Coordination to copper(I) and reaction with O
2
observed using Raman spectroscopy, only a weak Cu–N stretching
vibration was observed for the complex of the HDH-mimic (arrow
After this initial mapping of the coordination chemistry of these
mimics to Cu(II) ions, which showed close structural mimicry
of their biological counterparts, we studied their performance
in a biologically relevant reaction, namely the immobilization of
molecular oxygen by copper(I) complexes. Although the natural
ligand environments for this reaction consist solely of histidine
residues, we envisioned that the application of the current mimics
in this chemistry would be highly instructive to study the potential
application of these mimics in biologically relevant reactions.
Unfortunately, these mimics based on biologically relevant co-
ordinating moieties did not dissolve in the aprotic solvents that
are usually required for these studies.§ Therefore, the following
studies were performed in the aprotic but very hygroscopic DMSO
(
b), Fig. 3). The absence of this band in the complex of the
DHD-mimic can be explained by a weaker axial coordination
of the imidazole ring in that complex. Alternatively, one of the
imidazole rings of the HDH-mimic is weakly coordinating to an
axial position and the other one is more strongly coordinating to
an equatorial position, which is apparent from a weak intensity at
-1
4
70 cm . In addition, both mimics showed Cu–O bond vibrations
-1
by peaks just below 400 cm (arrows (c), Fig. 3). Concerning the
coordinating functional groups, histidyl imidazole rings appeared
to be coordinating by their N -nitrogen atoms, as was concluded
t
-1
10
from a band at 1600 cm in the Raman spectra (Fig. 3).
A
slightly more complicated picture was obtained for coordination of
the carboxylate functionalities. Although the mononuclear Cu(II)-
mimic complexes mostly showed monodentate coordination, these
dinuclear complexes could also have some bidentate bridging
coordination, as was inferred from absorbances between 1541 and
at room temperature. First, recrystallized [Cu(MeCN)
4
]PF and
6
the sodium salts of either the DHD- or the HDH-mimic was
mixed in stoichiometric ratios. Initially, only a vague blue colour
was formed even under exclusion of air, most likely caused by
oxidized Cu(I) due to traces of oxygen and water present in
DMSO. However, when air was bubbled through the solution
at set intervals a strong blue coloration of the mixture was
observed, corresponding to the generation of Cu(II) species. UV-
vis measurements of this solution at regular time intervals indeed
revealed an increase in absorption intensity and a small shift
in the position of the absorption maxima (lmax): for the HDH-
mimic a shift to shorter wavelengths was observed whereas for the
DHD-mimic a shift to longer wavelengths was observed (ESI†).
Interestingly, oxygenation of the Cu(I) complex was complete
within 90 min for the HDH-mimic and in 60 min for the DHD-
mimic (ESI†). Considering the conditions used formation of
-1
8
1
548 cm in the IR spectra (see ESI†). Apart from bidentate
coordination to the copper(II) ions, another possibility could
be hydrogen bond formation with a proximate hydroxo group
originating from the Cu
2
(OH) core. A combination of both mono-
2
and bidentate coordination is also possible.
9
dimeric bis(m-hydroxo) dicopper(II) complexes was most likely.
Indeed, an absorption maximum for the HDH-based complex at
7
20 nm corresponded with a square-pyramidal complex in which
the equatorial positions were occupied by the bridging hydroxo
groups, one of the two imidazole rings and the carboxylate group
and the remaining imidazole ring of the mimic was coordinating
7
axialy. Similarly, the DHD-mimic resulted in a complex with a
l
max of 740 nm, also in agreement with a square-pyramidal complex
with two hydroxo-groups and the two carboxylate groups of the
mimic positioned in the equatorial and the remaining imidazole
ring in one of the axial positions. In addition, both mimics
showed hydroxo-to-copper(II) charge-transfer bands around 425
nm (ESI†). A similar complex was previously uncovered for a
Fig. 3 Raman spectra of the bis(m-hydroxo) dicopper(II) complexes based
on the HDH- (top) or DHD-mimics (bottom). Arrows indicate the most
prominent features mentioned in the text. Asterisks mark positions of
peaks from residual DMSO.
7
Based on these spectroscopic findings, two bis(m-hydroxo)
dicopper(II) complexes in which each copper centre is surrounded
by one of the two mimics can be postulated (Fig. 4). That these
6
tris-histidine containing TAC-based mimic.
The samples of these Cu(I)–O studies were then condensed
2
under high vacuum and analyzed by infrared and Raman spec-
troscopy. From these measurements it was clear that indeed
dimeric bis(m-hydroxo) complexes were formed in which the bis(m-
hydroxo) dicopper(II) core was surrounded by either the HDH-
or the DHD-mimic. For instance, the infrared spectra showed
6
two complexes and the previously described “3-His” complex
can be useful to study metalloenzymes, can already be inferred
from the different rates observed for the reaction between Cu(I)
and molecular oxygen. The slower kinetics of oxygen binding of
the “3-His” variant when compared to the other two that are
currently described—i.e. 100 min for the HHH-mimic, 90 min
for the HDH-mimic and 60 min for the DHD-mimic—is likely
connected to the presence of only “3-His” sites in metalloenzymes
like the oxygen carrier hemocyanin and to the substrate converting
-
1
absorptions at 953 and 950 cm of the O–H bending vibrations of
§
Cu(I)–O
2
chemistry is usually performed in aprotic solvents and at low
◦
temperatures (typically around -80 C). See ref. 9
1
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the functionalization of the middle position, the Alloc groups
0
on the resin-bound precursors were removed using Pd and a
scavenger. To the liberated amine groups, Fmoc-His(Trt)-OH (for
mimic 2) and Fmoc-Asp(OtBu)-OH (for mimic 3) were coupled.
Acetylation of the N-terminal amine functionality was performed
after Fmoc removal. Resin-bound protected mimics were cleaved
and deprotected using the cleavage cocktail mentioned and the
released mimics were precipitated in cold MTBE and hexanes
◦
(
1 : 1, at -20 C). Lastly, the mimics were purified by column
chromatography (eluent: CHCl
(v/v)). Pure fractions were concentrated and lyophilized from
water with pH 7. Yields: 174 mg for 2 (226 mmol, 58%) and 185
3
/MeOH/25% NH OH 8 : 4 : 1.5
4
Fig. 4 Proposed structures of the two bis(m-hydroxo) dicopper(II) com-
plexes based on the two mimics. R represents the rest of the mimic; S is an
optional solvent molecule. Distortions from the perfect square-pyramidal
geometry as depicted above are likely to be present in the actual
compounds.
mg for 3 (234 mmol, 63%). R 0.33 for 2 and 0.58 for 3 and (eluent:
f
CHCl
mimic 2 = 13.58 min; t
= 792.3824 (calc. 792.3793 for [M + H] ); mimic 3 = 770.3450
3
/MeOH/25% NH
4
OH 60 : 45 : 20 (v/v)). RP-HPLC (C
8
):
t
R
R
mimic 3 = 13.94 min. HRMS: mimic
enzymes tyrosinase or catechol oxidase. Although this faster
binding by especially the DHD-mimic could be expected based
on general hard–soft acid–base principles, i.e. that the harder
Cu(II) will coordinate stronger to carboxylates and thereby pull
the oxygenation reaction, such subtle differences can now be
determined more precisely using these mimics.
+
2
+
(calc. 770.3473 for [M + H] ).
Coordination complexes with Cu(II) ions
For each of the ligands, stoichiometric mixtures of both ligand
and CuSO
1 N HCl. After recording the first UV-vis spectrum, the pH was
stepwise increased using 0.5–5% NaHCO solutions and at each
significant difference in pH value an absorption spectrum was
measured. For the d–d transition absorptions, 25 mM solutions
of the complexes were prepared by mixing equal amounts of
4
were prepared and the pH was adjusted to ~1.2 using
Conclusions
3
Two TAC-based mimics of frequently observed biological non-
heme metal-binding sites have been synthesized and their coor-
dination to copper ions has been characterized. Although none
of these complexes could be crystallized, UV-vis, ESI-MS, and
especially infrared and Raman spectroscopic analysis revealed
unambiguously that the coordination chemistry of these mimics
to Cu(II) and their behaviour in a biological relevant Cu(I)–O
reaction closely mimictheir counterparts found innature. Together
with a tris-histidine containing TAC-based mimic previously
5
0 mM solutions of both mimic and CuSO . The charge-transfer
4
spectra were obtained using the above procedure with 5 mM
solutions of the mimic-Cu(II) complexes. Spectra were recorded
on a 96-well microtiterplate suitable for UV measurements using
a microtiterplate reader measuring from 200–998 nm.
2
6
described by us, these three mimics represent three of the most
Infrared and Raman spectra
prominent non-heme metal-binding sites found in nature. We
anticipate that these and similar mimics will find useful synthetic
applications in the near future and will help the elucidation
of mechanisms in the often complicated enzymatic reactions
performed by non-heme metalloenzymes.
Lyophilized Cu(II)-mimic complexes and vacuum dried Cu(II) (m-
2
OH) -mimic complexes obtained after UV-vis experiments were
2
used for measuring infrared and Raman spectra. For measuring
the Raman spectra of both Cu(II)-mimic complexes a drop of water
had to be applied to the lyophilized powder in order to prevent
incineration of the powder by the laser beam. Reference spectra
of the sodium salts of the mimics were obtained using freshly
lyophilized samples of solutions of the mimics of which the pH
was set at ~7.5 using 1 N NaOH (aq).
Experimental
General remarks concerning the syntheses as well as detailed
description of procedures not described below can be found in
the supporting information†.
Reaction of Cu(I)-mimic complexes with molecular oxygen
Synthesis of “2-His-1-Asp” (2) and “1-His-2-Asp” mimic (3)
A solution of one of the mimics in DMSO (50 mM, 150 mL)
For the synthesis of these two mimics, the scaffold described in
the previous synthesis was coupled to 500 mg of PS-S RAM-
was added to a freshly prepared solution of [Cu(MeCN)
4
](PF ) in
6
DMSO (50 mM, 150 mL). Then, 300 mL of DMSO was added and
the resulting 12.5 mM solution was immediately analyzed. After
this, air was bubbled through the solution at regular intervals,
resulting in an increasing intensity of the blue colour.
-
1
Fmoc resin (loading: 0.78 mmol g ) using 1 equiv of the scaffold.
After overnight coupling, the remaining amine functionalities
were protected with the acetyl group using the capping reagent.
For coupling of the amino acids, 8 equiv were used compared
to the initial loading of the resin. After removal of the Fmoc
groups, the scaffold containing resin was divided in two equal
portions: one to construct mimic 2 and one for mimic 3. For
mimic 2 Fmoc-Asp(OtBu)-OH and for mimic 3 Fmoc-His(Trt)-
OH were coupled. After coupling, the N-terminal Fmoc protecting
group was removed and replaced by an acetyl group. For
Abbreviations
Alloc = allyloxycarbonyl; BOP = (benzotriazol-1-yloxy)-tris-
(dimethyl-amino)phosphonium hexafluorophosphate; DiPEA =
N,N-diisopropyl-ethylamine; Fmoc = 9-fluorenylmethyloxycar-
bonyl; tBu = tert-butyl; PTSA = para-toluenesulfinate anilinium;
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Trt = triphenylmethyl; TFA = trifluoroacetic acid; HOBt = 1-
hydroxybenzotriazole; TIS = triisopropylsilane.
5 See for example R. J. Radford, J. D. Brodin, E. N. Salgado and F. A.
Tezcan, Coord. Chem. Rev., 2011, 255, 790; H. Wade, S. E. Stayrook
and W. F. DeGrado, Angew. Chem., Int. Ed., 2006, 45, 4951.
6
H. B. Albada, F. Soulimani, B. M. Weckhuysen and R. M. J. Liskamp,
Chem. Commun., 2007, 4895.
E. Prenesti, P. G. Daniele, M. Prencipe and G. Ostacoli, Polyhedron,
1999, 18, 3233; E. Prenesti, P. G. Daniele and S. Toso, Anal. Chim.
Acta, 2002, 459, 323; E. Prenesti, P. G. Daniele, S. Berto and S. Toso,
Polyhedron, 2006, 25, 2815.
Notes and references
7
1
Biological Inorganic Chemistry, ed. I. Bertini, H. B. Gray, E. I. Stiefel
and J. S. Valentine, University Science Books, Sausalito, 1st edn,
2
007.
2
3
4
Activation of Small Molecules, ed. W. B. Tolman, Wiley, Weinheim, 1st
edn, 2006.
Concepts and Models in Bioinorganic Chemistry, ed. H.-B. Kraatz and
N. Metzler-Nolte, Wiley, Weinheim, 1st edn, 2007.
S. J. Lippard, Nat. Chem. Biol., 2006, 2, 504.
8 G. B. Deacon and R. J. Phillips, Coord. Chem. Rev., 1980, 33, 227.
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10 J. G. Mesu, T. Visser, F. Soulimani, E. E. van Faassen, P. de Peinder, A.
M. Baele and B. M. Weckhuysen, Inorg. Chem., 2006, 45, 1960.
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