New catalytic model systems of tyrosinase: Fine tuning of the reactivity with pyrazole-based N-donor ligands

Article abstract of DOI:10.1039/c3cc47888b

Two new Cu(I) complexes have been synthesized and investigated as model systems of the enzyme tyrosinase. The corresponding ligands are based on a combination of an imine function with two different pyrazole groups. The reactivity of the prepared systems with respect to the conversion of monophenols to the corresponding ortho-quinones is investigated. The resulting data are compared to results obtained for other catalytic model systems of tyrosinase. The Royal Society of Chemistry 2014.

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New catalytic model systems of tyrosinase: Fine tuning of the reactivity
with pyrazole-based N-donor ligands
Jessica Nadine Hamanna and Felix Tuczek*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000
(Scheme 1).1,6,10 In recent years, many lowꢀmolecular copper
Two new Cu(I) complexes have been synthesized and investi-
50 complexes have been synthesized as model systems of
tyrosinase.1,11 These compounds can be divided into systems
which hydroxylate the ligand framework12ꢀ16 and those which are
able to convert external monophenols to the corresponding orthoꢀ
quinones.11,17 The first model system exhibiting the latter
55 reactivity in a catalytic fashion was published by Réglier and
coworkers.18 This binuclear copper(I) complex generates 3,5ꢀdiꢀ
tertꢀbutylꢀoꢀquinone (DTBQ) from 2,4ꢀdiꢀtertꢀbutyl phenol
(DTBPꢀH) with a turnover number (TON) of 16. The formation
of orthoꢀquinone was detected by using UV/Vis spectroscopy,
60 because orthoꢀquinones show an intense absorption band in the
range of 400 ꢀ 420 nm.19 In 1991 a further system exhibiting
stoichiometric as well as weakly catalytic monooxygenation of
external phenols was published by Casella and coworkers.17 This
year HerresꢀPawlis et al. presented a new copper(I) catalyst
gated as model systems of the enzyme tyrosinase. The corres-
ponding ligands are based on a combination of an imine
function with two different pyrazole groups. The reactivity of
10 the prepared systems with respect to the conversion of
monophenols to the corresponding ortho-quinones is
investigated. The resulting data are compared to results
obtained on other catalytic model systems of tyrosinase.
The ubiquitous type 3 copper enzyme tyrosinase (Ty) catalyses
15 the formation of orthoꢀquinones, starting from monophenols. The
conversion of tyrosine to dopaquinone occurs in a twoꢀstep
process, starting with an aromatic hydroxylation which is
followed by twoꢀelectron oxidation.1,2 The formed dopaquinone
polymerises spontaneously to the important pigment melanin.3,4
20 The active site of tyrosinase contains a binuclear copper centre,
wherein each copper ion is coordinated by three histidine
residues.5,6 In 2006 Matoba and coworkers published the first
crystal structure of a tyrosinase derived from the bacterium
Streptomyces castaneoglobisporus.5 In the meantime more
25 tyrosinases have been structurally characterized.7
Hemocyanins (Hc) and catechol oxidases (CO) are also
important type 3 copper proteins, but exhibit reactivities different
to tyrosinase. Hemocyanins mediate the oxygen transport in
arthropods and molluscs whereas catechol oxidases are
30 responsible for the oxidation of catechols to the corresponding
orthoꢀquinones.6,8,9 All of these copper type 3 enzymes have very
similar active sites and bind dioxygen as peroxide in a typical
ꢁ²ꢀη²:η² (sideꢀon bridging) geometry. In the oxy form the
oxidation state of copper ions changes from +I to +II.1
65 mediating the hydroxylation of monophenols via
a wellꢀ
characterized peroxo intermediate.20
In 2010 our group published the first catalytic model system
based on a mononuclear copper(I) complex.11 This [Cu(I)Lpy1-
(CH3CN)2]PF6 system (cf Scheme 2) was found to generate
70 DTBQ from DTBPꢀH with a TON of 18 after 8 hours. More
recently we presented a second model system containing a
benzimidazole moiety.21 With the new Lbzm1 system, the TON
could be increased to 31, along with a significantly increased
turnover frequency.21 To further investigate the influence of the
75 heterocyclic group on the reactive properties of our system, we
developed two new pyrazole based copper(I) complexes as model
systems.
35
40
Scheme 2. a) Ligand Lpy1 of first mononuclear catalytic model
80 system, b) ligand Lbzm1 and ligands Lhpz1 and Lhpz2 of this study (c
and d).11,21
Scheme 1. Conversion of monophenolic substrates to orthoꢀ
quinones via catechols (monophenolase and diphenolase activity,
respectively).
45
The ligands of both systems contain an imine function terminated
by a tertꢀbutyl residue; they only differ with respect to the
85 substituents of pyrazole moiety (Lhpz1 and Lhpz2; Scheme 2).
Tyrosinase exhibits monophenolase and diphenolase activity. The
monophenolase cycle involves hydroxylation of monophenolic
substrates to catechols, which are released as orthoꢀquinones
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Lhpz2 counterpart (TON = 23, TOF = 0.62 minꢀ1). Both systems
form most of the quinone during the first 120 min.s and become
50 inactive after ~ 6 hours.
Synthesis and reactivity of the new Cu(I) complexes
The two ligands Lhpz1 and Lhpz2 were prepared in several steps
by using similar procedures (Scheme 3 and Supp. Inf.). For the
aminoethylation of the pyrazoles a modification of a literature
procedure was used.22 The corresponding copper(I) complexes
were prepared from tetrakis(acetonitrile)copper(I) hexafluoroꢀ
phosphate under anaerobic conditions.
5
10
15
20
55
Figure 2. Turnover frequency as function of time (15 min. < t < 6
60 hrs.) for the systems Lhpz1 and Lhpz2; Inset: Their turnover number
per dicopper unit as function of time.
In order to further prove the formation of DTBQ during the
reaction of DTBP with molecular oxygen NMR spectra of the
65 reaction mixture were recorded (Figure 3). To this end the
Scheme 3. Syntheses and metalation of the new ligands.
To investigate the catalytic activity of the pyrazoleꢀbased model
systems in situ UV/Vis spectroscopy was applied. To this end a
solutions were quenched with 6
minutes of oxygenation and extracted with dichloromethane to
eliminate the copper ions.
M hydrochloric acid after 30
500 ꢁM solution of the respective copper(I) complex in
dichloromethane was prepared and 50 eq. of DTBPꢀH and
25 100 eq. of triethylamine were added. Subsequent oxygenation at
ambient temperature was found to result in the formation of
DTBQ, as indicated by an absorption band appearing at 407 nm
(Figure 1).
70
75
80 Figure 3. 1HꢀNMR spectrum of the oxygenation solution after
quenching with HCl (organic phase) for the Lhpz1 system. Inset:
resonances in the aliphatic region.
In agreement with similar model systems the resulting NMR
85 spectra revealed signals from DTBQ, DTBPꢀH and the CꢀC
Figure 1. UV/Vis spectra of a 500 ꢁ
M solution of the complex
30 [Cu(I)Lhpz1(CH3CN)2]PF6 in CH2Cl2 after addition of 50 eq. DTBPꢀ
H, 100 eq. NEt3 and oxygenation between 15 min. and 6 h; l =
1 mm.
coupling
product
3,3’,5,5’ꢀtetraꢀtertꢀbutylꢀ2,2’ꢀbiphenol
("biphenol", Figure 3).11,21 The signals of DTBPꢀH, DTBQ and
the biphenol are found at a ratio of 51:18:31. This result agrees
with the TON of 18 measured after 30 minutes of oxygenation
90 (Figure 2). For the Lhpz2 system the corresponding ratio was
obtained as 70:15:15 (cf Supp. Inf.).
As evident from Figure 1, the complex [Cu(I)Lhpz1(CH3CN)2]PF6
35 catalyses the formation of 3,5ꢀdiꢀtertꢀbutylꢀoꢀquinone (DTBQ)
from the corresponding phenol DTBPꢀH. For the determination
Related smallꢀmolecule model systems of tyrosinase
investigated before (cf Scheme 2) have already indicated an
important influence of the heterocyclic Nꢀdonor group on the
95 catalytic activity.11,21 Specifically, the CuLbzm1 complex was
found to mediate the conversion of DTBPꢀH to DTBQ with a
TON of 31, 72 % higher than that of CuLpy1, and a TOF which
was about twice as large as that of the latter complex. This was in
part attributed to the wellꢀdocumented capability of copperꢀ
100 benzimidazole complexes to mediate the orthoꢀhydroxylation of
phenolic substrates.17 The new pyrazoleꢀbased systems range
between the original Lpy1 system and the Lbzm1 system; i.e., the
of the turnover number (TON), an extinction coefficient of ε =
ꢀ1
1830
M
cmꢀ1 at 407 nm was applied.19 Oxygenation of DTBPꢀH
occurred very fast during the first 100 minutes of reaction and
40 then slowed down, leading to a TON of 29 after 6 hours.
The oxygenation was also performed using the complex
[Cu(I)Lhpz2(CH3CN)2]PF6. In analogy to the Lhpz1 system, the
formation of DTBQ happened quickly during the first two hours,
reaching a TON of 23 after 6 hours. In Figure 2 the TON and
45 TOF (turnover frequency) of both systems are compared.
Importantly, the Lhpz1 system exhibits both a higher TON (29)
and a higher TOF (0.85 minꢀ1 after 15 min.s) as compared to its
2
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methylated Lhpz2 catalyst is somewhat more active than Lpy1 and 55 intermediate position (Table 1). There seems to be a clear
the unsubstituted Lhpz1 catalyst almost as active as Lbzm1. To
explain this trend we suggest that electronꢀrich Nꢀdonor ligands
promote the hydroxylation reaction by making the peroxo
intermediate less stable (Scheme 4, compound 3); i.e. the least
reactive pyridineꢀbased system would form the most stable
peroxo intermediate whereas the stability of the peroxo
intermediate is decreased for the more reactive benzimidazoleꢀ or
pyrazole based catalysts. In fact, we succeeded in observing the
10 peroxo adduct for the Lpy1 complex at low temperatures, in
contrast to the latter systems (cf Supp.Mat.). A second possible
influence on the reaction rate emerges from the twoꢀelectron
oxidation of the catecholate adduct 4, leading to the quinone.
correlation between TON and TOF; i.e., the faster the catalytic
reaction, the higher is the product yield. We assume that a higher
rate counteracts side reactions which destroy the catalyst, thus
leading to a higher catalytic performance.
5
60 Acknowledgements
The authors would like to thank Deutsche Forschungsꢀ
gemeinschaft (DFG), COST and CAU Kiel for financial support
as well as Holger Naggert for support with the measurements.
Notes and references
65 a Christian-Albrechts-Universität zu Kiel, Max-Eyth-Straße 2, 24118 Kiel,
Germany. Fax: +0049 (0)431 1520; Tel: +0049 (0)431 1410; E-mail:
ftuczek@ac.uni-kiel.de
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
70 DOI: 10.1039/b000000x/
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5
6
7
85
Scheme 4. Proposed catalytic cycle for investigated systems Lhpz
and Lhpz2 in analogy to model systems Lpy1 and Lbzm1.11,21
1
With respect to the system without substituted pyrazole (Lhpz1;
35 TON = 29), the TON decreases to 23 for the Lhpz2 system. In
agreement with the above considerations we attribute this effect
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Two new mononuclear copper(I) complexes containing ligands
with pyrazole groups were synthesized as model systems of the
enzyme tyrosinase and investigated regarding the conversion of
the external substrate DTBPꢀH to DTBQ, using UV/Vis and
45 NMR spectroscopy. Importantly, the Lhpz1 system (TON = 29)
was found to be more reactive than its Lhpz2 counterpart
(TON = 23; Table1)
100
105 15 A. De, S. Mandal, R. Mukherjee, J. Inorg. Biochem. 2008, 102, 1170.
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Lpy1
22
Lhpz
23
2
Lhpz
29
1
Lbzm
1
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31
TOF @15 min.
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0.62
0.85
0.98
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Table 1. All mononuclear tyrosinase model systems, ordered by
50 TON for the conversion of DTBPꢀH to DTBQ at ambient
temperature; grey marked new systems.11,21
115
In comparison with the model systems Lbmz1 (TON = 31) and
Lpy1 (TON = 22) published earlier, the new systems have an
22 K. Kenji, JP patent 2010-215610, 2010.
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