Galactose oxidase model complexes: Catalytic reactivities

Full text of DOI:10.1021/ja9621354

J. Am. Chem. Soc. 1996, 118, 13097-13098
13097
Galactose Oxidase Model Complexes: Catalytic
Reactivities
Yadong Wang and T. D. P. Stack*
Department of Chemistry, Stanford UniVersity
Stanford, California 94305
ReceiVed June 24, 1996
Protein-based radicals in enzymatic reactions are no longer
considered suspect, since numerous enzymes are now known
to function through organic side chain radicals.1,2 Such is the
case of galactose oxidase (GOase),3 a mononuclear copper
enzyme which uses a modified tyrosyl radical to facilitate the
two-electron oxidation of primary alcohols to aldehydes with
subsequent reduction of dioxygen to peroxide.1 This modified
tyrosine residue contains a covalent cross-link between an
aromatic carbon atom and a cysteine thiolate (Figure 1).4 The
hydroxyl group of the tyrosyl radical is ligated directly to the
copper center as revealed by spectroscopic5,6 and crystal-
lographic studies.4 In its fully oxidized form, the copper center
is EPR-silent, consistent with strong antiferromagnetic coupling
between a ligand-based radical and a d9 Cu(II) center. Mecha-
nistic proposals6,7 suggest that the square pyramidal coordination
of the copper in GOase serves to position the methylene
hydrogen of the substrate alcohol in close proximity to the
oxygen atom of the tyrosyl radical (Figure 1).
Figure 1. Proposed catalytic cycle of galactose oxidase.6
Our aim is to elucidate the structural features essential for
GOase reactivity through functional model chemistry. Many
structural and spectroscopic model complexes of GOase have
recently been reported;8,9 however, reactivity studies are pre-
cluded in many cases because the complexes exist as unreactive
phenolate-bridged dimers.9 One study has reported a nonsquare
planar copper complex that exhibits catalytic oxidase reactivity
under extremely basic conditions.10 Presented here are GOase
model complexes that reproduce many of the unique properties
of GOase, including stabilization of EPR-silent species obtained
by one-electron oxidation of Cu(II) complexes, and catalytic
conversion of an alcohol to an aldehyde in the presence of a
suitable oxidant. A correlation is found between Cu(II)
complexes that exhibit a moderately stable EPR-silent state and
those that exhibit oxidase reactivity.
Figure 2.15 Ligand synthesis. The key step is the ortho-lithiation of a
1,3-dimethylimidazolidine-protected salicylaldehyde14 followed by elec-
trophilic attack of a suitable disulfide. (Ligands H23, H25, and H26 do
not require steps i-iv.) Condensation of the aldehyde with diamine
followed by metal incorporation gives the complexes. Reagents: i.
dimethylethylenediamine, EtOH, 25 °C; ii. 2 equiv of n-BuLi, TMEDA,
Et2O, 25 °C; iii. R3-R3; iv. HCl (2 M), H2O; v. H2N-Rb-NH2, EtOH,
reflux; vi. Cu(OAc)2, MeOH, reflux.
(1) Whittaker, M. M.; DeVito, V. L.; Asher, S. A.; Whittaker, J. W. J.
Biol. Chem. 1989, 264, 7104-7106.
The ligands in Figure 2 provide a single metal ion with an
N2O2 coordination environment with various degrees of distor-
tion from a square planar geometry depending on the backbone
diamine. Two-carbon-bridged diimines generate nearly planar
Cu(II) complexes,11 while three-10 and four-carbon-bridged
diimines12 induce significant distortions toward a tetrahedral
coordination. Such tetrahedral distortions should enhance not
only the affinity of the metal center for a fifth ligand (potential
substrate molecule)13 but also the stability of the Cu(I) form. A
succinct, yet versatile synthesis of the appropriately substituted
salicylaldehydes is outlined in Figure 2. The modular nature
of this synthesis allows a systematic variation of the ligands to
(2) (a) Frey, P. A. Chem. ReV. 1990, 90, 1343-1357. (b) Harkins, T.
T.; Grissom, C. B. Science 1994, 958-960. (c) Picot, D.; Loll, P. J.;
Garavito, R. M. Nature 1994, 367, 243-249.
(3) Avigad, G.; Amaral, D.; Asensio, C.; Horecker, B. L. J. Biol. Chem.
1962, 237, 2736-2743.
(4) (a) Ito, N.; Phillips, S. E. V.; Stevens, C.; Ogel, Z. B.; McPherson,
M. J.; Keen, J. N.; Yadav, K. D. S.; Knowles, P. F. Nature 1991, 350,
87-90. (b) Ito, N.; Phillips, S.; Yadav, K.; Knowles, P. F. J. Mol. Biol.
1994, 238, 794-814.
(5) (a) Whittaker, M. M.; Whittaker, J. W. J. Biol. Chem. 1990, 265,
9610-9613. (b) Babcock, G. T.; Eldeeb, M. K.; Sandusky, P. O.; Whittaker,
M. M.; Whittaker, J. W. J. Am. Chem. Soc. 1992, 114, 3727-3734. (c)
Whittaker, M. M.; Chuang, Y. Y.; Whittaker, J. W. J. Am. Chem. Soc.
1993, 115, 10029-10035. (d) Clark, K.; Pennerhahn, J. E.; Whittaker, M.;
Whittaker, J. W. Biochemistry 1994, 33, 12553-12557. (e) Knowles, P.
F.; Brown, R. D.; Koenig, S. H.; Wang, S.; Scott, R. A.; McGuirl, M. A.;
Brown, D. E.; Dooley, D. M. Inorg. Chem. 1995, 34, 3895-3902.
(6) Whittaker, M. M.; Whittaker, J. W. Biophys. J. 1993, 64, 762-772.
(7) (a) Wachter, R. M.; Branchaud, B. P. J. Am. Chem. Soc. 1996, 118,
2782-2789. (b) Branchaud, B. P.; Montague-Smith, M. P.; Kosman, D. J.;
McLaren, F. R. J. Am. Chem. Soc. 1993, 115, 798-800.
(8) (a) Adams, H.; Bailey, N. A.; Campbell, I. K.; Fenton, D. E.; He, Q.
Y. J. Chem. Soc., Dalton Trans. 1996, 2233-2237. (b) Halfen, J. A.; Young,
V. G.; Tolman, W. B. Angew. Chem., Int. Ed. Engl. 1996, 35, 1687-1690.
(9) (a) Whittaker, M. M.; Duncan, W. R.; Whittaker, J. W. Inorg. Chem.
1996, 35, 382-386. (b) Adams, H.; Bailey, N. A.; Fenton, D. E.; He, Q.
Y.; Ohba, M.; Okawa, H. Inorg. Chim. Acta 1994, 215, 1-3.
(10) Kitajima, N.; Whang, K.; Moro-oka, Y.; Uchida, A.; Sasada, Y. J.
Chem. Soc., Chem. Commun. 1986, 1504-1505.
(11) The dihedral angle between the two O-Cu-N planes of the
cyclohexyldiamine Shiff-base complex is 15°. Bernardo, K.; Leppard, S.;
Robert, A.; Commenges, G.; Dahan, F.; Meunier, B. Inorg. Chem. 1996,
35, 387-396.
(12) O’Connor, M. J.; Ernst, R. E.; Holm, R. H. J. Am. Chem. Soc. 1968,
90, 4561-4568.
(13) Ligand exchange in four-coordinate complexes generally proceeds
through an associative mechanism. The idealized geometry of a five-
coordinate intermediate is trigonal bipyramidal. A 45° distortion from a
square planar array is necessary to achieve this geometry, a distortion similar
to that observed in the crystal structures reported.
(14) (a) Gray, M.; Parsons, P. J. Synlett 1991, 729-730. (b) Tamura,
E.; Kawasaki, K.; Mikame, D.; Katsuki, T. Synlett 1994, 609-610.
S0002-7863(96)02135-X CCC: $12.00 © 1996 American Chemical Society

13098 J. Am. Chem. Soc., Vol. 118, No. 51, 1996
Table 1. EPR Data and Reactivity Study Results
Communications to the Editor
oxidative
EPR signal
quenched
complex
peak potentialsa
turnovers22
CuII1
CuII2
CuII3
CuII4
CuII5
CuII7
1.00
0.92
1.08
yes
yes
no
no
yes
no
9.2 ( 0.8, 1.0 ( 0.2b
1
0
0
1.00
0.81,c 0.98d
1.00e
0, 1.2 ( 0.4b
0
a Potentials (vs SCE) were measured at -40 °C in MeCN with 0.1
M TBAP. b Stoichiometric reaction: 1 equiv each of copper complex,
c
d
(TPA+), and base. E1/2, ∆Ep ) 80 mV. E1/2, ∆Ep ) 67 mV.
e Estimated value.15
Figure 3. Electronic spectra for CuII1, its oxidized product (1 equiv
of TPA+), and the lithium benzenemethoxide adduct in toluene at -80
°C. Inset: EPR spectra for 1.1 mM CuII1 and its oxidized product in
toluene/acetonitrile solvents. Instrumental parameters: microwave
power, 13 dB; microwave frequency, 9.505 GHz; modulation amplitude,
identify the features which are necessary for oxidase reactivity.
The crystal structure of CuII215 confirms its existence as a
monomeric, four-coordinate, distorted square planar Cu(II)
complex.16 The tetrahedral distortion results from the binaphthyl
backbone rather than the steric interaction between the SPri
groups, as a similar distortion is observed in the unsubstituted
derivative CuII6.17 The EPR spectra of the Cu(II) complexes
are similar to that of GOase,5e whereas the electronic spectra
and the electrochemical properties significantly differ. These
Cu(II) complexes all display oxidative responses in the 0.80-
1.10 V range (Table 1),18 much higher than that of GOase
(E1/2, 0.23 V vs SCE).19 Addition of 1.1 equiv of the oxidant
tris(4-bromophenyl)aminium hexachloroantimonate, (TPA+)-
[SbCl6-],20 to these complexes at -40 °C yields varied results
as assessed by EPR (Table 1).15 Only the EPR signals of the
complexes with 3,5-disubstituted salicylates and a binaphthyl
backbone are quenched (e.g., Figure 3, inset), while the signals
of the other complexes are significantly altered.15 The three
EPR-silent species are moderately stable at room temperature.21
Associated with oxidation of these complexes is a change in
the absorption spectra (Figure 3). The spectra are further altered
by the addition of 1 equiv of lithium benzenemethoxide, which
suggests the binding of substrate to the oxidatively activated
species.
10 G; temperature, 77 K. The asterisk ( ) denotes excess (TPA+).
*
are used, only CuII1 displays catalytic behavior [9.2(( 0.8)
turnovers], consistent with the consumption of all oxidizing
equivalents (eq 1).24
Systematic variation of structural features of these model
complexes reveals that specific ligand attributes are necessary
for oxidase reactivity, namely, a nonsquare planar coordination
geometry and 3,5-disubstitution of the salicylate ring. The latter
likely precludes ligand radical coupling in the oxidized form,
while the former promotes substrate binding. These structural
attributes enable the generation of a moderately stable EPR-
silent species by one-electron oxidation of the Cu(II) complexes.
The stoichiometric turnover experiments indicate that the
oxidized forms must accept two electrons to accomplish the
oxidation of an alcohol to an aldehyde. This reactivity is
reminiscent of GOase. Although the complex that exhibits
catalytic behavior has similar coordination and structural
properties to GOase, the thioether linkage on the phenolate group
is not necessary for oxidase reactivity, as indicated by the
reactivity of CuII5 (Table 1).
The ability to oxidatively quench the Cu(II) EPR signal
directly coincides with the oxidase activity of these complexes
with benzyl alcohol (Table 1).22 The oxidized forms of both
CuII1 and CuII5 are capable of a stoichiometric conversion under
anaerobic conditions.23 When 20 equiv of oxidant and base
(15) See Supporting Information.
(16) Crystal data for CuII2: a small brownish green crystal from THF/
pentane; triclinic P1h (No. 2), a ) 10.4865(9) Å, b ) 15.996(1) Å, c )
17.986(2) Å, R ) 69.347(1)°, â ) 88.254(1)°, γ ) 81.377(1)°, V )
2790.2(4) Å3, Z ) 2.0; 3579 reflections (I > 4σ(I), 4.5° < 2θ < 45.5°),
R(Rw) ) 0.087(0.097). The dihedral angle between the two O-Cu-N planes
is 30°.
Acknowledgment. This research was supported by a grant from
NIH (GM50730) and a Shell Faculty Career Initiation Award (T.D.P.S.).
We thank the UCSF Mass Spectrometry Facility and others for
experimental assistance.
(17) Crystal data for CuII6: brownish green rhombic blocks from CH2-
Cl2/ether; monoclinic C2/c (No. 15), a ) 17.354(8) Å, b ) 17.479(6) Å, c
) 16.664(6) Å, â ) 100.66(4)°, V ) 4967(3) Å3, Z ) 8.0; 1974 reflections
(I > 3σ(I), 17° < 2θ < 36°), R(Rw) ) 0.049(0.043). The dihedral angle
between the two O-Cu-N planes is 35°.
Supporting Information Available: Crystallographic data for CuII2
and CuII6 and spectral data and synthetic details (19 pages). See any
current masthead page for ordering and Internet access instructions.
JA9621354
(18) These Cu(II) complexes all have similar spectroscopic properties
(UV-vis, EPR).
(22) Reaction conditions: under N2, 1 equiv of the copper complex is
dissolved in benzyl alcohol, cooled to -15 °C and 20 equiv of
(TPA+)[SbCl6-] is added followed by 20 equiv of n-BuLi. The solution is
stirred at 35 °C for 5 h, passed through a short silica column, and analyzed
by gas chromatography.15
(23) In the absence of copper complexes under otherwise identical
conditions, the oxidation of benzyl alcohol is negligible.
(24) Due to the limited solubility of the reactants, no more than 20 equiv
of oxidant and base were used.
(19) Dyrkacz, G. R.; Libby, R. D.; Hamilton, G. A. J. Am. Chem. Soc.
1976, 98, 626-628.
(20) The potential of tris(4-bromophenyl)aminium hexachloroantimonate,
(TPA+)[SbCl6-], is 1.15 V versus SCE in acetonitrile with 0.1 M
tetrabutylammonium perchlorate.
(21) Room temperature stable phenolic radicals ligated to metal centers
have been recently identified. See: Hockertz, J.; Steenken, S.; Wieghardt,
K.; Hildebrandt, P. J. Am. Chem. Soc. 1993, 115, 11222-11230.