Photomodulation of the conformation of cyclic peptides with azobenzene moieties in the peptide backbone

Article abstract of DOI:10.1002/(SICI)1521-3773(19990917)38:18<2771::AID-ANIE2771>3.0.CO;2-W

The cis?trans photoisomerization of the azobenzene building block 4-(4- aminophenylazo)benzoic acid incorporated in a cyclic peptide (see scheme) facilitated a two-state transition of the peptide chain from a rigid constrained conformation in the trans is

Full text of DOI:10.1002/(SICI)1521-3773(19990917)38:18<2771::AID-ANIE2771>3.0.CO;2-W

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‡
(CH2Cl2): nÄ(CO) ˆ 2004 (vs), 1947 (sh), 1931 cm 1 (vs). 3: MS (FAB ,
Photomodulation of the Conformation of
Cyclic Peptides with Azobenzene Moieties in
the Peptide Backbone**
‡
‡
CH2Cl2): m/z (%): 968 (100) [M ], 791 (92) [M
Cptt]; IR (CH2Cl2):
nÄ(CO) ˆ 2023 (vs), 1954 cm (vs); 1H NMR (300 MHz, CDCl3,
258C): rotamer 3a: d ˆ 7.18 (d, J(H,H) ˆ 2.4 Hz, 2H, H4 and H5),
6.60 (t, J(H,H) ˆ 2.4 Hz, 1H, H2), 5.94 (d, J(H,H) ˆ 2.4 Hz, 2H, H4
and H5), 5.83 (t, J(H,H) ˆ 2.4 Hz, 1H, H2, Cptt), 4.42 and 4.19 (m, 2H
1
Raymond Behrendt, Christian Renner,
Michaela Schenk, Fengqi Wang, Josef Wachtveitl,
Dieter Oesterhelt, and Luis Moroder*
ˆ
each, CH, cod), 2.7 ± 2.4 (m, 2H), 1.9 ± 2.2 (m, 4H), 1.8 ± 1.6 (m, 2H,
CH2, cod), 1.32 and 1.26 (s, 18H each, Cptt); rotamer 3b: d ˆ 6.69 and
6.38 (t, J(H,H) ˆ 2.4 Hz, 1H each, H2), 6.03 and 5.90 (d, J(H,H) ˆ
tt
ˆ
2.4 Hz, 2H each, H4 and H5, Cp ), 4.42 and 4.19 (m, 2H each, CH,
For photomodulation of conformational, physico-chemical,
and biological properties of peptides, proteins, and phospho-
lipids large use has been made of the cis/trans isomerization of
azobenzene moieties grafted to specific sites of such bioma-
terials or related model systems.[1, 2] Since the light-induced
isomerization of azobenzene is accompanied by significant
changes in geometry and polarity of the chromophore,[3] this
proved to be well suited for induction of local topochemical
changes in conformationally more restricted small sys-
tems.[4±10] Low-mass cyclic peptides are rigid structures that
are extensively exploited for the design of libraries of
conformers of defined bioactivities.[11, 12] Thus, incorporation
of the azobenzene moiety into the peptide backbone of such
cyclic peptides is expected to represent an ideal system to
probe the efficiency of this ªlight-switchº for induction of
conformational transitions. With 4-(4-aminophenylazo)benz-
oyl (APB) as amino acid residue in the peptide backbone we
succeeded in the present study to construct a very rigid,
conformationally constrained cyclic peptide in the trans
configuration which upon irradiation, relaxes into a largely
free conformational space.
The distance between the two para-carbon atoms of the
azobenzene unit in the trans configuration is 9 Š and in the cis
configuration 5 Š. To transfer optimally these changes in
geometry to the peptide backbone, H-APB-OH was synthe-
sized according to known methods[13] and fully characterized
in its photochemical properties.[14, 15] It was then incorporated
into an octapeptide related to the active site of thioredoxine
reductase (Scheme 1). Owing to the low nucleophilicity of the
para-amino group of H-APB-OH, its protection in acylating
the resin-bound peptide was not required, but difficulties
were encountered in the subsequent acylation of this group.
These were overcome by silylation and using the acid fluoride
method for the peptide-chain extension step. Mild acidic
cleavage from the resin produced the linear side chain
protected pseudo-nonapeptide 1, which was then cyclized by
the PyBOP/HOBt/DIEA procedure. Final acidic deprotec-
tion generated the target compound 2 cyclo(Ala-Cys(StBu)-
Ala-Thr-Cys(StBu)-Asp-Gly-Phe-APB).
cod), 2.7 ± 2.4 (m, 2H), 2.2 ± 1.9 (m, 4H), 1.8 ± 1.6 (m, 2H, CH2 cod),
1.31 and 1.30 (s, 18H each, Cptt); 13C{1H} NMR (75 MHz, CDCl3,
258C): rotamer 3a: d ˆ 177.7 (CO), 150.0 and 142.2 (C1 and C3), 127.6
(C2), 119.7 (C4 and C5), 110.0 (C2), 105.5 (C4 and C5, Cptt), 84.0 and
ˆ
80.6 (d, J(Rh-C) ˆ 11.5 Hz, CH, cod), 34.8 and 34.0 (CMe3), 32.7 and
31.8 (CH3, Cptt), 31.7 and 30.6 (CH2, cod); rotamer 3b: d ˆ 178.3
(CO), 147.2 and 147.1 (C1 and C3), 123.8 and 120.7 (C2), 109.5 and
tt
ˆ
108.8 (C4 and C5, Cp ), 85.9 and 80.6 (d, J(Rh,C) ˆ 11.5 Hz, CH,
cod), 34.5 and 34.4 (CMe3), 32.3 and 32.2 (CH3, Cptt), 31.6 and 30.2
(CH2, cod). 4: 1H NMR (300 MHz, CDCl3, 258C): d ˆ 6.72 and 6.62 (t,
J(H,H) ˆ 2.5 Hz, 1H each, H2), 6.16 and 6.11 (d, J(H,H) ˆ 2.5 Hz, 2H
each, H4 and H5), 1.31 and 1.30 (s, 18H each, Cptt); 13C{1H} NMR
(75 MHz, CDCl3, 258C): d ˆ 184.9 (d, J(Rh,C) ˆ 74 Hz, CO), 175.4
(CO), 149.4 and 149.1 (C1 and C3), 126.2 and 122.5 (C2), 110.1 and
‡
110.0 (C4 and C5), 34.9 and 34.8 (CMe3), 32.2 (CH3, Cptt); MS (FAB ,
‡
‡
CH2Cl2): m/z (%): 916 (46) [M ], 860 (100) [M
2CO], 739 (49)
Cptt]; IR (CH2Cl2): nÄ(CO) ˆ 2062 (s), 2031 (s), 1996 (m),
‡
[M
1971 cm 1 (m).
[9] P. Kalck, C. Serra, C. Machet, R. Broussier, B. Gautheron, G. Delmas,
Â
G. Trouve, M. Kubicki, Organometallics 1993, 12, 1021.
[10] Crystal data for 3: C36H54IrO2RhS2Zr, Mr ˆ 969.24, monoclinic, space
group P21/c; a ˆ 11.9123(9), b ˆ 21.5742(17), c ˆ 15.0796(12) Š, b ˆ
3
105.526(2)8, Vˆ 3734.0(5) Š3, Z ˆ 4, 1calcd ˆ 1.724 gcm
,
m ˆ
1
4.407 mm
.
Crystal dimensions 0.12 Â 0.16 Â 0.18 mm. Bruker
SMART CCD diffractometer, Tˆ 153(1) K, graphite-monochromat-
ed MoKa radiation (l ˆ 0.71073). A complete hemisphere of data was
scanned on w (0.308 per frame) with a run time of 20 s at the detector
resolution of 512 Â 512 pixel. Reflections were extracted by using the
SAINT program, and Lorentzian, polarization, and absorption
corrections were applied. Of 9398 reflections measured, 4506 were
unique (Rint ˆ 0.0657). The structure was solved by direct methods
(SHELXS-97) and refined by full-matrix least squares on F 2
(SHELXL-97). Anisotropic displacement parameters were used for
all non-hydrogen atoms. Hydrogen atoms were included in calculated
positions. 403 parameters, 18 restraints; R ˆ 0.0509 (3512 reflections,
F ꢀ 4sFo), Rw(F 2) ˆ 0.1007 (all reflections), and S ˆ 1.076. Max.
3
residual electron density 1.05 eŠ close to the Ir atom. Crystallo-
graphic data (excluding structure factors) for the structure reported in
this paper have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication no. CCDC-116623. Copies
of the data can be obtained free of charge on application to CCDC, 12
Union Road, Cambridge CB21EZ, UK (fax: (‡44)1223-336-033;
e-mail: deposit@ccdc.cam.ac.uk).
[11] A. A. Del Paggio, E. L. Muetterties, D. M. Heinekey, V. W. Day, C. S.
Day, Organometallics 1986, 5, 575.
[12] F. H. Antwi-Nsiah, O. Oke, M. Cowie, Organometallics 1996, 15, 1042.
[13] R. McDonald, M. Cowie, Inorg. Chem. 1990, 29, 1564.
[14] Z. Tang, Y. Nomura, Y. Ishii, Y. Mizobe, M. Hidai, Organometallics
1997, 16, 151.
Peptides 1 and 2 were characterized spectroscopically
(Table 1). Compared to H-APB-OH [lmax ˆ 420 nm (p ± p*)
[15] a) I. F. Urazowski, V. I. Ponomaryov, O. G. Ellert, I. E. Nifantꢁev,
D. A. Lemenovskii, J. Organomet. Chem. 1988, 356, 181; b) W. A.
[*] Prof. Dr. L. Moroder, Dipl.-Chem. R. Behrendt, Dr. C. Renner,
Dr. M. Schenk, Dr. F. Wang, Prof. Dr. D. Oesterhelt
Max-Planck-Institut für Biochemie
Á
King, S. Di Bella, A. Gulino, G. Lanza, I. L. Fragala, C. L. Stern, T.
Marks, J. Am. Chem. Soc. 1999, 121, 355.
[16] C. H. Winter, D. A. Dobbs, X.-X. Zhou, J. Organomet. Chem. 1991,
403, 145.
[17] a) J. Okuda, J. Organomet. Chem. 1988, 35b, C43; b) C. H. Winter,
X.-X. Zhou, M. J. Heeg, Inorg. Chem. 1992, 31, 1808.
Am Klopferspitz 18A, D-82152 Martinsried (Germany)
Fax : (‡ 49)89-8578-2847
E-mail: moroder@biochem.mpg.de
Dr. J. Wachtveitl
Institut für Medizinische Optik der Universität
Oettingenstrasse 67, D-80538 München (Germany)
[**] The study was supported by the SFB 533 (grant A8 Moroder/
Oesterhelt).
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Figure 1. Absorption spectra of 2 in DMSO (concentration 10 5 m, Tˆ
258C) before irradiation (0) and after irradiation (360 nm) with a light
2
intensity of 0.5 mWcm for 0.1, 0.25, 0.5, 0.6, 1, and 4 min. Quantitative
evaluation of the 1H NMR spectra yielded for the ground state a practically
100% trans-configured azobenzene and for the photostationary state (PSS)
a cis/trans isomer ratio of 78:22; the limits of error are Æ2 to Æ3%.
(Table 2). The difference in the activation energy of
1
24 kJmol represents the increased energy barrier of the
cis !trans transition resulting from the constrained conforma-
tional space of 2. The thermal isomerization of both peptides
Table 2. Rate constants kct of the cis !trans thermal isomerization of 1 and
2 in DMSO determined by 1H NMR spectroscopy at different temperatures
(peptide concentration 3 Â 10 3 m). The activation energy Ea was deter-
1
Scheme 1. Synthesis of 1 and 2: a) Couplings with HBTU/HOBt/DIEA (1/
1/2), except for Fmoc-Cys(StBu)-OH where HBTU/HOBt/DIEA (1/1/1.2)
was used; coupling of H-APB-OH without protection; Fmoc-removal with
20% piperidine in NMP; b) DIEA, CH3CON(SiMe3)2; Fmoc-Phe-F;
c) 20% piperidine in NMP; CH2Cl2/trifluoroethanol/AcOH/triethylsilane
(8/1/1/0.15); d) PyBOP/HOBt/DIEA (1/1/2); trifluoroacetic acid/CH2Cl2/
triethylsilane (95/3.5/1.5). HBTU ˆ O-(benzotriazol-1-yl)-N,N,N',N'-tetra-
methyluronium hexafluorophosphate, HOBt ˆ 1-hydroxybenzotriazole,
DIEA ˆ ethyldiisopropylamine, NMP ˆ N-methylpyrrolidone, PyBOP ˆ
benzotriazolyl-1-oxytris(pyrrolidino)phosphonium hexafluorophosphate.
mined from Arrhenius plots; the limits of error are Æ10 kJmol
.
1
1
Compound
kct [h
408C
]
Ea [kJmol ]
228C
308C
458C
508C
1
2
0.057
0.011
0.16
0.042
0.57
0.20
0.91
0.38
1.6
0.72
96
120
at 228C is markedly slower than that of the free azobenzene
derivative H-APB-OH, in which this process is completed in
about 10 min. Thus, the slow thermal relaxation of compound
1
Table 1. Spectroscopic parameters of 1 and 2 in DMSO. The extinction
coefficients (m 1 dm3 cm 1) were calculated from two isomeric mixtures; the
different cis/trans isomer ratios were determined quantitatively from
1H NMR spectra.
2 (t1/2 ˆ 62 h at 228C) allowed H NMR experiments to be
performed for the conformational analysis of the cis isomer.
The octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-
Gly-Phe-OH of the active site of thioredoxin reductase
retains in the oxidized form the identical 310-helix turn
structure as in the intact enzyme.[16] This indicates an intrinsi-
cally high tendency of the sequence portion Ala-Thr-Cys-Asp
for a turn conformation.[17] The main goal of our work was to
obtain a photoswitchable two-state system with a transition
from a rigid to a flexible peptide conformation. Therefore this
bis(cysteinyl)-peptide was selected for our model studies to
eventually allow for further conformational restriction
through disulfide bridging to the bicyclic form, if required.
As shown in Figure 2, already the monocyclic peptide with
trans-configured azobenzene moiety is fully constrained in its
conformational space. The azobenzene moiety is planar and
the peptide backbone displays an overall extended rigid
structure; only residue 5 exhibits dihedral angles of the a-
helical type. While in the trans isomer primarily a 1808 flip-
flop of the azobenzene moiety is observed, in the cis isomer
the free rotation of the two phenyl rings is restricted by
mutual hindrance and by steric effects of the peptide moiety.
Com-
pound
Configuration of the
azobenzene unit
p ± p*
lmax [nm] e  10
n ± p*
lmax [nm] e  10
4
3
1
1
2
2
trans
cis
trans
cis
370
260
370
260
2.0
1.3
3.4
2.4
450
450
450
450
4.7
5.2
2.8
3.6
and 440 nm (n ± p*)][14] the p ± p* transition in both the linear
and cyclic peptide is significantly blue-shifted, while the n ± p*
transition is located at similar wavelengths. The evolution of
the cis/trans photoisomerization upon irradiation at 360 nm
and 450 nm, respectively, shows for both the peptide 1 and 2
two isosbestic points at 330 and 440 nm which indicate that the
photoreaction follows a first-order kinetic, as exemplified for
2 in Figure 1. A comparison of the thermal isomerization rates
of the cyclic peptide 2 with those of the linear peptide 1
indicates an enhanced hindrance of the cis !trans isomer-
ization by the peptide backbone in the cyclized form
2772
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‡
5/95 to 80/20 in 30 min): tr ˆ 28 min; FAB-MS: m/z: 1320 [M‡Na ]; Mr ˆ
1297.4 calcd for C60H87N11O13S4; amino acid analysis (6m HCl; 1108C; 24 h):
Asp 1.00 (1) Thr 0.75 (1) Gly 1.00 (1) Ala 1.94 (2) Cys 1.64 (2) Phe 0.94 (1);
peptide content: 95%
2: The linear peptide 1 was cyclized with PyBOP/HOBt/DIEA in DMF and
the resulting compound was deprotected with trifluoroacetic acid/CH2Cl2/
triethylsilane (95/3.5/1.5). The crude product was precipitated with diethyl
ether and purified by extensive washings with MeOH; HPLC (Nucleosil
300/C18 (Machery & Nagel, Düren), linear gradient of acetonitrile/2%
H3PO4 from 5/95 to 80/20 in 30 min): tr ˆ 24.35 min; ESI-MS: m/z: 1168.4
‡
[M‡H ]; Mr ˆ 1167.2 calcd for C52H69N11O12S4; amino acid analysis (6m
HCl; 1108C; 24 h): Asp 1.01 (1) Thr 0.85 (1) Gly 1.00 (1) Ala 1.96 (2) Cys
1.65 (2) Phe 0.99 (1); peptide content: 93%.
UV spectra were recorded at a peptide concentration of 10 5 m in DMSO
on a Lambda 19 spectrometer (Perkin Elmer). A xenon lamp 450 XBO
(Osram, München) was used for irradiation at 360 nm (filter from Itos,
2
Mainz) with a light intensity of 0.5 mWcm
.
The 1H NMR experiments (500.13 MHz, 295 K) were recorded on a Bruker
AMX 500 spectrometer equipped with pulsed-field-gradient (PFG)
accessories. The signals were assigned according to the method of
Wüthrich.[19] The 2D TOCSY spectra were recorded with a spin-lock period
of 70 ms using the MLEV-17 sequence for isotropic mixing.[20] Experimen-
tal distance constraints (trans-azo: 44; cis-azo: 41) were extracted from 2D
NOESY experiments[21] with mixing times of 50 ms and 200 ms and from
2D ROESY experiments with a mixing time of 100 ms. Angle constraints
(trans-azo: 18; cis-azo: 12) were extracted from 2D DQF-COSY[22] and
simple 1H-1D spectra. Structure calculations and evaluations were
performed with the INSIGHTII (version 97.0) software package from
MSI by simulating the solvent DMSO with a dielectric constant of 46.7.
Structures were first generated by a distance geometry algorithm and
subsequently refined with a short simulated annealing molecular dynamics
protocol. The experimental constraints were applied at every stage of the
calculation. To check for the stability of the resulting structures, molecular
dynamics simulations over a period of 1 ns were performed for represen-
tative members of the structure ensembles. No significant violations of
experimental constraints occurred for any of the calculated structures.
Figure 2. Ensembles of the ten structures of lowest energy for cyclo(Ala-
Cys(StBu)-Ala-Thr-Cys(StBu)-Asp-Gly-Phe-APB) (2) in the trans (A) and
cis configuration (B).
Even the peptide backbone is relaxed in the cis isomer into a
significantly less constrained conformational space (Fig-
ure 2B). In its C-terminal peptide sequence a g-turn centered
on Gly-7 is largely populated, which fluctuates in an uncon-
certed mode with the rest of the peptide. The remaining part
of the peptide chain bulges into a loose turnlike structure with
a strongly 310-helical residue in position 4 and less helical Ala-
3 and Cys-5 residues, which reflects the intrinsic tendency of
this peptide portion to form an ordered fold. This makes the
cis isomer a frustrated system consisting of an ensemble of
energetically degenerate states. The N-terminus of the pep-
tide part finally forms a short turn to the azobenzene moiety.
By using 4-(4-(aminomethyl)phenylazo)benzoic acid as
building block in the cyclic octapeptide, as previously
proposed by Ulysse et al.,[9] the cis > trans photoisomeriza-
tion leads again to a conformational transition which, how-
ever, is less pronounced. In fact, under identical conditions the
peptide moiety, already in the trans-configured azobenzene,
exhibits two different conformations in a ratio of 3:1, as
determined by NMR conformational analysis. Conversely, by
using the chromophore APB that lacks the flexible methylene
spacer we obtained a peptide template, whose photoisome-
rization proceeds in a two-state transition. It therefore
represents a promising system for spectroscopic analysis of
this conformational transition on different time scales, also
because of the high quantum yields (Ftc ꢁ 20% and Fct ꢁ
50% for the n-p* transition).[18] Correspondingly, this tem-
plate should also allow photomodulation of biological proper-
ties of peptidic receptor ligands.
Received: February 25, 1999 [Z13081IE]
German version: Angew. Chem. 1999, 111, 2941 ± 2943
Keywords: azo compounds ´ conformation analysis ´ isomer-
izations ´ peptides ´ solid-phase synthesis
[1] I. Willner, S. Rubin, Angew. Chem. 1996, 108, 419 ± 439; Angew. Chem.
Int. Ed. Engl. 1996, 35, 367 ± 385.
[2] I. Willner, Acc. Chem. Res. 1997, 30, 347 ± 356.
[3] H. Rau in Studies in Organic Chemistry: Photochromism, Molecules
and Systems, Vol. 40 (Eds.: H. Dürr, H. Bonas-Laurent), Elsevier,
Amsterdam, 1990, pp. 165 ± 192.
[4] H.-W. Losensky, H. Spelthann, A. Ehlen, F. Vögtle, J. Bargon, Angew.
Chem. 1988, 100, 1225 ± 1227; Angew. Chem. Int. Ed. Engl. 1988, 27,
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[5] K. H. Neumann, F. Vögtle, J. Chem. Soc. Chem. Commun. 1988, 520 ±
522.
[6] M. S. Voller, T. D. Clark, C. Steinem, M. Z. Ghadiri, Angew. Chem.
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[8] T. Nagasaki, S. Tamagaki, K. Ogino, Chem. Lett. 1997, 717 ± 718.
[9] L. Ulysse, J. Cubillos, J. Chmielewski, J. Am. Chem. Soc. 1995, 117,
8466 ± 8467.
[10] S. Rudolph-Böhner, M. Krüger, D. Oesterhelt, L. Moroder, T. Nägele,
J. Wachtveitl, J. Photochem. Photobiol. A 1997, 105, 235 ± 248.
[11] G. Müller, M. Gurrath, H. Kessler, J. Comput. Aided Mol. Des. 1994, 8,
709 ± 730.
[12] H. R. Haubner, D. Finsinger, H. Kessler, Angew. Chem. 1997, 109,
1440 ± 1456; Angew. Chem. Int. Ed. Engl. 1997, 36, 1375 ± 1389.
Experimental Section
1: The linear peptide was synthesized by applying Fmoc/tBu chemistry on
chlorotrityl resin (PepChem, Tübingen) and was cleaved from this with
CH2Cl2/trifluoroethanol/AcOH/triethylsilane. HPLC (Nucleosil 300/C18
(Machery & Nagel, Düren), linear gradient of acetonitrile/2% H3PO4 from
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COMMUNICATIONS
residue (Tyr272), which is directly coordinated to a Cu2‡ ion
at the equatorial position, is covalently bound to the sulfur
atom of the adjacent Cys228, constituting a new organic
cofactor (Scheme 1).[2] The active form of GO has recently
been suggested to be the CuII ± phenoxyl radical of this
organic cofactor, which is converted into the two-electron
reduced species [CuI(phenol)] in the oxidation of alcohols to
aldehydes.[3±6]
[13] K. H. Schündehütte in Houben-Weyl, Methoden Org. Chem. (Hou-
ben-Weyl) 4th ed. 1965 ± , Vol. 10/3, p. 240.
[14] J. Wachtveitl, T. Nägele, B. Puell, W. Zinth, M. Krüger, S. Rudolph-
Böhner, D. Oesterhelt, L. Moroder, J. Photochem. Photobiol. A 1997,
105, 283 ± 288.
[15] T. Nägele, R. Hoche, W. Zinth, J. Wachtveitl, Chem. Phys. Lett. 1997,
272, 489 ± 495.
[16] L. Moroder, D. Besse, H.-J. Musiol, S. Rudolph-Böhner, F. Siedler,
Biopolym. Pept. Sci. 1996, 40, 207 ± 234.
[17] J. Kuriyan, T. S. R. Krishna, L. Wong, B. Guenther, A. Pahler, C. H.
Williams, Jr., P. Moldel, Nature 1991, 352, 172 ± 174.
[18] J. Wachtveitl, T. Nägele, A. J. Wurzer, M. Schenk, L. Moroder in
Springer Series in Chemical Physics, Vol. 63 (Eds.: T. Elsaesser, J. G.
Fujimoto, D. A. Wiersma, W. Zinth), Springer, Stuttgart, 1998,
pp. 609 ± 611.
[19] K. Wüthrich, NMR of Proteins and Nucleic Acids, Wiley, New York,
1986.
[20] A. Bax, D. G. Davis, J. Magn. Reson. 1985, 65, 355 ± 360.
[21] J Jeener, B. H. Meier, P. Bachman, R. R. Ernst, J. Chem. Phys. 1979,
71, 4546 ± 4553.
[22] M. Rance, O. W. Sorensen, G. Bodenhausen, G. Wagner, R. R. Ernst,
K. Wüthrich, Biochem. Biophys. Res. Commun. 1983, 117, 479 ± 485.
Scheme 1. Representation of the coordination at the CuII center of
galactose oxidase (left; the unlabeled O atom is from an acetate ion or
water), the organic cofactor (center), and the protonated form of the ligand
used in the model complexes 1 (right; py ˆ 2-pyridyl).
Extensive efforts have been made recently to develop
synthetic model complexes that can mimic the catalytic
function and the spectroscopic characteristics of the CuII ±
phenoxyl radical species of the native enzymes.[7±10] Wieghardt
et al. have succeeded in developing a very unique functional
model of GO using 2,2'-thiobis(4,6-di-tert-butylphenol) (LH2)
as the ligand; this complex can oxidize alcohols under aerobic
conditions.[7b] In this system the active species has been shown
Oxidation of Benzyl Alcohol with CuII and ZnII
Complexes of the Phenoxyl Radical as a Model
of the Reaction of Galactose Oxidase**
Shinobu Itoh,* Masayasu Taki, Shigehisa Takayama,
Shigenori Nagatomo, Teizo Kitagawa, Norio Sakurada,
Ryuichi Arakawa, and Shunichi Fukuzumi*
.
to be the dimeric phenoxyl radical complex [CuI2I(L )2]2‡, in
Redox interactions between a transition metal ion and a
redox-active amino acid side chain such as the phenol group
of tyrosine have been recognized to play a crucial role in
several biologically important processes.[1] Recently, a tyrosyl
radical directly coordinated to a CuII center has been
discovered in the active site of galactose oxidase (GO,
EC 1.1.3.9), which catalyzes the oxidation of d-galactose and
primary alcohols to the corresponding aldehydes, coupled to
the reduction of O2 to H2O2 [Eq. (1)].[1, 2] The crystal structure
of GO at 1.7-Š resolution clearly shows that the tyrosine
which only the radicals act as oxidation sites and no redox
reaction occurs at the CuII site. Now the important question
remains as to the essential role of the CuII site.
We report herein two synthetic model complexes whose
structures and spectroscopic features are similar to those of
the active form of GO. One is a CuII ± phenoxyl radical
complex that can accept two electrons, and the other is a
ZnII ± phenoxyl radical complex that can accept only one
electron. To our surprise, both model complexes can oxidize
benzyl alcohol to yield the same product, benzaldehyde. The
important difference between the two model complexes is
found to lie in the different kinetic formulation for the
oxidation of the alcohol. These features of our model
complexes provide valuable insights into the catalytic mech-
anism of GO.
RCH2OH ‡ O2 ! RCHO ‡ H2O2
(1)
[*] Dr. S. Itoh, Prof. S. Fukuzumi, M. Taki, S. Takayama
Department of Material and Life Science
Graduate School of Engineering, Osaka University
2 ± 1 Yamada-oka, Suita, Osaka 565-0871 (Japan)
Fax: (‡81)6-6879-7370
Addition of (NH4)2[CeIV(NO3)6] (ceric ammonium nitrate,
CAN) to a solution of the dimeric copper(ii) ± phenolate
complex [CuI2I(1)2](PF6)2 (2.5 Â 10 4 m in CH3CN)[11] caused an
immediate decrease in the absorption band at 522 nm due to
the dimeric copper(ii) ± phenolate complex (ligand-to-metal
charge-transfer (LMCT) transition from the phenolate to the
Cu2‡ ion), accompanied by the concomitant increase in the
E-mail: fukuzumi@chem.eng.osaka-u.ac.jp
Dr. S. Nagatomo, Prof. T. Kitagawa
Institute for Molecular Science
Myodaiji, Okazaki 444-8585 (Japan)
N. Sakurada, Prof. R. Arakawa
Department of Applied Chemistry
Faculty of Engineering, Kansai University
3-3-35 Yamate-cho, Suita, Osaka 564-8680 (Japan)
1
new absorption bands at 415 nm (e ˆ 1790m 1 cm ) and
1
867 nm (e ˆ 550m 1 cm ) at 258C (Figure 1). A similar
[**] This study was financially supported in part by a Grant-in-Aid for
Scientific Research on Priority Area (Molecular Biometallics:
10129218; Electrochemistry of Ordered Interface: 10131242) and a
Grant-in-Aid for General Scientific Research (08458177) from the
Ministry of Education, Science, Culture, and Sports of Japan.
spectrum was obtained in the oxidation of [ZnII(1)-
[11]
(CH3CN)]PF6
by CAN under the same experimental
conditions (418 nm (e ˆ 1250m 1 cm ) and 887 nm (e ˆ
1
510m 1 cm ) in CH3CN at 258C). The absorption bands in
1
2774
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
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Angew. Chem. Int. Ed. 1999, 38, No. 18