Isotopic probing of molecular oxygen activation at copper(I) sites

Article abstract of DOI:10.1021/ja074620c

Copper-dioxygen (CuO₂) adducts are frequently proposed as intermediates in enzymes, yet their electronic and vibrational structures have not always been understood. [Cu(η¹-O₂)TMG ₃tren]⁺ (TMG₃-tren = 1,1,1-tris{2-[N ²-(1,1,3,3-tetramethylguanidino)]ethyl}amine) features end-on (η¹) O₂ coordination in the solid state. Described here is an investigation of the compound's solution properties by nuclear magnetic resonance spectroscopy, density functional calculations, and oxygen isotope effects. The study yields two major findings. First, [Cu(η¹-O₂)TMG₃tren]⁺ is paramagnetic due to a triplet electronic structure; this is in contrast to other copper compounds where O₂ is bound in a side-on manner. Second, the oxygen equilibrium isotope effect upon O₂ binding to copper(I) ( ¹⁸O EIE = K(¹⁶O¹⁶O)/K(¹⁶O ¹⁸O) = 1.0148 ± 0.0012) is significantly larger than those determined for iron and cobalt η¹-O₂ adducts. This result is suggested to reflect greater ionic (Cu^II-O₂ ^-I) character within the valence bond description. A revised interpretation of the physical origins of the ¹⁸O EIEs upon O ₂ binding to redox metals is also advanced along with experimental data that should be used as benchmarks for interpreting ¹⁸O kinetic isotope effects upon enzyme reactions.

Full text of DOI:10.1021/ja074620c

Published on Web 10/26/2007
Isotopic Probing of Molecular Oxygen Activation at Copper(I)
Sites
Michael P. Lanci,† Valeriy V. Smirnov,† Christopher J. Cramer,‡
Ekaterina V. Gauchenova,§ Jo¨rg Sundermeyer,§ and Justine P. Roth*,†
Contribution from the Department of Chemistry, Johns Hopkins UniVersity, 3400 North Charles
Street, Baltimore, Maryland 21218, Department of Chemistry, Supercomputer Institute and
Center for Metals in Biocatalysis, UniVersity of Minnesota, 207 Pleasant Street Southeast,
Minneapolis, Minnesota 55410, and Fachbereich Chemie, Philipps-UniVersita¨t Marburg,
Hans-Meerwein-Strasse, 35032 Marburg, Germany
Received June 24, 2007; E-mail: jproth@jhu.edu
Abstract: Copper-dioxygen (CuO2) adducts are frequently proposed as intermediates in enzymes, yet
their electronic and vibrational structures have not always been understood. [Cu(η1-O2)TMG3tren]+ (TMG3-
tren ) 1,1,1-tris{2-[N2-(1,1,3,3-tetramethylguanidino)]ethyl}amine) features end-on (η1) O2 coordination in
the solid state. Described here is an investigation of the compound’s solution properties by nuclear magnetic
resonance spectroscopy, density functional calculations, and oxygen isotope effects. The study yields two
major findings. First, [Cu(η1-O2)TMG3tren]+ is paramagnetic due to a triplet electronic structure; this is in
contrast to other copper compounds where O2 is bound in a side-on manner. Second, the oxygen equilibrium
isotope effect upon O2 binding to copper(I) (18O EIE ≡ K(16O16O)/K(16O18O) ) 1.0148 ( 0.0012) is
significantly larger than those determined for iron and cobalt η1-O2 adducts. This result is suggested to
reflect greater ionic (CuII-O2-I) character within the valence bond description. A revised interpretation of
the physical origins of the 18O EIEs upon O2 binding to redox metals is also advanced along with experimental
data that should be used as benchmarks for interpreting 18O kinetic isotope effects upon enzyme reactions.
1. Introduction
instance, in enzymes CuO2 species have been proposed to
oxidize C-H bonds.4,5 Protein crystallography has provided
support for such intermediates, although assessing their involve-
ment during catalysis has remained a challenge.8,9
The coordination of molecular oxygen to a reduced iron or
copper site is a primary step in aerobic respiration,1 sensing of
cellular redox status,2 and catalysis by a wide variety of
enzymes.3-5 Mononuclear copper-dioxygen (CuO2) intermedi-
ates have only fairly recently been characterized, and much
remains to be understood regarding their reactivity.6-9 For
Our efforts have been concentrated on developing isotope
fractionation techniques to probe oxidation reactions of metal-
loenzymes and inorganic compounds.10-12 Presented here are
findings which demonstrate the ability to predict and identify
structures relevant to metal-mediated O2 activation. Preliminary
† Johns Hopkins University.
‡ University of Minnesota.
§ Philipps-Universita¨t Marburg.
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9
10.1021/ja074620c CCC: $37.00 © 2007 American Chemical Society
J. AM. CHEM. SOC. 2007, 129, 14697-14709
14697

A R T I C L E S
Lanci et al.
studies have suggested that oxygen equilibrium isotope effects
(18O EIEs) can be used to differentiate end-on (η1) superoxide
(O2-I) and side-on (η2) peroxide (O2-II) species;13,14 however,
the theoretical basis for these observations had not been
established.10,13
the same spin associated with the superoxide ligand. Another
possibility, although less commonly accepted, is copper(I) bound
to oxygen containing two unpaired electrons of the same spin.
The isotope fractionation technique is extended to the
characterization of CuO2 species, which are currently the focus
of synthetic modeling efforts.6,7,15,16 The relationship of experi-
mentally determined to computationally determined isotope
effects is described. Analysis within the context of the accepted
quantum mechanical theory sheds new light on the physical
origins of the 18O EIEs. The results can be used to interpret the
oxygen kinetic isotope effects (18O KIEs) which have now been
measured for a number of copper-containing enzymes.11,12
The oxygen isotope effect upon the equilibrium in eq 1 (18O
EIE ) K16,16O2/K16,18O2) is determined by the change in
vibrational frequencies of the reactant relative to the product.
We describe here how the experimental value determined for
[Cu(η1-O2)TMG3tren]+ can be reproduced remarkably well
using density functional calculations. The agreement between
theory and experiment validates our basic approach of using
isotope effects together with computational methods to identify
activated oxygen intermediates during enzyme catalysis.
Calculations are performed for other synthetic mononuclear
CuO2 compounds where the irreversible nature of O2 binding
has precluded 18O EIE measurements. On the basis of the
predicted values, end-on CuO2 structures should be easily
distinguished from the side-on structures. The analysis of
isotope-dependent partition functions reveals the underlying
enthalpic and entropic causes of the different 18O EIEs. The
emerging model provides a framework for future studies of
heavy-atom isotope effects on transition metal-mediated activa-
tion of various small molecules.11,12,22,23
We have examined the only synthetic copper compound
shown by X-ray crystallography to contain molecular oxygen
bound in an end-on fashion.15,16 The solid-state structure of [Cu-
(η1-O2)TMG3tren](SbF6) (TMG3tren ) 1,1,1-tris{2-[N2-(1,1,3,3-
tetramethylguanidino)]ethyl}amine) was recently determined
using cryogenic methods.15 These experiments were quite
challenging as the compound readily loses O2 in the solid state
at temperatures above 210 K. Through solution studies of the
reversible O2 binding equilibrium (eq 1) by nuclear magnetic
resonance (NMR) spectroscopy, we have discovered that [Cu-
(η1-O2)TMG3tren]+ is paramagnetic. Magnetic susceptibility
measurements on analytically pure samples at low temperatures
in N,N-dimethylformamide (DMF) indicate the presence of two
unpaired electrons. This observation is consistent with a triplet
ground state, although it is presently unclear where exactly the
electrons are localized. This unusual electronic structure for a
metal-oxygen adduct, although proposed on the basis of
computational studies,17-20 has not, to our knowledge, been
conclusively demonstrated by experiment.21 The triplet state is
most readily attributed to ferromagnetic coupling of an unpaired
electron associated with copper(II) and an unpaired electron of
2. Experimental Section
2.1. General. Specialty chemicals were obtained from Sigma-Aldrich
unless noted otherwise. Compressed gases were obtained from Airgas
in the highest purity available. “Anhydrous” brand DMF was obtained
from Burdick and Jackson and sparged with N2 prior to use. Other
solvents were obtained from EMD Chemicals and purified following
standard protocols.24 Deuterium-labeled compounds (tetramethylurea-
d12) and NMR solvents were obtained from Cambridge Isotope
Laboratories.
(13) (a) Lanci, M. P.; Roth, J. P. J. Am. Chem Soc. 2006, 128, 16006-16007.
(b) Lanci, M. P.; Brinkley, D. W.; Stone, K. L.; Smirnov, V. V.; Roth, J.
P. Angew. Chem., Int. Ed. 2005, 44, 7273-7276.
(14) The first indication that 18O EIEs could serve as probes of activated oxygen
structures derived from O2 was published several years earlier: Tian, G.;
Klinman, J. P. J. Am. Chem. Soc. 1993, 115, 8891-8897.
Copper compounds were synthesized as the triflate (OTf ) trifluo-
romethanesulfonate) salts according to the modified literature procedures
presented below and in the Supporting Information.25 Purity of the
compounds was assessed by elemental analysis (Desert Analytics,
Tucson, AZ), spectrophotometric determination of molar extinction
coefficients, and NMR spectroscopy. Samples for 18O EIE measure-
ments were prepared using a home-built vacuum apparatus that allowed
concurrent pressure measurements (manometry) on the O2 binding
reactions. The apparatus and procedures used to determine competitive
(15) Wurtele, C; Gaoutchenova, E.; Harms, K.; Holthausen, M. C.; Sundermeyer,
J.; Schindler, S. Angew. Chem., Int. Ed. 2006, 45, 3867-3869.
(16) Schatz, M.; Raab, V.; Foxon, S. P.; Brehm, G.; Schneider, S.; Reiher, M.;
Holthausen, M. C.; Sundermeyer, J.; Schindler, S Angew. Chem., Int. Ed.
2004, 43, 4360-4363.
(17) Most relevant to the present work are density functional calculations which
have predicted a ground-state triplet structure for a copper-oxygen adduct
isoelectronic and isostructural to that described in the present study. (a) de
la Lande, A.; Moliner, V.; Parisel, O. J. Chem. Phys. 2007, 126, 035102/
1-035102/7. (b) de la Lande, A.; Gerard, H.; Moliner, V.; Izzet, G.;
Reinaud, O.; Parisel, O. J. Biol. Inorg. Chem. 2006, 11, 593-608.
(18) An early ab initio study predicted a ground-state triplet for the product
formed upon reacting nickel(0) bis(alkyl)isocynanides with O2. Jørgensen,
K. A.; Swanstrøm, P. Acta Chem. Scand. 1992, 46, 82-86.
(19) The electronic structure proposed in ref 18 was later re-evaluated using
electron correlation methods and a ground-state singlet with low-lying triplet
states were found, consistent with the available experimental data. (a)
Bytheway, I.; Hall, M. B. Inorg. Chem. 1995, 34, 3741-3746. (b)
Bytheway, I.; Hall, M. B. Chem. ReV. 1994, 94, 639-658.
(22) Examples include isotopic studies of nitrogen fixation and O2 evolution
by photsystem II: (a) Sra, Amandeep, K.; Hu, Y.; Martin, G. E.; Snow, D.
D.; Ribbe, M. W.; Kohen, A. J. Am. Chem. Soc. 2004, 126, 12768-12769.
(b) Metzner, H.; Fischer, K.; Bazlen, O. Biochim. Biophys. Acta 1979, 548,
287-295. (c) Guy, R. D.; Fogel, M. L.; Berry, J. A. Plant Phys. 1993,
101, 37-47. (d) Burda, K.; Bader, K. P.; Schmid, G. H. Biochim. Biophys.
Acta: Bioenerg. 2003, 1557, 77-82.
(23) (a) Purdy, M. M.; Koo, L. S.; Ortiz de Montellano, P. R.; Klinman, J. P.
Biochemistry 2006, 45, 15793-15806. (b) Mure, M.; Mills, S. A.; Klinman,
J. P. Biochemistry 2002, 41, 9269-9278. (c) Stahl, S. S.; Francisco, W.
A.; Merkx, M.; Klinman, J. P.; Lippard, S. J. J. Biol. Chem. 2001, 276,
4549-4553. (d) Francisco, W. A.; Tian, G.; Fitzpatrick, P. F.; Klinman, J.
P. J. Am. Chem. Soc. 1998, 120, 4057-4062. (e) Tian, G.; Berry, J. A.;
Klinman, J. P. Biochemistry 1994, 33, 226-234.
(24) See the Supporting Information.
(25) Raab, V.; Kipke, J.; Burghaus, O.; Sundermeyer, J. Inorg. Chem. 2001,
40, 6964-6971.
(20) Palladium(I) η1-superoxide complexes, isoelectronic to the copper and nickel
dioxygen compounds in refs 17-19 and 21, have been proposed as
intermediates in the oxygenation of palladium(0) ligand complexes. On
the basis of density functional calculations these species are proposed to
have ground-state triplet structures. (a) Landis, C. R.; Morales, C. M.; Stahl,
S. S. J. Am. Chem. Soc. 2004, 126, 16302-16303. (b) Popp, B. V.;
Wendlandt, J. G.; Landis, C. R.; Stahl, S. S. Angew. Chem., Int. Ed. 2007,
46, 601-604.
(21) Chen, P.; Root, D. E.; Campochiaro, C.; Fujisawa, K.; Solomon, E. I. J.
Am. Chem. Soc. 2003, 125, 466-474. This reference describes the
possibility of a low-lying triplet excited state present in a solid-state sample
of a side-on bound CuO2 complex.
9
14698 J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007

Molecular Oxygen Activation at Copper(I) Sites
A R T I C L E S
oxygen isotope effects on reactions of natural abundance O2 have been
described elsewhere.10
H, H-CH), 65.3 (br s, 3 H, H-CH), 7.2 (br s, 3 H, H-CH), and -5.2
(br s, 3 H, H-CH); H NMR (76.8 MHz, DMF, 213 K) δ ) 20.8 (br
2
s, 9 H, CH3), 16.2 (br s, 9 H, CH3), 14.4 (br s, 9 H, CH3), and 6.2 (br
s, 9 H, CH3). The 2H resonances in [Cu(η1-O2)TMG3tren]+-d36 are
sharpened by approximately a factor of 7 relative to the 1H resonances
of [Cu(η1-O2)TMG3tren]+ at the same field. This sharpening is due to
the decreased gyromagnetic constant associated with deuterium.
2.3. Solution Magnetic Susceptibility Measurements.24,27 A stan-
dard solution containing a known concentration of internal standard
(HMDS) was prepared in DMF-d7 inside a N2-filled glove box. This
solution was partitioned and a portion used to dissolve a weighed
amount of [CuITMG3tren](OTf). Solutions with and without copper
were placed in separate vials, sealed with rubber septa under N2, and
chilled to the experimental temperature. The solutions were then
oxygenated by rapid stirring under air or O2 for at least 1 h.
The standard solution without copper was transferred by cannula
into a prechilled NMR tube. The coaxial insert (New Era Enterprises)
was subsequently placed inside this tube under the appropriate
atmosphere. The equilibrated [Cu(η1-O2)TMG3tren]+ solution was then
transferred into the coaxial insert and the tube sealed at 1 atm. Care
was taken to ensure that the samples remained at constant temperature
(213 K or 247 K) during all manipulations to avoid changes in the
concentration of O2.
The magnetic susceptibility, øg (mL/g), was calculated from the
frequency shift of HMDS (∆ν in Hz) and the concentration of [Cu-
(η1-O2)TMG3tren]+ (m in g/mL) according to eq 2.28 Control experi-
ments with tert-butanol and 1,4-dioxane as the internal standards
confirmed the nonspecific nature of the paramagnetic shift.29 The
concentration of [Cu(η1-O2)TMG3tren]+ was calculated on the basis
of the concentration of O2 at the experimental temperature30 and the
equilibrium constant for O2 binding to [CuITMG3tren]+ (KO2). The KO2
was determined independently by manometry as described below. The
temperature dependence of the DMF-d7 density was taken into
consideration through the empirically derived expression: d(g/mL) )
1.344 - 0.001012 × T(K).
NMR spectra were recorded using Varian Unity Inova 500 MHz
and Bruker Avance 300 MHz spectrometers. The temperature inside
the spectrometer was checked using Van Geet’s method.26 NMR
samples were prepared in tubes equipped with resealable Teflon valves
(J. Young). Solutions were saturated with the appropriate gas mixture
at the experimental temperature and ambient pressure. Magnetic
susceptibility measurements were performed following Evans’ method27
as detailed below. Chemical shifts are quoted in ppm versus the residual
solvent signals, and integrated peak areas are relative to an internal
standard of known concentration; hexamethyldisiloxane (HMDS) was
typically used for this purpose. Electron paramagnetic resonance (EPR)
spectra were recorded on X-band Bruker EMX and ESP 300E
spectrometers at temperatures between 4 and 230 K.
2.2. Synthesis and Characterization of Compounds. 2.2.1.
[CuITMG3tren](OTf). [CuITMG3tren]1+ was prepared by reacting bis-
[triflate copper(I)]‚benzene (90%) (231 mg, 458 mmols) with TMG3-
tren (430 mg, 989 mmols) in anhydrous, deoxygenated acetonitrile.
The copper(I) triflate solution (8 mL) was added dropwise to the ligand
solution (8 mL) at room temperature. The reaction mixture, after stirring
for 45 min, appeared pale yellow. This solution was filtered through
Celite and concentrated to 4 mL. The filtrate was layered with 30 mL
of diethyl ether. Slow diffusion of the ether layer afforded colorless
crystals in 79% yield. 1H NMR (300 MHz, DMF-d7, 298 K) δ ) 3.26
(vt, 6 H, CH2), 2.78 (s, 18 H, CH3), 2.71 (s, 18 H, CH3), 2.64 (vt, 6 H,
CH2); 13C{1H} NMR (75.5 MHz, DMF-d7, 298 K) δ ) 160.0 (CN3),
50.7 (CH2), 47.1 (CH2), 37.5 (CH3), 37.4 (CH3). Elemental analysis:
calcd for C22H48N10O3F3SCu: 40.45 C, 7.41 H, 21.44 N; found: 40.58
C, 7.07 H, 21.35 N.
[CuITMG3tren](OTf)-d36 was prepared from TMG3tren-d36 as de-
scribed above. The isolated yield based upon copper was 81%; 1H NMR
(500 MHz, DMF-d7, 298 K) δ ) 3.26 (vt, 6 H, CH2), 2.63 (vt, 6 H,
2
CH2); H NMR (76.8 MHz, DMF, 298 K) δ ) 2.73 (s, 18 H, CH3),
2.65 (s, 18 H, CH3); 13C{1H} NMR (75.5 MHz, DMF-d7, 298 K) δ )
160.1 (CN3), 120.2 (quartet, 1JC-F ) 322.7 Hz, S-C-F3), 50.8 (s, CH2),
47.0 (s, CH2), 37.5 (septet, 1JC-D ) 20.8 Hz, CD3), 37.4 (septet, 1JC-D
) 20.7 Hz, CD3). Elemental analysis: calcd for C22H12D36N10O3F3-
SCu: 38.32 C, 7.02 D, 20.31 N; found: 38.04 C, 6.78 D, 20.31 N.
This analytical method does not distinguish deuterium from protium.
2.2.2. [CuIITMG3tren](OTf)2. [CuIITMG3tren]2+ was prepared using
CuII(OTf)2 and the procedure outlined for [CuITMG3tren](OTf). Emerald
green crystals were obtained in 80% yield. Elemental analysis: calcd
for C25H51N10O6F6S2Cu: 35.60 C, 6.09 H, 18.27 N; found: 35.97 C,
6.18 H, 18.24 N. The following extinction coefficients were determined
in DMF at 298 K: ꢀ388 nm ) 960 ( 170 M-1 cm-1 (shoulder), ꢀ773 nm
3
4π
∆ν 1
øg ) -
(2)
(
)( )( )
ν
m
The molar magnetic susceptibility, øm (mL/mol), was calculated from
øg and the molecular weight of [Cu(η1-O2)TMG3tren]+ (536.24 g/mol).
Corrections for the diamagnetic contribution, ødm, associated with [Cu-
(η1-O2)TMG3tren]+, [CuITMG3tren]+, and the triflate counterions, were
applied in order to obtain the molar paramagnetic susceptibility, øpm.31
The øpm values were then used to estimate the effective magnetic
moment, µeff, in units of the Bohr magneton (â), according to eq 3; the
calculation includes the temperature (T), Boltzmann’s constant (k), and
Avogadro’s number (NA).
) 73 ( 15 M-1 cm-1 and ꢀ1056 ) 300 ( 53 M-1 cm-1
.
nm
2.2.3. [Cu(η1-O2)TMG3tren](OTf). [Cu(η1-O2)TMG3tren]+ was
generated in situ by reacting a solution of analytically pure [CuITMG3-
tren]1+ with O2 at low temperature. In a typical experiment, a weighed
amount of the copper(I) compound was dissolved in the appropriate
solvent under N2 and cooled to the experimental temperature. An NMR
spectrum was obtained, and the diamagnetic signals were quantified
versus an internal standard. Exposure to O2 caused the solution to turn
from colorless to green. Analysis of the NMR spectrum at this point
revealed paramagnetically shifted and broadened signals with the
following integral areas: 1H NMR (300 MHz, DMF-d7, 213 K) δ )
301 (br s, 3 H, H-CH), 67.1 (br s, 3 H, H-CH), 21.9 (br s, 9 H,
CH3), 16.2 (br s, 9 H, CH3), 15.2 (br s, 9 H, CH3), 7.2 (br s, 3 H,
H-CH), 6.2 (br s, 9 H, CH3), and -5.5 (br s, 3 H, H-CH).
3kTøpm
µeff
)
(3)
N â
x
A
2.4. Procedure for Measuring Equilibrium Isotope Effects. The
oxygen equilibrium isotope effect is defined as the ratio of equilibrium
constants for the binding of 16,16O2 and 16,18O2 to [CuITMG3tren]+
according to eq 1, i.e., K(16O16O)/K(16O18O). This ratio was determined
by analyzing the isotope composition of the O2 released from [Cu(η1-
O2)TMG3tren]+ using an isotope ratio mass spectrometry method. The
detailed procedure has been published10,11,13,14 and a brief description
is provided below.
Spectral assignments were confirmed by analysis of [Cu(η1-
O2)TMG3tren]+-d36 in which the methyl groups were labeled with
(28) Sur, S. K. J. Magn. Reson. 1989, 82, 169-173.
(29) Bertini, I.; Luchinat, C.; Turano, P.; Battaini, G.; Casella, L. Chem. Eur.
J. 2003, 9, 2316-2322.
(30) The solubility of O2 was determined at varying temperatures in DMF as
described in ref 13a.
1
deuterium. H NMR (500 MHz, DMF-d7, 213 K) δ ) 294.9 (br s, 3
(26) Van Geet, A. L. Anal. Chem. 1970, 42, 679-680.
(27) Evans, D. F. J. Chem. Soc. 1959, 2003-2005.
(31) O’Connor, C. J. Progress in Inorganic Chemistry; Lippard, S. J., Eds.;
Wiley & Sons: New York, 1982; pp 203-213.
9
J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007 14699

A R T I C L E S
Lanci et al.
Samples of [CuITMG3tren]OTf (0.6-8.0 mM) were pre-equilibrated
in air-saturated DMF solutions at low temperatures. The O2 was isolated
before and after adding the copper(I) compound. Thus, the 18O:16O
content of the O2 derived from [Cu(η1-O2)TMG3tren]+ could be
determined relative to the 18O:16O present in air at natural abundance
levels. The solutions containing copper reached equilibrium rapidly,
yet they were typically maintained at the experimental temperature for
1-5 h prior to collection of the O2. Quantitative isolation of O2 was
achieved by sparging a fixed volume of solution with helium under
dynamic vacuum. The O2 in the helium carrier gas was passed through
a series of traps cooled with liquid nitrogen to remove volatile solvent,
water vapor, and CO2. The O2 was collected on 5 Å molecular sieves,
and the helium was removed by evacuation. The O2 was then
quantitatively converted to CO2 by combustion at 900 °C over graphite
with a platinum catalyst. The pressure of the CO2 was determined using
a capacitance manometer, and then the gas was flame sealed in a dry
glass sample tube for later isotope ratio mass spectrometry (IRMS)
analysis. Isotope effects were calculated according to eq 4. The IRMS
afforded 18O:16O versus a standard (e.g., standard mean ocean water,
SMOW) with a typical error of (0.0002 (0.2 ppm).
The approach of Bigeleisen and Goeppert-Mayer32 was used to
calculate the 18O EIEs from the partition functions associated with the
16O16O and 16O18O isotopologues (eq 7). These isotope-dependent
partition functions corresponding to zero-point energy (ZPE), vibrational
excited-state energy (EXC), and the mass and moments of inertia (MMI)
can be expressed in terms of stretching frequencies (eqs 8-10). The
definition of MMI as a vibrational product (VP) derives from the
Redlich-Teller product rule; the application of which requires using
all of the vibrational modes.33 MMI can also be calculated according
to a classical expression (eq 11) which is based only on the masses
(M) and the rotational moments of inertia (I). The latter values as well
as the stretching frequencies were determined from density functional
calculations on fully optimized geometries.
The precision of the measurements owes to the fact that the 18O
EIE comes from a ratio of ratios. In eq 4, Rt is the 18O:16O in the “total”
O2 isolated from the [Cu(η1-O2)TMG3tren]+-containing solutions. Ru
is the 18O:16O of the “unbound” O2 isolated from solutions that do not
contain copper. The 1 - f is the fraction of the total O2 which is bound
to copper at equilibrium. Measurements were performed using solutions
freshly prepared from different lots of solvent and [CuITMG3tren](OTf).
The 18O EIE at each temperature was determined from multiple
independent measurements and reported with an error reflecting one
standard deviation about the mean.
18O EIEcalc ) MMI × EXC × ZPE
(7)
(hνjB*/2kT)
3N-6 exp
∏
(hνjB/2kT)
[
[
]
]
j
exp
ZPE )
(8)
(hνiA*/2kT)
3N-5 exp
∏
(hνiA/2kT)
i
exp
K(16O16O)
1 - f
3N-6
B*/kT)
1 - exp-(hν
1 - exp-(hν
18O EIE )
)
(4)
j
K(16O18O)
Rt/Ru - f
∏
B
[
[
]
]
/kT)
j
j
EXC )
(9)
2.5. Calculation of Equilibrium Isotope Effects. For the end-on
binding of O2 to copper(I) in eq 1 where K(O2) )[CuO2]/[CuI][O2] the
18O EIE is defined according to eq 4. Under the experimental conditions,
the 16,16O2 and 16,18O2 isotopologues of O2 react competitively with
copper(I). Thus, the 18O EIE is determined from the equilibrium
concentrations of “bound” and “unbound” O2 according to:
-(hνiA*/kT)
3N-5 1 - exp
∏
A
1 - exp-(hν
/kT)
i
i
3N-6
(νjB/νjB*
)
∏
[Cu16,16O2] [Cu16,18O2] [Cu16,16O2][16,18O2]
j
18O EIE )
)
(5)
MMI ) VP )
(10)
(11)
16,16O2]
[
16,18O2]
[Cu16,18O2][16,16O2]
3N-5
[
/
(νiA/νiA*
)
∏
i
The right part of eq 5 expresses the equilibrium constant for an isotope
exchange reaction, such as that shown in eq 6a. When a side-on CuO2
product is formed and the oxygen nuclei are equivalent by symmetry,
only one isotope exchange reaction is required.
nrot
3/2
1/2
1/2
M16-16
Ii,16-16
∏
(
)
(
)
[
]
M16-18
Ii,16-18
i
final
MMI )
nrot
3/2
M16-16
M16-18
Ii,16-16
∏
(
[
)
(
)
]
Ii,16-18
i
initial
When an end-on CuO2 product is formed, it is necessary to consider
two isotope exchange reactions, as shown in eqs 6b and 6c.32 The initial
and final states of the O2 are designated A and B, respectively, with
the asterisk marking the site of the heavy isotope. The two equilibria
differ with respect to the identity of B*, i.e. whether O2 is bound to
copper through 16O or 18O. Throughout this study, the simplifying
assumption is made that B* consists of a 50/50 mixture of Cu-16O18O
and Cu-18O16O. Neglecting a small (ca. 1%) isotope effect due to the
preferred coordination of 16O to the copper causes the calculated 18O
EIE to be slightly underestimated, the variation occurring in the fourth
decimal place.
2.6. Density Functional Calculations. Molecular geometries were
fully optimized at the density functional level of theory (DFT) using
the exchange and correlation functionals of Perdew and co-workers34
as modified by Adamo and Barone (mPWPW91).35 Atomic orbital basis
functions were taken for Cu from the compact relativistic effective core
potential basis CEP-31G,36 for N and O from the 6-311G* basis, for C
from the 6-31G basis, and for H from the minimal STO-3G basis.37
(33) Wilson, E. B., Jr.; Decius, J. C.; Cross, P. C. Molecular Vibrations;
Dover: New York, 1980; pp 182-193.
(34) (a) Perdew, J. P.; Wang, Y. Phys. ReV. B 1986, 33, 8800-8802. (b) Perdew,
J. P. In Electronic Structure of Solids ’91; Ziesche, P., Eschrig, H., Eds.;
Akademie Verlag: Berlin, 1991; pp 11-20.
(32) Bigeleisen, J.; Goeppert-Mayer, M. J. Chem. Phys. 1947, 15, 261-267.
(35) Adamo, C.; Barone, V. J. Chem. Phys. 1998, 108, 664-675.
9
14700 J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007

Molecular Oxygen Activation at Copper(I) Sites
A R T I C L E S
Figure 1. 1H NMR spectrum (500 Mhz) of [CuI(TMG3tren)]1+ in DMF-d7 at 298 K.
Unrestricted Kohn-Sham (KS) DFT was used for the triplet states
as well as for the broken-symmetry (BS) states that mix singlet and
triplet character for equal numbers of R and â electrons (such states
are sometimes called unrestricted singlets, but this is a misnomer
since the spin state is usually far from pure).38 In order to eliminate
triplet spin contamination from the broken-symmetry KS self-consistent
field (SCF) energies, purified singlet energies, were computed according
to:39
3. Results
3.1. Characterization of Reversible O2-Binding. [CuITMG3-
tren]+ undergoes rapid and reversible oxygenation in solvents
of varying polarity. The O2 affinity increases in proportion to
the solvent dielectric constant with KO2 ∼14 times greater in
DMF (ꢀ ) 36.7) than in chlorobenzene (ꢀ ) 2.7) at 247 K.43
Similar behavior has been reported for cobalt(II) Schiff’s base
and porphyrin compounds which also form η1-O2 structures.44,45
In the present study, NMR spectral changes reflecting the
formation of the paramagnetic [Cu(η1-O2)TMG3tren]+ com-
pound were correlated to O2 uptake using manometry.
2E )0 - S2 E
Sz
Sz )1
Esinglet
)
(12)
2 - S2
3.1.1. 1H and 2H NMR Studies. There are eight chemically
inequivalent protons on each arm of the tripodal ligand in the
static structure of [CuI(TMG3tren)]+, yet the 1H NMR spectrum
in DMF-d7 at 293 K reveals only four resonances: two broad
multiplets and two singlets integrating 6:6 and 18:18, respec-
tively (Figure 1). The spectrum arises from rapid equilibration
of the two sets of diastereotopic methylene protons and methyl
groups on the NMR time scale. Consistent with this explanation,
lowering the temperature results in broadening of the signals
and eventual decoalescence. The diastereotopic methylene
protons exhibit an a,a′,b,b′ pattern, and the methyl groups appear
as a c,c′,d,d′ pattern at 213 K.24 The [CuI(TMG3tren)]+-d36
compound with deuterium labeling at each methyl group
behaves in the same manner, as indicated by analysis of the 1H
The triplet energy is computed for the single-determinantal high-spin
configuration Sz ) 1 (at the BS geometry), and S2 is the expectation
value of the total-spin operator applied to the KS determinant for the
unrestricted BS Sz ) 0 calculation.
For comparison purposes, restricted KS DFT was also carried out
for singlet states; the restricted KS SCF solutions were unstable to spin-
symmetry, breaking in every case. Vibrational frequencies were
calculated analytically within the harmonic oscillator approximation.38
All minima were characterized by real frequencies24 unless noted
otherwise.
Solvation effects were included in certain calculations using the
polarized continuum model40 (PCM) in its integral-equation formalism41
(IEF-PCM). Default parameters for acetone as implemented in Gaussian
0342 were chosen because the experimental resonance Raman data for
[Cu(η1-O2)TMG3tren]+ have been reported in this solvent.16
2
and H NMR spectra.24
(36) Stevens, W. J.; Krauss, M.; Basch, H.; Jasien, P. G. Can. J. Chem. 1992,
70, 612-629.
Exposure of the copper(I)-containing DMF-d7 solution to O2
(37) Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab Initio Molecular
Orbital Theory; Wiley: New York, 1986.
1
at 273 K results in the disappearance of the characteristic H
NMR spectrum due to [CuITMG3tren]+ and the appearance of
broad signals in the diamagnetic region. Lowering the temper-
ature shifts the equilibrium to favor [Cu(η1-O2)TMG3tren]+,
revealing several signals between -6 and 300 ppm at 213 K
(Figure 2). Assignments of these signals were made through
comparisons to the compound with deuterium-labeled methyl
(38) Cramer, C. J. Essentials of Computational Chemistry, 2nd ed.; Wiley:
Chichester, 2004.
(39) (a) Ziegler, T.; Rauk, A.; Baerends, E. J. Theor. Chim. Acta 1977, 43,
261-271. (b) Noodleman, L.; Norman, J. G. J. Chem. Phys. 1979, 70,
4903-4906. (c) Yamaguchi, K.; Jensen, F.; Dorigo, A.; Houk, K. N. Chem.
Phys. Lett. 1988, 149, 537-542. (d) Lim, M. H.; Worthington, S. E.; Dulles,
F. J.; Cramer, C. J. In Chemical Applications of Density Functional Theory;
Laird, B. B., Ross, R. B., Ziegler, T., Eds.; American Chemical Society:
Washington, DC, 1996; Vol. 629, pp 402-422. (e) Isobe, H.; Takano, Y.;
Kitagawa, Y.; Kawakami, T.; Yamanaka, S.; Yamaguchi, K.; Houk, K. N.
Mol. Phys. 2002, 100, 717-727.
1
groups on the ligand. The H spectrum of [Cu(η1-O2)TMG3-
tren]+-d36 consists of four diastereotopic methylene protons, and
(40) (a) Miertus, S.; Scrocco, E.; Tomasi, J. Chem. Phys. 1981, 55, 117-129.
(b) Tomasi, J. In Structure and ReactiVity in Aqueous Solution; Cramer,
C. J., Truhlar, D. G., Eds.; American Chemical Society: Washington, DC,
1994; Vol. 568, pp 10-23.
(43) Lide, D. R. CRC Handbook of Chemistry and Physics, 74th ed.; CRC
Press: Boca Raton, 1993. The dielectric constants of DMF and acetone
are 38.9 and 21, respectively, at 298.15 K.
(44) Trovog, B. S.; Kitko, D. J.; Drago, R. S. J. Am. Chem. Soc. 1976, 98,
5144-5153.
(45) Walker, F. A. J. Am. Chem. Soc. 1973, 95, 1154-1159.
(41) (a) Cance`s, E.; Mennucci, B.; Tomasi, J. J. Chem. Phys. 1997, 107, 3032-
3041. (b) Mennucci, B.; Tomasi, J. J. Chem. Phys. 1997, 106, 5151-5158.
(c) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. J. Chem. Phys. 2002,
117, 43-54.
(42) Frisch, M. J.; et al. Gaussian 03; Gaussian, Inc.: Pittsburgh, PA, 2003.
9
J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007 14701

A R T I C L E S
Lanci et al.
Figure 2. 1H NMR spectra (300 Mhz) revealing the increased concentration of [Cu(η1-O2)TMG3tren]+ in O2-saturated DMF-d7 as the temperature is
lowered from 273 to 213 K. The internal standard, hexamethyldisiloxane (HMDS) is noted. Only the region between 28 and -9 ppm is shown. Additional
signals are observed at ∼65 and 300 ppm.
Figure 3. (Top) 2H NMR spectrum (76.8 MHz) of [Cu(η1-O2)TMG3tren]+-d36 in DMF at 213 K. (Bottom) 1H NMR spectrum (500 MHz) of the same
compound in DMF-d7 at 213 K. Hexamethyldisiloxane (HMDS) is noted. The asterisks designate residual deuterium in DMF.
2
the H spectrum consists of four diastereotopic methyl groups
at 213 K (Figure 3). The latter signals are approximately 7 times
sharper than those of the per-protio analogue (as expected, due
spectra of [CuITMG3tren]+ and [Cu(η1-O2)TMG3tren]+ are
unchanged by the addition of [CuIITMG3tren]2+ 24
In addition,
.
[Cu(η1-O2)TMG3tren]+ exhibits no discernible EPR signal at
X-band frequency, under conditions where [CuIITMG3tren]2+
can be readily quantified.24,46 While the absence of an EPR
signal is a negative result, it is consistent with the proposed
triplet state of [Cu(η1-O2)TMG3tren]+. Integer spin systems can
be difficult to observe at X-band frequency due to the large
zero-field splitting associated with dipole-dipole interaction of
the unpaired electrons.47
2
1
to smaller gyromagnetic constant of H relative to H).
Importantly, the spectral changes associated with forming
[Cu(η1-O2)TMG3tren]+ are completely reversed upon removing
O2 from the sample.24 Integration of the NMR signals versus
an internal standard reveals that [CuITMG3tren]+ is recovered
in 94 ( 6% yield. This observation indicates that a hypothetical
CuII-containing decomposition product can be ruled out as the
cause of the paramagnetically shifted and broadened NMR
spectra (cf. Figures 2 and 3). The NMR results were cor-
roborated by manometry under the analogous conditions. For
instance, samples prepared at low O2 concentration (0.2 mM)
contained resolvable signals due to [Cu(η1-O2)TMG3tren]+ and
[CuITMG3tren]+.24 The equilibrium constant determined by
NMR integration, KO2 ) 3030 ( 1200 M-1 agreed reasonably
well with the KO2 ) 4340 ( 600 M-1 determined from the O2
pressure measurements.
3.1.2. Solution Magnetic Susceptibility. Measurements
employing Evans’ method27,28 indicate that [Cu(η1-O2)TMG3-
tren]+ exhibits a magnetic moment that is relatively insensitive
to temperature (Figure 4). Values of µeff ) 3.21 ( 0.26 µB (213
K) and 3.15 ( 0.25 µB (247 K) were obtained by measuring
the paramagnetic shift associated with the oxygenated copper
compound. Analyses were performed in the presence of O2 and
the diamagnetic [CuITMG3tren]+ as outlined in Experimental
Section 2.3. The paramagnetic susceptibility of [Cu(η1-O2)TMG3-
tren]+ is only slightly smaller in the absence of the diamagnetic
correction: µeff,uncorr ) 3.12 ( 0.27 µB at 213 K and 3.04 (
The presence of a significant concentration of [CuIITMG3-
tren]2+, which could potentially form together with unbound
superoxide (O2•-) upon oxygenation of [CuITMG3tren]+, can
also be ruled out as the cause of the paramagnetic NMR
spectrum. Evidence to this effect includes the observation that
the peak positions as well as the line widths within the 1H NMR
(46) Gauchenova, E. V. Chemie superbasischer Chelatliganden fu¨r H+, Cu+ und
Mn2+. Ph.D. Thesis, Fachbereich Chemie, Philipps-Universita¨t Marburg,
Hans-Meerwein-Strasse, 35032 Marburg, Germany 2006.
(47) Drago, R. S. Physical Methods for Chemists, 2nd ed.; Surfside Scientific
Publishers: Gainesville, 1992; pp 390-392.
9
14702 J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007

Molecular Oxygen Activation at Copper(I) Sites
A R T I C L E S
Figure 5. Oxygen equilibrium isotope effects (18O EIEs) upon the formation
of [Cu(η1-O2)TMG3tren]+ in DMF (eq 1). The red curve represents values
calculated in the gas phase, and the blue curve represents values calculated
using the polarized continuum model (PCM) and eqs 7-10.
Figure 4. Determination of the solution magnetic moment of [Cu(η1-
O2)TMG3tren]+. Data at 213 K and 247 are shown on one correlation line
to illustrate the temperature insensitivity of µeff.
0.26 µB at 247 K. The average magnetic moment is within error
of the spin-only value of 2.83 µB calculated for a ground-state
triplet species. A similar µeff ) 3.02 ( 0.25 µB has been
estimated in acetone at 190 K under the assumption that [Cu-
(η1-O2)TMG3tren]+ is formed quantitatively from [CuITMG3-
tren]+ and O2.46
conservative error limits, reflecting the precision of all manipu-
lations,10 were estimated from repeated measurements upon
solutions containing natural abundance O2. Data collected
between 213 and 273 K indicate an average 18O EIE ) 1.0148
( 0.0012. This value is within the errors of the individual 18
O
3.1.3. Temperature Dependence of the Equilibrium Con-
stant. Equilibrium constants corresponding to the formation of
[Cu(η1-O2)TMG3tren]+ were determined by manometry.24 KO2
at each temperature was extracted from several independently
prepared samples. Pressure changes were related to the moles
of O2 taken up upon addition of a known concentration of [CuI-
TMG3tren]+ to air-saturated DMF. The O2 obtained from the
pre-equilibrated solutions varied in response to the concentration
of [CuITMG3tren]+ as expected for a 1:1 adduct. The manometry
results could not be fitted to the equilibrium expression for a
2:1 adduct, consistent with [Cu(η1-O2)TMG3tren]+ as the only
copper-oxygen adduct present in solution. This observation is
also consistent with the NMR analysis described above.
The temperature dependence of KO2 was analyzed between
213 and 273 K. The ∆H ) -8.4 ( 0.8 kcal mol-1 and ∆S )
-23 ( 3 cal mol-1 K-1 (at 1 atm O2) appear to be typical for
formation of η1-O2 structures irrespective of the solvent
polarity.13a,48 The reaction exothermicity and competing unfa-
vorable reaction entropy accounts for the common observation
that end-on O2 binding to copper(I) and cobalt(II) is rendered
favorable by lowering the temperature.44,45,48
3.1.4. Equilibrium Isotope Effect upon O2 Binding. 18O
EIEs were determined from the 18O-enrichment of [Cu(η1-
O2)TMG3tren]+ in solutions equilibrated with natural abundance
O2 (Figure 5). Measurements were performed by equilibrating
samples at the experimental temperature and then isolating all
of the O2 from the copper-containing solutions. The yield of
O2 in these experiments was typically 97 ( 9% based upon the
initial concentration of [Cu(η1-O2)TMG3tren]+ estimated from
the NMR spectrum.
EIEs determined at the temperature extremes.
3.2. Density Functional Calculations. Optimized structures
of [Cu(η1-O2)TMG3tren]+ were determined for the unrestricted
triplet, restricted singlet, and unrestricted broken-symmetry
states. Calculations assuming a restricted singlet state had
previously been used to corroborate the assignment of the
resonance Raman spectrum (see below).16 Frequency calcula-
tions on the energy-minimized structures afforded several
isotopically sensitive vibrations which were used in eqs 8-10
without scaling. The full sets of the calculated frequencies are
provided in the Supporting Information.
The calculated energy of the triplet [Cu(η1-O2)TMG3tren]+
is 16.3 kcal mol-1 lower than that of the restricted singlet state
and 7.3 kcal mol-1 lower than that of the broken-symmetry state.
The biradical nature of a Cu(II) superoxide species makes the
accurate calculation of the state-energy splitting quite difficult
because the biradical singlet cannot be represented by a single
Kohn-Sham determinant.49-52 Therefore, while experiment
clearly indicates the triplet to be dominant at the temperatures
studied, it is unlikely that the DFT calculations provide a
reliable, quantitative measure of that preference.
Calculations were performed on the compounds in Figure 6
for comparison to [Cu(η1-O2)TMG3tren]+. Relevant structural
results are presented in Table 1 for the mixed and triplet states.
The calculations indicate longer O-O bond distances and lower
O-O stretching frequencies (νO-O) for the side-on structures
relative to those of the end-on structures.53 The results are
arranged to reflect the extent of O-O reduction, with [Cu(η1-
The measurements of 18O EIEs rely upon precise determi-
nation of O2 pressure changes and the 18O:16O ratios (cf. eq 4).
The error in the IRMS analysis is very small ((0.0002). More
(49) Gherman, B. F.; Cramer, C. J. Inorg. Chem. 2004, 43, 7281-7283.
(50) Aboelella, N. W.; Kryatov, S.; Gherman, B. F.; Brennessel, W. W.; Young,
V. G.; Sarangi, R.; Rybak-Akimova, E.; Hodgson, K. O.; Hedman, B.;
Solomon, E. I.; Cramer, C. J.; Tolman, W. B. J. Am. Chem. Soc. 2004,
126, 16896-16911.
(51) Gherman, B. F.; Heppner, D. E.; Tolman, W. B.; Cramer, C. J. J. Biol.
Inorg. Chem. 2006, 11, 197-205.
(48) (a) Rybak-Akimova, E. V.; Otto, W.; Deardorf, P.; Roesner, R.; Busch, D.
H. Inorg. Chem. 1997, 36, 2746-2753. (b) Drago, R. S.; Cannady, J. P.;
Leslie, K. A. J. Am. Chem. Soc. 1980, 102, 6014-6019. (c) Fry, H. C.;
Scaltrito, D. V.; Karlin, K. D.; Meyer, G. J. Am. Chem. Soc. 2003, 125,
11866-11871.
(52) Kinsinger, C. R.; Gherman, B. F.; Gagliardi, L.; Cramer, C. J. J. Biol.
Inorg. Chem. 2005, 10, 778-789.
(53) Cramer, C. J.; Tolman, W. B.; Theopold, K. H.; Rheingold, A. L. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 3635-3640.
9
J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007 14703

A R T I C L E S
Lanci et al.
O2)TMG3tren]+ by the polar solvents. Although difficult to
quantify, the increased charge transfer within CuII-O2-I would
be expected to lower the νO-O due to population of antibonding
orbitals.
To probe the influence of solvent dielectric, we recomputed
the frequencies for a triplet structure of [Cu(η1-O2)TMG3tren]+
reoptimized while including continuum acetone solvation effects
within the polarized continuum model (PCM). Consistent with
our hypothesis, the polar solvent enhanced charge transfer to
O2 which was manifested in an increased bond length (0.006
Å) and decreased stretching frequency (now 1183 cm-1
compared to 1218 cm-1 in the gas phase). While this does not
completely resolve the difference between theory and experi-
ment, it does substantially improve the agreement between the
predicted O-O stretching frequency as well as the predicted
EIE (vide infra). The remaining error might be associated with
ion-pairing to the counterion, incompleteness of the one-particle
basis set, and inaccuracy in the density functional.
Figure 6. Mononuclear end-on (η1) and side-on (η2) CuO2 complexes.
Best agreement with the experimental EIE for [Cu(η1-
O2)TMG3tren]+ is obtained using the frequencies obtained
including the effects of continuum acetone solvation as shown
in the Figure 5. Polarization effects associated with solvent are,
to first order, proportional to (1 - ꢀ-1) where ꢀ is the dielectric
constant of the solvent.38 In the case of acetone, the value of (1
- ꢀ-1) is 0.952. For DMF, the corresponding value is 0.974,
which differs from acetone by about 2%.43 Thus, insignificant
changes in electronic structure would be expected between
structures optimized in continuum models for these two solvents,
and it is not surprising that there is good agreement between
the computed and experimental EIEs.
Table 1. Comparison of Calculated O-O Stretching Frequencies
and Bond Lengths to the Available Experimental Data for
Mononuclear CuO2 Compounds
1
νO
(∆
-O
16,18) (cm-
)
bond length (Å)
exp
calcd
exp
calcd
O2
1556.3(43.8)b 1550(44)
1.21b
n.d.
1.22
1.27
1.28
1.29
1.33
1.38
a
[Cu(η1-O2)TEPA]+ c
[Cu(η1-O2)TMPA]+ c
n.d.
n.d.
1279(29)
1251(33)
1218(32)
1124(31)
1013(28)
n.d.
[Cu(η1-O2)TMG3tren]+ a 1117(28)d
1.28e
1.22g
1.39i
Cu(η2-O2)tBu-Tpc
Cu(η2-O2)âDKc
1112(26)f
968(25)h
a Unrestricted triplet calculation. b Reference 56. c Unrestricted singlet
calculation. d Reference 16. e Reference 15. f Reference 21. g Reference 7a.
h Reference 57. i Reference 7b.
3.3. Calculations of Oxygen Equilibrium Isotope Effects.
18O EIEs were calculated at varying temperatures using eqs
7-10 which represent the gas-phase partition functions. The
gas-phase vibrational frequencies for O2 and the various
oxygenated copper compounds were obtained from the density
functional calculations described above. The mPWPW91 func-
tional with the 6-311G* basis set on oxygen (previously used
for other metal-O2 complexes52,53) is particularly appropriate
for this purpose as it reproduces the experimental O-O
stretching frequency, isotope shift, and bond length of free O2.
Other methods (for example one employed by Popp et al. in a
recent study of a palladium dioxygen complex20b) can fail in
this respect, leading to potential errors in comparing experi-
mental and theoretical isotope effects.
O2)TMG3tren]+ lying between the proposed54 end-on com-
pounds, [Cu(η1-O2)TEPA]+ and [Cu(η1-O2)TMPA]+, and the
structurally characterized side-on species, Cu(η2-O2)-
tBu-Tp and Cu(η2-O2)âDK.7a,b
In some cases, the experimental parameters differ significantly
from the calculated ones. For example, the crystallographically
determined bond length for a trispyrazolylborate Cu(η2-O2)
compound analogous to Cu(η2-O2)tBu-Tp is likely in error,
owing to the librational motion of the O2 fragment and an
erroneously short, fitted bond length.53 Another example
involves [Cu(η1-O2)TMG3tren]+, which exhibits a νO-O in
acetone solution that is ∼100 cm-1 less than the value calculated
in the gas phase.
In the original report, close matching of the observed and
calculated νO-O was reported for the restricted singlet structure
of [Cu(η1-O2)TMG3tren]+.16 While we have reproduced this
calculation,24 a revised treatment is clearly in order in view of
the present results which establish that [Cu(η1-O2)TMG3tren]+
is paramagnetic.55 The lack of agreement between the calculated
and experimental stretching frequencies could, in principle,
reflect enhancement of the ionic character within [Cu(η1-
The maximum 18O EIEs derived from calculations of isotopic
partition functions at different temperatures are provided in
Table 2. For [Cu(η1-O2)TMG3tren]+, the measured and calcu-
lated 18O EIEs agree quite well even without the continuum
dielectric correction. Use of the experimentally determined νO-O
) 1556.3 cm-1 and 16,18∆ ) 43.8 cm-1 for O2
gave 18O
14,56
EIEcalc of 1.0125 which is quite close to the 1.0128 estimated
from the νO-O ) 1550 cm-1 and 16,18∆ ) 44 cm-1 calculated
for gas phase O2 in this study. The νO-O calculated in acetone
is 1537 cm-1 and 16,18∆ ) 43 cm-1. The use of unscaled values
gives reasonably good agreement with the experimentally
determined 18O EIE ) 1.0148.58 Comparisons of the experi-
mental to computational 18O EIEs for the η2-CuO2 species in
Figure 6 have not been possible due to the lack of reversibility
in the reactions with O2.
(54) Smirnov, V. V.; Roth, J. P. J. Am. Chem. Soc. 2006, 128, 3683-3695.
(55) Similar results have been obtained in magnetic circular dichroism studies
(Schindler, S. Personal communication.) and will be the subject of a
forthcoming manuscript.
(56) Chase, M. W., Jr.; Curnutt, J. L.; Downey, J. R., Jr.; McDonald, R. A.;
Syverud, A. N.; Valenzuela, E. A. J. Phys. Chem. Ref. Data 1982, 11,
695-940.
(57) Spencer, D. J.; Aboelella, N. W.; Reynolds, A. M.; Holland, P. L.; Tolman,
W. B. J. Am. Chem. Soc. 2002, 124, 2108-2109.
9
14704 J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007

Molecular Oxygen Activation at Copper(I) Sites
A R T I C L E S
Table 2. Oxygen Equilibrium Isotope Effects (18O EIEcalc) and Partition Functions Calculated from Eqs 7-11; Tabulated Values Correspond
to the Temperatures at Which the Maximum 18O EIEs Are Observed
18O EIEcalc
ZPE
EXC
MMI (eq 10)
MMI
×
EXC
MMI (eq 11)
[(η1-O2)CuTEPA]+ a
[(η1-O2)CuTMPA]+ a
[(η1-O2)CuTMG3tren]+ d
(η2-O2)CutBu-Tpa
1.0107b
1.0128c
1.0129b
1.0239e
1.0251f
0.9597
0.9644
0.9589
0.9529
0.9558
0.9192
0.9207
0.9174
0.9367
0.9337
1.1457
1.1406
1.1515
1.1472
1.1487
1.0531
1.0502
1.0564
1.0746
1.0725
1.1452
1.1420
1.1509
1.1485
1.1507
(η2-O2)CuâDKa
b
c
e
f
a Unrestricted singlet (mixed-state) calculation. Tmax ) 243 K. Tmax ) 222 K. d Unrestricted triplet calculation. Tmax )173 K. Tmax ) 197 K.
For the triplet state, the 18O EIEcalc passes through a maximum
of 1.0128 at 243 K. For the mixed and singlet states, the maxima
at 1.0134 and 1.0144 occur at 255 and 247 K, respectively.19
The gas phase νO-O associated with these structures vary from
1218 cm-1 (triplet) to 1198 cm-1 (unrestricted singlet) to 1140
cm-1 (singlet).24
Discernible differences in the temperature dependences of
the 18O EIEcalc are predicted for the end-on and side-on
structures. The maximum values predicted for [Cu(η1-O2)-
TEPA]+ (1.0107) and [Cu(η1-O2)TMPA]+ (1.0128) occur at 243
and 222 K, respectively, similar to that of [Cu(η1-O2)TMG3-
tren]+. In contrast, the maximum 18O EIEcalc for Cu(η2-O2)-
tBu-Tp (1.0239) and Cu(η2-O2)âdk (1.0251) are at 173 and 197
K. The observation that the maximum isotope effects occur at
lower temperatures for the side-on structures than for the end-
on structures is consistent with a less significant contribution
to the 18O EIE from the low-frequency modes. For example in
the case of the triplet structure for [Cu(η1-O2)TMG3tren]+,
truncating the set of frequencies such that only vibrations above
100 cm-1 are used in the calculations, results in a slight change
in the isotope effect from 1.0129 to 1.0136 and shifting of the
maximum from 243 to 231 K.
Figure 7. Calculation of the gas-phase partition functions and 18O EIE
upon formation of [Cu(η1-O2)TMG3tren]+ from 16,16O2 and 16,18O2.
The temperature dependences of the equilibrium isotope
effects as well as the contributing isotopic partition functions
were calculated to obtain insights as to the theoretical origins
of the 18O EIEs. The results obtained for [Cu(η1-O2)TMG3tren]+
are shown in Figure 7. The 18O EIEcalc begins as an inverse
value at the lowest temperatures and then passes through a
normal maximum as the temperature increases. This is the result
of a negative exponential relationship of the ZPE to temperature
and the compensating influence of MMI × EXC. In all cases,
the 18O EIEs are predicted to be relatively insensitive to
temperature over the experimentally accessible range. Similar
results are indicated in the calculations using the polarized
continuum model.24 At the highest temperatures, the 18O EIEcalc
approaches unity as the inverse ZPE effect vanishes and EXC
approaches 1/MMI.
4. Discussion
Ever since Pauling’s assignment of oxyhemoglobin as an end-
0
on ferrous dioxygen adduct (FeII-O2 ),1a the electronic structures
of metal-bound dioxygen species have been subject to
debate.1a,6a,17-19,59-63 Studies of the cobalt-reconstituted proteins
have provided perhaps the most compelling experimental
support for the alternative ferric superoxide (FeIII-O2
)
-I
description of oxyhemoglobin and related systems.59-61 Electron
paramagnetic resonance (EPR) spectroscopy has elegantly been
applied to assess the degree of internal electron transfer and,
hence, the valence bond contributions to transition-metal-
activated oxygen structures.62 The EPR analysis has largely been
limited to O2 adducts of cobalt where nuclear hyperfine
interactions are observed between the unpaired electron and the
59Co- or 17O-labeled oxygen.59,60,63
The dependence of 18O EIEcalc upon temperature is essentially
the same for all of the CuO2 compounds.24 The enthalpic
contribution to the isotope effect, represented by ZPE, is only
slightly different for the η1-species (0.9589-0.9644) and the
η2-species (0.9529, 0.9558). Similar behavior is indicated for
the EXC terms: η1-species (0.9174-0.9207) and η2-species
(0.9337, 0.9367). The temperature independent MMI terms are
calculated to be large for both types of products, ranging from
1.1420 to 1.1509 without a discernible trend. The entropic
contribution, EXC × MMI, apparently differentiates the end-
on (1.0502-1.0564) from the side-on (1.0725, 1.0746) struc-
tures.
(59) (a) Hoffman, B. M.; Petering, D. H. Proc. Natl. Acad. Sci. U.S.A. 1970,
67, 637-643. (b) Jones, R. D.; Summervill, D. A.; Basolo, F. Chem. ReV.
1979, 79, 139-179.
(60) (a) Ikeda-Saito, M.; Horui, H.; Inubushi, T.; Yonetani, T. J. Biol. Chem.
1981, 256, 10267-10271 and refs therein. (b) Gupta, R. K.; Mildvan, A.
S.; Yonetani, Takashi; Srivastava, T. S. Biochem. Biophys. Res. Commun.
1975, 67, 1005-1012.
The temperature dependences of 18O EIEcalc are also similar
for the different electronic structures of [Cu(η1-O2)TMG3tren]+.
(61) Lever, A. B. P.; Ozin, G. A.; Gray, H. B. Inorg. Chem. 1980, 19, 1823-
1824.
(62) (a) Herman, Z. S.; Loew, G. H. J. Am Chem. Soc. 1980, 102, 1815-1821.
(b) Jensen, K. P.; Ryde, U. J. Biol. Chem. 2004, 279, 14561-14569. (c)
Jensen, K. P.; Roos, B. J.; Ryde, U. J. Inorg. Biochem. 2005, 99, 45-54.
(d) Jensen, K. P.; Roos, B. J.; Ryde, U. J. Inorg. Biochem. 2005, 99, 978.
(63) (a) Tovrog, B. S.; Kitko, D. J.; Drago, R. S. J. Am. Chem. Soc. 1976, 98,
5144-5153. (b) Hoffman, B. M.; Szymanski, T.; Brown, T. G.; Basolo, F.
G. J. Am. Chem. Soc. 1978, 100, 7523-7259. (c) Drago, R. S.; Corden, B.
B. Acc. Chem. Res. 1980, 13, 353-360.
(58) While the analysis of EIEs using scaled quantum mechanical force constants
may seem to be a more rigorous approach for comparing theory to
experiment, such a procedure would require a large number of experimental
data, including several vibrational frequencies for each of the isotopologues,
in order to scale a reasonable portion of the quantum mechanical Hessian
matrix.
9
J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007 14705

A R T I C L E S
Lanci et al.
Formal oxidation-state assignments de-emphasize the impor-
tance of both ionic and covalent character within the bonds of
various types of metal-dioxygen species.61 The 18O EIEs
determined here for CuO2 species, together with those deter-
mined previously for group IX transition metals, provide
experimental support for a bonding continuum, from dioxygen
to superoxide to peroxide-like structures.6a,7c,49,53,54 It is interest-
ing to consider how differing extents of ionic and covalent
bonding influence reactivity. For example, the stable iron-
different from those proposed for most other types of metal-
oxygen adducts,17-21,57 including those derived from heme.59,62
That [Cu(η1-O2)TMG3tren]+ does not exhibit an X-band EPR
spectrum is consistent with the compound having either a triplet
or singlet structure. The paramagnetic NMR spectrum, together
with the solution magnetic susceptibility, provides evidence for
the former at least under the experimental conditions. The
presence of two unpaired spins is most readily attributed to
ferromagnetic coupling of an electron associated with copper-
(II) and another associated with the superoxide (O2-I) ligand.
As mentioned above, another possibility could involve copper-
(I) bound to O20 containing two unpaired electrons of the same
spin.
oxygen adducts derived from O2-transport and O2-sensing heme
•- 64
proteins are known to dissociate O2
.
In contrast, the
mononuclear non-heme iron- and copper active sites appear
more likely to generate high-energy oxygen adducts that readily
dissociate O2. This is but one reason why such species can be
difficult to detect spectroscopically.65,66 In the enzymes, the
kinetics of O2 binding and subsequent substrate oxidation must
be finely tuned to prevent oxidative damage to the surrounding
protein.67 The oxygen isotope effects provide a way to identify
catalytic intermediates, which may be spectroscopically unde-
tectable, and to correlate the vibrational structures of these
species to reactivity.
The combined use of experiment and theory, as described in
this work, has for many years been the cornerstone of isotope
effect studies, with a significant portion of this work employing
heavy atom isotope fractionation methods.68-71 We add that
the comparison of experimental to predicted oxygen isotope
effects upon reactions of O2 has been limited to but a few
examples.13a,69h,72,73
The thermal instability of the isolated compound has unfor-
tunately precluded magnetic susceptibility measurements in the
crystalline state.46 The characterization of the O2 binding
equilibria reported in this work can, however, provide a basis
for future investigations of the magnetic properties.55
The paramagnetic [Cu(η1-O2)TMG3tren]+ exhibits a dominant
contribution from the ionic (CuII-O2-I) structure, as indicated
by the magnitude of the 18O EIE (see below). Evidence for a
tightly bound, inner-sphere complex rather than a weak as-
sociation complex of [CuIITMG3tren]2+ and O2 comes from
•-
the following observations: (i) [CuITMG3tren]1+ is regenerated
in 94 ( 6% yield upon removal of O2 from a sample of [Cu-
(η1-O2)TMG3tren]+, (ii) no NMR spectral change is detected
upon addition of [CuIITMG3tren]2+ to samples of either
[CuITMG3tren]1+ or [Cu(η1-O2)TMG3tren]+, and (iii) no EPR
signal due to [Cu(η1-O2)TMG3tren]+ is observable at X-band
frequency, under conditions where [CuIITMG3tren]2+ is readily
quantified. The very favorable electrostatic interaction energy
4.1. Electronic Structures of End-on CuO2 Species. [Cu-
(η1-O2)TMG3tren]+ exhibits an electronic structure that is
(64) (a) Gilles-Gonzalez, M. A.; Gonzalez, G.; Perutz, M. F.; Kiger, L.; Marden,
M. C.; Poyart, C. Biochemistry 1994, 33, 8067-8073. (b) Gonzalez, G.;
Gilles-Gonzalez, M. A.; Rybak-Akimova, E. V.; Buchalova, M.; Busch,
D. H. Biochemistry 1998, 37, 10188-10194. (c) Shikama, K. Chem. ReV.
1998, 98, 1357-1373.
(65) For a possible exception of a stable FeO2 species in a non-heme iron enzyme
see: Brown, C. D.; Neidig, M. L.; Neibergall, M. B.; Lipscomb, J. D.;
Solomon, E. I. J. Am. Chem. Soc. 2007, 129, 7427-7438 and refs therein.
(66) High-energy, end-on dioxygen adducts have also been proposed to form
at diiron active sites. See: Wei, P.; Skulan, A. J.; Wade, H.; DeGrado, W.
F.; Solomon, E. I. J. Am. Chem. Soc. 2005, 127, 16098-16106.
(67) Klinman, J. P. Acc. Chem. Res. 2007, 40, 325-333.
(68) Two historical overviews are provided by Bigeleisen, J.; Wolfsberg, M. In
Isotope Effects in Chemistry and Biology; Kohen, A., Limbach, H. H., Eds.;
CRC Press: Boca Raton, 2006; pp 1-39, 89-117.
(69) For examples related to reactions of O2 see: (a) Nahm, K.; Li, Y.; Evanseck,
J. D.; Houk, K. N.; Foote, C. S. J. Am. Chem. Soc. 1993, 115, 4879-
4884. (b) Burger, R. M.; Tian, G.; Drlica, K. J. Am. Chem. Soc. 1995,
117, 1167-1168. (c) Andrews, L.; Chertihin, G. V.; Ricca, A.; Bauschlicher,
C. W., Jr. J. Am. Chem. Soc. 1996, 118, 467-470. (d) Danset, D.;
Manceron, L.; Andrews, L. J. Phys. Chem. A 2001, 105, 7205-7210. (e)
Palmer, A. E.; Lee, S. K.; Solomon, E. I. J. Am. Chem. Soc. 2001, 123,
6591-6599. (f) Singleton, D. A; Hang, C.; Szymanski, M. J.; Meyer, M.
P.; Leach, A. G.; Kuwata, K. T.; Chen, J. S.; Greer, A.; Foote, C.S.; Houk,
K. N. J. Am. Chem. Soc. 2003, 125, 1319-1328. (g) Smirnov, V. V.; Roth,
J. P. J. Am. Chem. Soc. 2006, 128, 16424-16425.
(70) For relevant studies of equilibrium isotope effects see: (a) Saunders, M.;
Laidig, K. E.; Wolfsberg, M. J. Am. Chem. Soc. 1989, 111, 8889-8994.
(b) Bender, B. J. Am. Chem. Soc. 1995, 117, 11239-11246. (c) Slaughter,
L. M.; Wolczanski, P. T.; Klinckman, T. R. Cundari, T. R. J. Am. Chem.
Soc. 2000, 122, 7953-7975. (d) Abu-Hasanayn, F.; Goldman, A. S.; Krogh-
Jesperson, K. J. Phys. Chem. 1993, 97, 5890-5896.
estimated for [CuIITMG3tren]2+ and O2 (∆G°298K ) -21
•-
kcal mol-1) from the redox potentials15,74 and the calculated
KO2 further argues against an outer-sphere complex.75
Qualitatively, the paramagnetism of [Cu(η1-O2)TMG3tren]+
could partly be attributable to the η1 versus η2 coordination and
partly to the weak electron-donating nature of the ligand relative
to those of the anionic Tp and âDK ligands. These features
effectively eliminate the singlet copper(III) peroxide dianion
character in the valence bond description of the compound.
Calculations have predicted that end-on coordination substan-
tially reduces the stability of the singlet relative to that of the
triplet state in CuO2 species, irrespective of ligand,50-52 and
have established that the ligands of lesser electron-donating
ability stabilize end-on structures relative to side-on structures.76
Within the context of the density functional calculations,
ferromagnetic coupling suggests decreased covalency within the
Cu/O2 interaction. This diminished covalency would more likely
be the case for end-on oxygen adducts than for a side-on species.
Ground-state singlet structures have been reported for
(71) For several leading examples of combined experimental/computational
methods for the determination of kinetic isotope effects on organic reactions,
see: (a) Ralph, E. C.; Hirschi, J. S.; Anderson, M. A.; Cleland, W. W.;
Singleton, D. A.; Fitzpatrick, P. F. Biochemistry 2007, 46, 7655-7664.
(b) Gustin, D. J.; Mattei, P.; Kast, P.; Wiest, O.; Lee, L.; Cleland, W. W.;
Hilvert, D. J. Am. Chem. Soc. 1999, 121, 1756-1757. (c) Meyer, M. P.;
DelMonte, A. J.; Singleton, D. A. J. Am. Chem. Soc. 1999, 121, 10865-
10874. (d) DelMonte, A. J.; Haller, J.; Houk, K. N.; Sharpless, K. B.;
Singleton, D. A.; Strassner, T.; Thomas, A. A. J. Am. Chem. Soc. 1997,
119, 9907-9908. (e) Beno, B.R.; Houk, K. N.; Singleton, D. A. J. Am.
Chem. Soc. 1996, 118, 9984-9985.
(73) Reference 20b describes what may be one of the first uses of density
functional methods to calculate oxygen isotope effects relevant to
those measured in refs 13a and 13b. There is disagreement between
the experimental and calculated values for reasons that are not yet
understood.
(74) Using E1/2 ) 0.26 V versus NHE for [Cu(TMG3Tren]2+/1+ in acetone (ref
15) and E1/2 ) -0.62 V versus NHE for O2 in DMF, as quoted in Sawyer,
D. T.; Sobkowiak, A.; Roberts, J. L., Jr. Electrochemistry for Chemists,
2nd ed.; Wiley: New York, 1995; pp 364-372.
(72) (a) Roth, J. P.; Wincek, R.; Nodet, G.; Edmondson, D. E.; McIntire, W.
S.; Klinman, J. P. J. Am. Chem. Soc. 2004, 126, 15120-15131. (b)
References 13a, 13b, and 54 describe measured and calculated oxygen
isotope effects on O2 binding to transition metal compounds.
(75) Slightly larger values have been estimated for Co-O2 complexes. See:
Taube, H. Prog. Inorg. Chem. 1986, 34, 607-625.
(76) Heppner, D. E.; Gherman, B. F.; Tolman, W. B.; Cramer, C. J. Dalton
Trans. 2006, 4773-4782.
9
14706 J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007

Molecular Oxygen Activation at Copper(I) Sites
A R T I C L E S
Figure 8. Singly occupied molecular orbitals (SOMOs) for the calculated triplet ground state of [Cu(η1-O2)TMG3tren]+. See the text for descriptions.
Cu(η2-O2)tBu-Tp and Cu(η2-O2)âDk.21,49,77 The magnetic prop-
erties of the proposed54 [Cu(η1-O2)TMPA]+ and its dimethy-
lamino derivative7h have yet to be evaluated and should provide
interesting comparisons to [Cu(η1-O2)TMG3tren]+ due to the
isostructural nature of the compounds.7d
1.38 units of spin on the O2 fragment and the remaining 0.62
units on the Cu and coordinating N atoms, reflecting the degree
of charge transfer associated with covalent bonding between
the Cu and O2.
Few transition-metal compounds have been reported to exhibit
ground-state triplet electronic structures. A recent example79 is
a copper(II) compound containing an arylnitroxyl ligand. This
species exists predominantly in the triplet state at temperatures
between 100 and 300 K. The preference for the triplet electronic
structure has been suggested to arise from the high-spin density
on the nitroxyl, orthogonal (in the geometric sense) to the orbital
containing the unpaired electron on copper. The computational
results for [Cu(η1-O2)TMG3tren]+ raise the possibility that
orbital orthogonality may not be a requirement for observable
ferromagnetic coupling, given that this is the origin of the
observed paramagnetism in the present study. In support of the
calculations described in this work, “spin-flip” time-dependent
density functional calculations have also predicted triplet ground
states for CuO2 compounds structurally related to [Cu(η1-
O2)TMG3tren]+.17a
4.2. Interpretation of Equilibrium Isotope Effects. 4.2.1.
Vibrational Structures of End-on and Side-on CuO2 Species.
The first 18O equilibrium isotope effects upon the formation of
mononuclear CuO2 complexes from natural abundance O2 have
been determined. The O2 is bound in an end-on manner in the
one case where isotope effects can be both measured and
calculated. The structure of [Cu(η1-O2)TMG3tren]+ may be
analogous to those of intermediates proposed to effect C-H
oxidation in enzymes.4,5 In such instances, competitive 18O
kinetic isotope effects have been suggested as evidence that the
CuO2 species is the reactive oxidant.11,12 The rigorous applica-
tion of 18O isotope effects to the identification of such
intermediates has been limited, however, due to the absence of
benchmarks and computational methods for predicting activated
oxygen structures at the requisite level of detail.
The calculations performed on [Cu(η1-O2)TMG3tren]+ dis-
cussed in Results (section 3.2.) suggest a large singlet vs triplet
gap (ca. 10 kcal mol-1). A full theoretical analysis of this Cu-
O2 system will be the subject of a forthcoming study.78 Here
we suggest simply that the triplet ground state can be attributed
2
to a relatively weak interaction between the filled dz orbital on
copper and the singly occupied π* molecular orbital on dioxygen
with which it overlaps, possibly because of energy mismatch
resulting from the geometric constraints and the weak electron-
donating ability of the TMG3tren ligand. Splitting of the O2 π*
2
levels due to hybridization of one of these orbitals with the dz
is apparently too weak to overcome the exchange energy
required to pair the electrons in the lower-energy noninteracting
O2 π*.
Given such limited hybridization, the DFT calculations
suggest that the complex has CuI-O20 character. The covalency
of the Cu-O interaction must, however, permit some charge
transfer to the O2 fragment with concomitant buildup of
ferromagnetically coupled spin within the CuII-O2 species.
-I
This description is fully consistent with the solution magnetic
susceptibility measurements presented herein, the previously
reported O-O stretching frequency and bond distances which
•- 15,16
are intermediate of O2 and O2
,
as well as the optical
spectrum which shows a strong band ca. 440 nm typically
assigned as a ligand to metal charge transfer.6b,7d,16
The two singly occupied molecular orbitals (SOMOs, see
Figure 8) are consistent with this analysis. The lower-lying
SOMO1 would ideally be associated with the noninteracting
π* orbital of O2. The additional contributions from the TMG3-
tren ligand and the Cu dxy orbitals are commonly observed for
molecules of this size. The higher-energy SOMO2 is the hybrid
Interestingly, the 18O EIE of 1.0148 which characterizes [Cu-
(η1-O2)TMG3tren]+ is significantly larger than those seen for
other end-on oxygen adducts. Small 18O EIEs (1.0041-1.0066)
have been reported for O2 binding to hemes14 as well as for
synthetic cobalt compounds.13a Meanwhile, the isotope effect
2
π*-dz orbital, also with additional contributions from the
TMG3tren ligand, especially the tertiary amine trans to the
dioxygen ligand. A Mulliken analysis of the spin distribution
for the structure computed including solvation effects places
for [Cu(η1-O2)TMG3tren]+ is significantly smaller than the 18
O
EIEs calculated for side-on CuO2 compounds (1.024, 1.025) as
well as those measured for side-on peroxide compounds of other
(77) Reynolds, A. M.; Gherman, B. F.; Cramer, C. J.; Tolman, W. B. Inorg.
Chem. 2005, 44, 6989-6997. Sarangi, R.; Aboelella, N.; Fujisawa, K.;
Tolman, W. B.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem.
Soc. 2006, 128, 8286-8296.
(78) Cramer, C. J.; Gour, J. R.; Wloch, M.; Piecuch, P.; Rehaman Moughal
Shahi, A.; Gagliardi, L. Manuscript in preparation.
(79) Osanai, K.; Okazawa, A.; Nogami, T.; Ishida, T. J. Am. Chem. Soc. 2006,
128, 14008-14009.
9
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A R T I C L E S
Lanci et al.
transition metals (1.020-1.028). These results support a con-
tinuum bonding description where the extent of O-O reduction
is correlated to, yet does not alone determine, the magnitude of
the 18O EIE. We have now confirmed that measured 18O EIEs
can be reproduced using vibrational frequencies obtained from
the appropriate density functional methods.
In contrast to the ZPE effect, the EXC × MMI appears to
differentiate the two classes of CuO2 adducts: η1 (1.0502-
1.0564) and η2 (1.0725, 1.0746). Similar differences have also
been calculated for the η1-superoxide and η2-peroxide structures
derived from group IX transition metals;82 except in the case
of the Co(η1-O2) structures the EXC × MMI terms are
somewhat smaller than for the Cu(η1-O2) structures. In future
studies, we will attempt to experimentally delineate the enthalpic
and entropic contributions by examining the temperature
dependence of the isotope effects over ranges where they are
predicted to vary significantly and the ZPE terms are predicted
to transition from normal to inverse.82
4.3. Implications for Enzymatic O2 Activation. The 18O
EIEs in the present study provide boundary conditions for the
interpretation of 18O KIEs upon enzymatic reactions.4,10-12,23,69g
By virtue of being competitive measurements, the latter probe
all steps beginning with the association of the enzyme and O2
up to and including the first kinetically irreversible step. The
18O EIE is often assumed to define the upper limit to the 18O
KIE for a reaction involving a single, irreversible step. This
relationship, which derives from Transition State Theory and
the formulation of the reaction coordinate as classical with a
transition state that has a vibrational structure intermediate
between the reactant and the product, has not yet been rigorously
tested. This assumption has, however, served as the basis for
interpreting heavy atom kinetic isotope effects for many years.83
Nevertheless, the isotope effects described in this study are
recommended as benchmarks for the characterization of inter-
mediates formed in the pre-equilibrium steps of O2 reduction
mechanisms.
4.2.2. Physical Origins of the Isotope Effects. Analysis of
isotopic stretching frequencies within the formalism of Big-
eleisen and Goeppert-Mayer provides valuable insights concern-
ing the origins of 18O EIEs upon O2 binding to reduced metal
centers. We have performed calculations of isotope-dependent
partition functions at different temperatures to determine how
enthalpy and entropy contribute to the 18O EIEs.
The coordination of O2 is entropically unfavorable but
enthalpically driven by the formation of metal-O bonds, at the
expense of weakening the O-O bond. Extensive enthalpy-
entropy compensation is expected for O2 binding to transition
metals because of the low-frequency vibrational modes created
which connect the isotopic sites within the products.80 Similar
behaviors have been reported by Parkin and co-workers to
characterize the formation of organometallic σ complexes of
H2 and CH4. Unlike O2, in these examples, the bound substrates
are not believed to undergo extensive reduction.81
The isotopic partition functions in Table 2 would seem to
suggest that the primary contribution to the 18O EIE is entropic
(EXC × MMI) rather than enthalpic (ZPE) in origin; however,
this is not meant to suggest that changes in force constants are
unimportant.68 In all of the reactions analyzed, the ZPE terms
are inverse and span a relatively narrow range (0.953-0.964).
This observation argues against a dominant contribution from
reduction of the O-O bond, as assumed in an earlier model;14
in this case the accompanying change in force constant would
result in a normal (>1) effect.
The inverse ZPE terms indicate a net increase in bonding
within the product relative to free O2. This observation is
consistent with the favorable ∆H ) -8.4 kcal mol-1 determined
for the formation of [Cu(η1-O2)TMG3tren]+. The absence of a
trend among the ZPE terms for η1- and η2-O2 structures implies
that strengthening of the Cu-O bond results in a commensurate
weakening of the O-O bond and Vice Versa. In support of this
view, similar ZPE terms have been estimated for the formally
η1-superoxide and η2-peroxide compounds derived from group
IX transition metals.82
4.3.1. Copper Monooxygenases. Dopamine â-monooxyge-
nase (DâM) and peptidylglycine R-hydroxylating monooxyge-
nase (PHM) are structurally homologous enzymes which activate
O2 for the biosynthesis of hormones and neurotransmitters. An
outstanding question has been the identity of the species
responsible for substrate C-H oxidation by H• abstraction.4,5,84
The remarkably similar 18O KIEs determined for the enzyme
reactions have been cited as evidence that a copper species, with
an intact O-O bond, is the active oxidant. A copper(II)
superoxide species has been proposed on the basis of observa-
tions with deuterium-labeled substrates4 where 18O KIEs vary
from 1.0197 ( 0.0003 to 1.0256 ( 0.0003 in DâM and from
1.0173 ( 0.0009 to 1.0212 ( 0.0018 in PHM upon replacing
2
1H with H at the reactive position.
The entropic contribution to the isotope effect, EXC × MMI,
reflects the mass-dependent population difference arising from
rotations and translations of free O2 which are converted to low-
frequency vibrations within the product. Large normal MMI
terms (1.14-1.15) are calculated representing the loss of mass
discrimination among translational and rotational modes in free
O2 upon its coordination to the heavy metal fragment. The MMI
terms are offset by inverse EXC terms (0.9174-0.9367)
originating from the increased number of vibrational modes
within the product. The thermal population of these modes is
greater for the heavier isotopologue than for the lighter one due
to the more closely spaced energy levels.
The 18O EIE determined for [Cu(η1-O2)TMG3tren]+ (1.0148)
is consistent with the proposal of the pre-equilibrium formation
of the copper(II) end-on superoxide intermediate in DâM and
PHM.85 The side-on structure5 cannot be rigorously excluded,
however, since the 18O EIEs for Cu(η2-O2)tBu-Tp and
Cu(η2-O2)âDK are close to the upper limits defined by the range
of KIEs. A slight increase in the 18O KIE observed for the
enzyme reaction relative to the 18O EIE is anticipated due to
(83) The heart of the issue is the reaction coordinate frequency and its isotope
sensitivity. The assumptions with respect to heavy atom kinetic isotope
effects upon O2 binding are discussed in refs 11, 13b, 68 pp 18-25, ref 80
pp 44-47, and in the classic monograph: Melander, L.; Saunders, W. H.,
Jr. Reaction Rates of Isotopic Molecules; Wiley: New York, 1980; pp 45-
49, 64-83, 272-275.
(84) Crespo, A.; Marti, M. A.; Roitberg, A. E.; Amzel, L. M.; Estrin, D. A. J.
Am. Chem. Soc. 2006, 128, 12817-12828.
(85) The end-on geometry has also been supported by recent density functional
calculations in ref 49.
(80) Huskey, W. P. In Enzyme Mechanism from Isotope Effects; Cook, P. F.,
Ed.; CRC Press: Boca Raton, 1991 pp 37-72.
(81) (a) Janak, K. E.; Parkin, G. J. Am. Chem. Soc. 2003, 125, 13219-13224.
(b) Janak, K. E.; Parkin, G. J. Am. Chem. Soc. 2003, 125, 6889-6891.
(82) Lanci, M. P.; Smirnov, V. V.; Roth, J. P. Manuscript in preparation.
9
14708 J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007

Molecular Oxygen Activation at Copper(I) Sites
A R T I C L E S
the subsequent step where the O-O bond is reduced further
upon H• transfer.
implies differences in the low-frequency stretching modes which
characterize the metal-oxygen interaction. The elevated isotope
effect suggests increased ionic character within the valence bond
description. Precisely how this relates to the finding that [Cu-
(η1-O2)TMG3tren]+ is paramagnetic is not yet understood;
however, density functional calculations suggest a triplet ground
4.3.2. Copper Amine Oxidases. The 18O EIE determined
for [Cu(η1-O2)TMG3tren]+ is also relevant to the mechanism
by which copper- and topaquinone(TPQ)-containing amine
oxidases reduce O2 to H2O2. This reaction, in structurally
homologous enzymes from a variety of sources, provides the
oxidizing equivalents needed to convert primary amines to
aldehydes. Two proposals which differ with respect to the role
of the copper in activating O2 have been considered.86 Dooley
and co-workers originally proposed that O2 binds to copper(I)
in the initial step of O2 reduction. Klinman and co-workers have
put forth an alternative mechanism where O2 reacts directly with
TPQreduced by outer-sphere electron transfer.23b,87
2
state due to the lack of significant interaction between the dz
orbital on copper and the π* orbitals on dioxygen.
The agreement observed between the measured and calculated
isotope effects for [Cu(η1-O2)TMG3tren]+ validates the applica-
tion of the theoretical formalism and density functional methods
to calculating oxygen isotope effects for ground-state, metal-
activated oxygen structures. The importance of using the
appropriate functional and basis set cannot be overemphasized
since the isotope effects are very sensitive to small frequency
shifts. Validating the quantitative utility of such calculations is
a vital step toward applying DFT to predict transition-state
structures and the accompanying kinetic isotope effects upon
enzymatic oxidation reactions.
The 18O KIEs determined for copper amine oxidases have
been difficult to interpret in the absence of boundary conditions
for structurally defined CuO2 species. The 18O KIEs reported
for the enzymes from yeast and from bovine serum range from
1.008 to 1.011,23b,87a,88 well outside the limits expected for outer-
sphere electron transfer.11,12,69g,72a
Larger 18O KIEs from 1.014 to 1.018 have recently been
determined for the amine oxidases from pea seedling and
Arthrobacter globiformis.89 That these values are close to the
18O EIE determined for [Cu(η1-O2)TMG3tren]+ raises the
possibility that a copper(II) superoxide species is formed in a
pre-equilibrium step. Thus, the electron transfer from TPQsemi-
quinone to Cu(η1-O2) may be rate-determining in the reaction
of copper amine oxidases with O2.12 Such a mechanism,
involving reversible O2 binding prior to electron or coupled
electron/proton transfer to CuO2, is similar to that proposed for
the copper monooxygenases. The observation in the yeast and
bovine amine oxidase, where the 18O KIE is somewhat smaller
than the expected 18O EIE, further implies an internal commit-
ment factor, where formation of the Cu(η1-O2) species contrib-
utes to the rate-limiting step.12,90
Analyzing the isotope effects in light of the constituent
isotopic partition functions has provided new insights concerning
the physical origins. Calculations at varying temperatures reveal
extensive enthalpy-entropy compensation; this can be under-
stood in terms of the binding of O2 concomitant with the
formation of new isotopically sensitive low-frequency modes
unique to the product. An isotope effect is thus observed to be
characteristic of the dioxygen-, superoxide-, or peroxide-like
structure. This view is in contrast to an earlier model where the
isotope effect was assumed to arise primarily from the change
in the O-O force constant. While this is most certainly a
contributing factor to the observed trends, its influence cannot
be deconvolved from that of the new metal-oxygen bonds
formed.
5. Conclusions
The results described above have important implications for
interpreting isotope effects upon oxidation reactions, particularly
when O2 is activated by inner-sphere electron transfer. In these
cases, the oxygen EIEs for structurally defined compounds
provide relevant benchmarks for identifying O2-derived inter-
mediates during enzyme catalysis.
On the basis of oxygen isotope effect measurements together
with density functional methods we have outlined a new
approach to characterizing unstable O2-derived species. Proof-
of-principle is provided by (i) the demonstration that the
calculated and observed oxygen equilibrium isotope effects are
in good agreement for [Cu(η1-O2)TMG3tren]+ and (ii) com-
parisons of calculated values which reveal a trend where the
18O EIEs increase in direct proportion to the level of O-O
reduction, from oxygen-like to superoxide-like to peroxide-like
structures.
The first competitive isotope effects upon reversible O2
binding to form a structurally defined CuO2 species have been
determined. Interestingly, the 18O EIE upon [Cu(η1-O2)TMG3-
tren]+ formation is significantly larger than those measured for
structurally analogous end-on oxygen adducts derived from iron
and cobalt. Analysis within the context of the accepted theory
Acknowledgment. We acknowledge funding provided by a
Martin and Mary Kilpatrick Fellowship (M.P.L.), the National
Science Foundation: CAREER award CHE-0449900 (J.P.R)
and CHE-0610183 (C.J.C.), the Research Corporation Cottrell
Scholar Award CS1461 (J.P.R.) and the Alfred P. Sloan
Foundation (J.P.R.). We thank Dr. Charles A. Long for technical
support and Siegfried Schindler and Jim Mayer for helpful
discussions.
Supporting Information Available: Additional synthetic and
procedural details, spectroscopic results, stretching frequencies
from density functional calculations, graphs showing the
calculated temperature dependence of oxygen isotope effects,
and a full list of authors for ref 42. This material is available
free of charge via the Internet at http://pubs.acs.org.
(86) Juda, G. A.; Shepard, E. M.; Elmore, B. O.; Dooley, D. M. Biochemistry
2006, 45, 8788-8800.
(87) (a) Mills, S. A.; Goto, Y.; Su, Q.; Plastino, J.; Klinman, J. P. Biochemistry
2002, 41, 10577-10584. (b) Takahashi, K.; Klinman, J. P. Biochemistry
2006, 45, 4683-4694.
(88) Su, Q.; Klinman, J. P. Biochemistry 1998, 37, 12513-12525.
(89) Mukherjee, A.; Lanci, M. P.; Brown, D. E.; Shepard, E. M.; Dooley, D.
M.; Roth, J. P. Manuscript in preparation.
(90) Welford, R. W. D.; Lam, A.; Mirica, L. M.; Klinman, J. P. Biochemistry
2007, 46, 10817-10827.
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