Direct Copper(III) Formation from O2 and Copper(I) with Histamine Ligation

Article abstract of DOI:10.1021/jacs.6b05538

Histamine chelation of copper(I) by a terminal histidine residue in copper hydroxylating enzymes activates dioxygen to form unknown oxidants, generally assumed as copper(II) species. The direct formation of copper(III)-containing products from the oxygenation of histamine-ligated copper(I) complexes is demonstrated here, indicating that copper(III) is a viable oxidation state in such products from both kinetic and thermodynamic perspectives. At low temperatures, both trinuclear Cu(II)₂Cu(III)O₂ and dinuclear Cu(III)₂O₂ predominate, with the distribution dependent on the histamine ligand structure and oxygenation conditions. Kinetics studies suggest the bifurcation point to these two products is an unobserved peroxide-level dimer intermediate. The hydrogen atom reactivity difference between the trinuclear and binuclear complexes at parity of histamine ligand is striking. This behavior is best attributed to the accessibility of the bridging oxide ligands to exogenous substrates rather than a difference in oxidizing abilities of the clusters.

Full text of DOI:10.1021/jacs.6b05538

Article
pubs.acs.org/JACS
Direct Copper(III) Formation from O2 and Copper(I) with Histamine
Ligation
J. Brannon Gary,† Cooper Citek,† Timothy A. Brown,† Richard N. Zare,† Erik C. Wasinger,‡
and T. Daniel P. Stack*,†
†Department of Chemistry, Stanford University, Stanford, California 94305, United States
‡Department of Chemistry and Biochemistry, California State University, Chico, California 95929, United States
S
* Supporting Information
ABSTRACT: Histamine chelation of copper(I) by a terminal
histidine residue in copper hydroxylating enzymes activates
dioxygen to form unknown oxidants, generally assumed as
copper(II) species. The direct formation of copper(III)-
containing products from the oxygenation of histamine-ligated
copper(I) complexes is demonstrated here, indicating that
copper(III) is a viable oxidation state in such products from
both kinetic and thermodynamic perspectives. At low temperatures, both trinuclear Cu(II)2Cu(III)O2 and dinuclear Cu(III)2O2
predominate, with the distribution dependent on the histamine ligand structure and oxygenation conditions. Kinetics studies
suggest the bifurcation point to these two products is an unobserved peroxide-level dimer intermediate. The hydrogen atom
reactivity difference between the trinuclear and binuclear complexes at parity of histamine ligand is striking. This behavior is best
attributed to the accessibility of the bridging oxide ligands to exogenous substrates rather than a difference in oxidizing abilities of
the clusters.
Terminal histamine ligation (a “histidine brace”) in copper
enzymes is an emerging theme.1,10−16 It has been most recently
characterized in mononuclear polysaccharide monooxygenases,
capable of oxidative degradation of cellulose.10,11 Similar
terminal histamine ligation exists in the proposed dicopper
active sites of particulate methane monooxygenases (pMMO),
which convert methane to methanol,12−14 and, by structural
homology, ammonia monooxygenases (AMO), which convert
ammonia to hydroxylamine.15,16 As this last reaction is essential
in the global nitrogen cycle, it is performed on a massive scale
on this planet by bacteria and archaea with the associated
consumption of dioxygen.17−19
While the histamine brace ligation motif has been established
in biological systems, the nature of solution reactivity by
histamine-ligated copper(I) complexes with dioxygen is
unknown. Two ligands containing primary amine substituents
have been reported that allow for transiently observed copper-
dioxygen adducts by direct oxygenation using stopped-flow
experiments, although their stability has restricted character-
ization.20,21 Given these current limitations, synthetic model
systems that use primary amine ligation in direct oxygenation
reactions could inform on this new and unique ligation
environment to add insight into its possible use and function in
biology.
1. INTRODUCTION
Copper enzymes in biology are capable of dioxygen activation
at mono-, bi-, and trimetallic active sites.1 Given this diversity,
defining the structure−reactivity relationships between Cu−O2
species is foundational to the understanding of critical redox
transformations central to aerobic life. Synthetic systems that
faithfully reproduce biological copper(I) ligation and oxygenate
to discrete, characterizable species provide chemical precedents
for potential reactivity operative in biological systems. While
nature typically uses imidazole ligation via the histidine amino
acid,1 synthetic systems are dominated by pyridine, pyrazole,
and alkylated amine ligation.2,3 Several studies of the direct
oxygenation of copper(I) imidazole-ligated complexes suggest
that μ-η2:η2-peroxodicopper(II) SP and trans-1,2-
peroxodicopper(II) TP species are stabilized by imidazole
subunits (Figure 1).4−9 By comparison, direct oxygenation of
copper(I) complexes with imidazole ligation to bis(μ-oxide)-
dicopper(III) O species or the more elusive trinuclear bis(μ3-
oxide)Cu(II)2Cu(III) T species is heretofore unknown, raising
the question of whether imidazole ligation is compatible with
direct oxygenation to a copper(III) oxidation state.
To alleviate problematic issues of a direct oxygenation with
primary amine ligation to Cu(I) complexes, our group recently
reported a ligand substitution strategy on a preformed copper-
Figure 1. Characterized synthetic dimeric and trimeric copper
complexes derived from oxygenation of Cu(I) complexes.
Received: May 30, 2016
© XXXX American Chemical Society
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dioxygen species (i.e., core capture).22,23 Thus, histamine and
simple primary amine ligation of dimeric Cu(III) O species are
accessible indirectly from more easily oxygenated Cu(I)
complexes. Herein, we expand on the species formed by direct
oxygenation of copper(I) histamine-ligated complexes that
yield both dimeric O and trimeric T species (Figure 1). These
results mark the first example of imidazole copper(I) ligation
that are kinetically and thermodynamically competent to
oxygenate directly to form copper(III) species, highlighting
that, from a chemical perspective, there is no reason to exclude
copper(III) in binuclear or trinuclear copper sites in biological
systems that activate dioxygen.
procedure. These modifications allow for study of primary and
tertiary amine analogues of histamines along with variable
imidazole alkylation, all key properties associated with the
biological histamine brace.1,10−16
2.2. Direct Oxygenation of Histamine Copper(I)
Complexes. Injection of a 1:1 molar solution of LMe3 and
copper(I) into an O2-saturated 2-methyltetrahydrofuran (2-
MeTHF) solution at −125 °C yields a distinct but new optical
spectrum after 5 min (Figure 2), suggestive of the formation of
2. RESULTS
2.1. Histamine Ligands. The role of histamine ligation in
copper-dioxygen chemistry is expanding due to the recent
observations of the histamine brace ligation in copper-
dioxygen-dependent enzymes. While this motif is observed in
resting forms of the enzymes, the nature of the oxygenated
intermediates is unknown experimentally. Oxygenation of
synthetic histamine-ligated copper(I) complexes with O2 to
known intermediates is also unrealized. To address this issue, a
series of substituted histamine ligands were synthesized
(Scheme 1).
Figure 2. Optical absorption spectra of OMe3 (core capture-black) and
the new species (direct oxygenation-red). For comparative purposes,
the extinction coefficients are based upon the initial Cu concentration
(0.9 mM Cu, 2MeTHF, 1 mm path length, SbF6 counterion, −125
°C).
−
a
Scheme 1. Synthesis of Alkylated Histamine Derivatives
a new copper-dioxygen species. By contrast, the formation of a
histamine-ligated dimeric copper(III)-bis-oxide species is
possible using a core capture strategy (i.e., ligand substitution)
at −125 °C; addition of 2 equiv of LMe3 to the preformed
binuclear copper(III) bis-oxide OTMPD precursor (Scheme 2
and Figure 2) efficiently yields an OMe3 species, previously
characterized.19
Scheme 2. Core Capture vs Direct Oxygenation with LMe3
Histamine Ligation
a
Reaction conditions: (a) paraformaldehyde, NaBH3CN; (b) carbon-
yldiimidazole; (c) n-butyl iodide; (d) 12 M HCl; (e) MeI; (f) 12 M
HCl; (g) paraformaldehyde, NaBH3CN.
Eschweiler−Clarke methylation of the amine of histamine is
ineffective in producing LMe2, but reductive amination with
paraformaldehyde and cyanoborohydride results in an efficient,
direct synthesis of the tertiary-amine histamine analogue on
gram scale. This amount allows distillation of ligand from
calcium hydride, which assures maximal formation of the
copper oxygenated intermediates. Methylation of LMe2 to LMe3
using standard methylating agents, however, results in difficult-
to-separate mixtures of the Nτ- versus Nπ-methyl-imidazoles. An
alternative synthetic route to pure LMe3 initially protects the Nπ-
imidazole position as a cyclic urea, followed by Nτ-alkylation,
deprotection, and a reductive amination with paraformaldehyde
and cyanoborohydride. Though longer, each step is efficient
and reproducible, yielding gram quantities of LMe3. Additionally,
functionalization of the cyclic urea intermediate with other alkyl
substituents is possible, which proved critical for the
subsequent purification of LnBu through an organic extractive
With access to the family of histamine derivatives (LMe3, LnBu
,
and LMe2), the generalization of the direct oxygenation of
copper(I) with histamine ligation was optimized. Addition of
1:1 histamine (LMe3, LnBu, and LMe2) to copper(I) mixtures to
an O2-saturated 2-MeTHF solution at −95 °C yielded, within 3
min, similar spectra for all three histamine derivatives (Scheme
3). LMe2 results in lower intensity absorption features,
presumably a result of the lower solubility observed for this
ligand and copper mixture. All of these optical spectra are again
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spectra observed for other bis-oxide trinuclear species, T, one
example of which has been characterized structurally with a
localized valence Cu(III)Cu(II)Cu(II) core (Figure 1).25 All
previously observed T species exhibit two intense UV and two
lower-intensity visible features.25−29 Consistent with other
reported systems, TnBu, TMe3, and TMe2 exhibit at least two
intense UV and two lower-intensity visible features (Figure
3),25−29 although blue-shifted compared to other reported
complexes (Table 1). Specifically, optical transitions of TMe3
and TMe2 are on the high-energy side of reported T species. In
fact, only the anionically ligating β-diketiminate ligand disclosed
by Gupta et al. has a more blue-shifted absorption spectrum.28
With the significant blue-shifting of TnBu, an additional much
less intense visible feature is also observed at 686 nm.
Scheme 3. Trinuclear Complex Formation
distinctly different from those of their corresponding bis-oxide
dimeric O species previously synthesized by core capture.19
The O species formed through core capture (e.g., OMe3) and
the new products of direct oxygenation show markedly different
thermal stabilities. For example, using the LMe3 ligand creates a
new species that is stable at −80 °C with a half-life (t1/2) of ca.
6 min at −40 °C. By comparison, dimeric OMe3 is less thermally
stable, with a t1/2 of 50 min at −80 °C. The thermal stability
difference is even more striking with the primary amine
containing n-butyl histamine ligand (LnBu); the direct oxygen-
ation product is stable at −80 °C, but decays with a t1/2 of 60
min at −40 °C. This relatively high thermal stability is not
observed with the comparable bis-oxide OnBu that thermally
decomposes quickly at −80 °C (t1/2 = 1 min). Thus, the
distinctly different optical absorption spectra and thermal
stabilities indicate that core capture and direct oxygenation
produce distinctly different but low-temperature stable copper-
dioxygen species.
2.3.3. Dinuclear O to Trinuclear T Cluster Conversion.
Further evidence of the T cluster formation during direct
oxygenation results from the addition of 1 equiv of [Cu(I)
L
Me3]SbF6 to a deoxygenated solution of OMe3, formed by core
capture at −125 °C (Scheme 4). This sequence cleanly
converts the OMe3 optical spectrum to that of TMe3 in 30 min
(Figure 4). This conversion corroborates the simplest assign-
ment of TMe3 as a cluster with three copper centers.
The slow rate of this conversion is striking given that the
direct oxygenation of [Cu(I)LMe3]SbF6 at −125 °C is complete
in 5 min. Addition of 1 equiv of [Cu(I)LMe3]CF3SO3 to OMe3
-
SbF6 accelerates the rate of conversion 3-fold (10 min). With
2.3. Trinuclear Cluster Characterization. 2.3.1. O2
Uptake Experiments. In an effort to establish the Cu-to-O2
ratio in the direct oxygenation of copper(I) with histamine
ligation, the product formation was monitored as a function of
substoichiometric equivalents of O2. Using a 10 mM [Cu(I)
−
exclusive use of CF3SO3 anions for both reactants, the
conversion is accelerated 30-fold (1 min) (Table 2).
2.3.4. Cu K-Edge X-ray Absorption Spectroscopy (XAS).
The only crystallographically characterized T cluster, reported
nearly 20 years ago, shows a valence-localized Cu(III)Cu(II)-
Cu(II) formulation with C2 symmetry, with 2.64 Å Cu(II)−
Cu(II) and 2.70 Å Cu(III)−Cu(II) distances, along with short
Cu(III)−O distances at 1.84 Å.25 The Cu K-edge EXAFS
model of TMe3 (Figure 5) requires each copper to have two
copper scatterers at an averaged distance of 2.67 Å, consistent
with a DFT-optimized structure (Cu−Cuaverage 2.67 Å − vide
infra) and the previous crystal structure.25 EXAFS resolution
does not allow separation of the Cu−Cu distances, though the
coordinating ligand shells can be separated into one short Cu−
N/O shell at 1.92 Å and three Cu−N/O scatters at 2.03 Å. The
Cu K-edge pre-edge is dominated by the Cu(II) feature at 8979
eV, obscuring the less intense Cu(III) feature typically found
near 8981 eV.30,31
LnBu]SbF6 solution in 2-MeTHF, dioxygen-saturated tetrahy-
drofuran (10 mM)24 was added in 0.1 equiv aliquots, and the
cluster formation was monitored optically. Maximal cluster
formation for the new species using LnBu was observed at 0.33
equiv of O2 or a Cu-to-O2 ratio of 3.0:1.0. The simplest
interpretation of this stoichiometry is that the direct oxygen-
ation product is a trinuclear copper cluster.25−29 Analogous O2
titrations with 10 mM [Cu(I)LMe3]SbF6 solutions in 2-MeTHF
yielded maximal absorbances with 0.31 equiv of O2 or a Cu to
O2 ratio of 3.2:1.0, again consistent with characterization of a
trinuclear copper cluster.25−29 The direct oxygenated products
with LnBu, LMe3, and LMe2 will be designated as TnBu, TMe3, and
T
Me2, respectively.
2.3.2. UV−Vis Spectroscopy. The resulting optical spectra of
2.3.5. High-Resolution Mass Spectrometry. In an effort to
further characterize the cluster composition, high-resolution
mass spectrometry was used to establish the atomic
composition of the copper clusters. Using this technique,
the newly formed species (Figure 3) are similar to the optical
T
nBu is observed as a monocation with two SbF6− anions at m/z
of 1195.99001 (theoretical 1195.99189) with a distinct isotope
pattern, which is characteristic of three copper and two
antimony atoms (Figure 6). TMe3 is also observed as a
−
monocation with two SbF6 anions at m/z of 1153.94277
(theoretical 1153.94494) and matching isotope pattern.
2.3.6. Trinuclear Cluster Formation Yields. The formation
of a trinuclear cluster produces a distinct optical spectrum from
other copper-dioxygen-derived thermally sensitive intermedi-
ates. The molar extinction coefficients of TnBu, TMe3, and TMe2
are in line with other reported trinuclear clusters (Table 1) but
do not establish a yield for their formation. TnBu, TMe3, and
TMe2 react rapidly with decamethylferrocene (Scheme 5),
providing a convenient titration method to assess their
formation yields by following the disappearance of their
Figure 3. Observed optical absorption spectra of direct oxygenation
products of the Cu(I) complexes of LnBu (blue), LMe3 (red), and LMe2
(green) assuming a trinuclear copper composition (0.9 mM Cu,
−
2MeTHF, 1 mm path length, SbF6 counterion, −95 °C).
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Table 1. Optical Features of Trinuclear Clusters TnBu, TMe3, TMe2, and Literature Trinuclear Clusters
λmax (nm) and ε (mM−1 cm−1
)
a
b
c
optical transition
TnBu
TMe3
TMe2
T
T
T
1
2
3
4
250 (12.1)
300 (12.6)
443 (2.5)
580 (1.3)
285 (14.7)
332 (13.5)
507 (1.4)
655 (0.9)
264 (12.0)
330 (9.0)
505 (0.9)
658 (0.6)
290 (12.6)
355 (15.0)
480 (1.4)
620 (0.8)
N/A
N/A
345 (12.6)
500 (1.3)
620 (0.9)
342 (12.0)
515 (1.0)
685 (0.8)
a
b
c
N,N,N′,N′-(R,R)-1,2-Cyclohexanediamine.25 N,N,N′,N′-Tetramethylethylenediamine.27 N,N-Dimethyl-2-(2-pyridyl)ethylamine.26
Scheme 4. O-to-T Cluster Conversion
Figure 6. Electrospray high-resolution mass spectra of TnBu: top,
experimentally observed isotope pattern; bottom, theoretical predicted
isotope pattern for [(LnBuCu)3O2(SbF6)2]+.
Figure 4. Optical absorption time trace of OMe3 (red) converting to
TMe3 (blue) by the addition of 1 equiv of [Cu(I)LMe3]SbF6 (1.5 mM
total Cu, 2MeTHF, 1 mm path length, −125 °C).
Scheme 5. Trinuclear Cluster Formation Yields by Redox
Titrations
Table 2. Addition of Cu(I) to Preformed OMe3 Species
electron oxidant, the titration of TnBu suggests greater than 70%
formation. A similar optical titration of the permethylated
analogue TMe3 quantifies its formation at 80% and the N−H
imidazole derivative (TMe2) at 40%. The presence of the amine
N−H groups impacts not only the formation yield but also the
thermal stability of the trinuclear cluster (vide supra).
X−
Y−
formation time (min)
−
−
SbF6
SbF6
30
10
1
−
−
−
SbF6
CF3SO3
CF3SO3
CF3SO3
−
2.4. Modified Reaction Conditions: Temperature and
Concentration. In the standard formation conditions
described above, the total copper concentration is ca. 1 mM,
and the reaction temperature is −95 °C. Under these
conditions, only the trinuclear clusters TnBu and TMe3 are
discernible products from the optical spectra in the direct
oxygenation reactions. In an effort to modulate cluster
formation, temperature and concentration were adjusted to
ascertain their effect on cluster formation. Oxygenation of 1
mM [Cu(I)LnBu]SbF6 at −125 °C results in a more complex
optical spectrum, which is resolvable to a 5:1 ratio of the TnBu
and OnBu species (Scheme 6); this analysis is possible using the
known optical spectra of TnBu and OnBu species, which each
possess characteristically intense ligand-to-metal charge transfer
(LMCT) optical bands.22 By contrast, 1 mM [Cu(I)LMe3]SbF6
cleanly oxygenates to TMe3 at −125 °C. Lowering the
concentration of [Cu(I)LMe3]SbF6 to 0.1 mM, however, yields
Figure 5. Fourier transform fit of the TMe3 EXAFS data.
characteristic UV and visible absorption features (see
Supporting Information). Assuming the Cu3O2 core as a one-
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Scheme 6. Trinuclear/Binuclear Mixture Formation
Scheme 8. Isodesmic Comparisons of Trinuclear and
Binuclear Clusters for Electron and H-Atom Transfers
a spectrum resolvable to a 3.5:1 mixture of trinuclear TMe3 and
dimeric OMe3. These results indicate that concentration,
temperature, and ligand steric demands impact the product
distribution of the direct oxygenation reaction; ligands with
lesser steric demands require colder temperatures and lower
copper concentrations to form any appreciable O species.
These results provide possible insights into cluster formation
mechanisms operative during direct oxygenation conditions
(vide infra).
2.5. Comparative Cluster Reactivity with Exogenous
Substates. With access to both O and T species at parity of
histamine ligation, reactivity comparison with a hydrogen atom
donor substrate could be investigated (Scheme 7). TMe3 (0.33
(DKH2) single-point energy,37 a methodology that successfully
correlates and theoretical free energies of Cu(II) and Cu(III)
species.19 The optimization of TnBu as a triplet reproduces the
valence-localized Cu(III)Cu(II)Cu(II) electronic structure,
consistent with previous calculations, and the previously
structurally characterized trinuclear cluster.25,38−40 The relative
positioning of the histamine ligands on the T cluster generates
several isomers (Figure 7), the most stable of which positions
Scheme 7. Trinuclear/Binuclear Relative Proton-Coupled
Electron-Transfer Reactivity
Figure 7. Possible histamine isomers in trinuclear complexes.
mM) undergoes first-order decay with 50 equiv of 5,6-
isopropylidene ascorbic acid with a kobs of 0.02 min−1; the
rate of reaction with OMe3 is estimated to be at least 106 faster.
The latter estimate required using 10-fold less of the ascorbic
acid substrate and more dilute reaction conditions.32 The
dramatic reaction rate difference between clusters is confirmed
in the competitive experiment of adding substoichiometric
amounts of ascorbic acid to OMe3/TMe3 mixtures; at −125 °C
only selective reduction of the optical features of the OMe3
species is observed. After selective OMe3 reduction, OMe3/TMe3
ratios are unchanged with time, indicating no appreciable
interconversion of O and T clusters. A similar reactivity
behavior is observed for OnBu/TnBu mixtures, except that the
difference in their reaction rates is measured to be only 100-
fold. The faster absolute rates of OnBu and TnBu species limits
the ability to differentiate these complexes.32 Trinuclear species
react with substrates at significantly slower rates than their
related binuclear species.
the two Cu(II) ligands in a trans orientation; this isomer is 3−5
kcal mol−1 more stable. The lowest energy one-electron and H-
atom reduced species are calculated to be pseudo-C3 symmetric
quartet species with a Cu(II)Cu(II)Cu(II) electronic structure.
The trans arrangement of the histamine ligands in the O species
is also preferred energetically, although the energy difference is
small (<1 kcal mol−1). Both the one-electron and H-atom
reduced species are calculated to be valence-localized doublet
species.
The isodesmic reaction of the reduced, mixed-valent OMe3
species with the TMe3 species is predicted to have a favorable
free energy of −6.2 kcal mol−1 evaluated at 298 °C, suggesting
that the TMe3 species is a more powerful oxidant. Comparing
the electron reduction affinity between OnBu and TnBu, the
reduced TnBu is favored by ca. 4 kcal mol−1. The favorable
predicted formation of TMe3 and TnBu indicate that these
clusters are 200−300 mV stronger one-electron oxidants than
their binuclear analogues. Comparison of the H-atom affinity of
the binuclear O versus trinuclear T species through isodesmic
reactions is also illustrated in Scheme 8. The H-atom reduced T
clusters are effectively isoenergetic with their O analogues,
suggesting compariable H-atom affinities.
The comparison of the reaction rates between related
species, differentially ligated, is also interesting at −125 °C.
T
nBu, a less sterically encumbered species compared to TMe3,
reacts 10 000 times faster with 5,6-isopropylidene ascorbic acid.
By contrast, access to the dimeric cores is similar, as OnBu reacts
22
only 10-fold faster than OMe3
.
It is difficult to decouple the inherent reactivity of the O and
T species from effects attributable to steric demands. In an
effort to deconvolute the steric and electronic components
controlling reduction of O and T species, the Marcus self-
In an attempt to understand the relative substrate reactivity
of the O and T clusters, a series of isodesmic reactions for
comparison of electron and H-atom affinities were examined
computationally (Scheme 8). Optimizations of all clusters used
a M06 functional, tzvp basis set, and an SMD solvation
model,33−37 followed by a second-order relativistic corrected
exchange reorganizational energy (λ) values of TMe3, TnBu
,
O
Me3, and OnBu were estimated from their difference between
the electronic ground state and the excited state. The electronic
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ground-state energy is estimated as the sum of the cluster and
its one-electron reduced optimized forms, while the excited
state energy is estimated from two single-point calculations in
which ground-state optimized geometries have exchanged
electronic configurations. One-quarter of this reorganization
energy (λ/4) approximates the electronic component or
intrinsic barrier of the one-electron or H-atom reduction of
the copper clusters.41
The calculated self-exchange energy for one-electron
reduction for all four species is ca. 20 kcal mol−1 and differs
by less than 1.7 kcal mol−1 between all four self-exchange
reactions (Table 3). The estimated reaction barriers are small,
in both T and O clusters from external substrates. Previous
calculations by our group suggest that linear attack of a C−H
bond along the O−O bond vector of the O species is the lowest
energy pathway to C−H activation, presumably the same
mechanism of attack operative in the T clusters.22
3. DISCUSSION
The role of the copper(III) oxidation state in biology has been
discounted generally because of the lack of known ligands
present in biology that can stabilize a synthetic copper(III)
containing cluster or spectroscopic observation of copper(III)
in biological systems. The very positive reduction potentials for
mononuclear copper(III) centers ligated by biologically
relevant ligands generally argue against its role in biology.42,43
This work provides a new perspective given the propensity for
histamine-ligated copper(I) species to react with dioxygen to
form copper(III)-containing trinuclear T clusters and, under
specialized conditions, copper(III)-containing O species with
biological histamine ligation. This result highlights that
imidazole ligation is viable to facilitate the formation of high-
valent copper(III) species. Remarkably, this system tolerates
the generally oxidatively susceptible primary amine ligation of
Table 3. Estimated Self-Exchange Reorganizational Energies
a
for Electron and H-Atom Transfers by DFT
electron transfer
H-atom transfer
λ
λ/4
λ
λ/4
TnBu
OnBu
TMe3
OMe3
19.0
19.4
20.7
19.5
4.8
4.8
5.2
4.9
23.1
24.5
24.8
25.3
5.8
6.1
6.2
6.3
a
Energies are given in kcal mol−1. n-Butyl groups of TnBu and OnBu are
L
nBu, which, to our knowledge, is the first example of the direct
modeled as methyl groups.
oxygenation to a stable and characterizable copper-dioxygen
adduct. Previous reports by Schindler have illustrated that
tetradentate ligation containing primary amines are viable to
at ca. 5 kcal mol−1, with a variation of only 0.4 kcal mol−1.
Although slightly higher, the calculated self-exchange energy for
reduction by an H-atom for all four species is ca. 24 kcal mol−1,
which translates to a reaction barrier of 5.8−6.3 kcal mol−1
(Table 3). Assuming similar solvation reorganizational energies
for both T and O clusters, the electronic component to the
barrier of reduction appears to be a minor contributor and
cannot account for the 104−106 rate differences experimentally
observed.
Space-filling models of the four clusters studied for reactivity
(TMe3, TnBu, OMe3, and OnBu) with a trans disposition of ligands
provide a clear perspective of the access to the copper-oxide
cores of each cluster (Figure 8). Histamine methylation in TMe3
clearly imparts significant steric protection of the oxide ligands
T
form superoxide or P species transiently, observable by stop
flow methods, but these species have not been further
characterized.20,21 By adjusting the concentration and temper-
ature, synthetic histamine-ligated copper(I) species are
competent kinetically to oxygenate directly to dimeric OnBu
or OMe3 species with potential relevance to the binuclear active-
site oxidants of pMMO and AMO.22 In this study, the
formation of OnBu or OMe3 is complicated by the thermody-
namic preference for trinuclear formation, a complication not
encountered in pMMO or AMO due to the site-isolation of
their dimeric copper active sites.
In general, the trinuclear cluster has been a minority species
in copper-dioxygen chemistry due to the dearth of ligand
systems that are known to form and stabilize these very
compact cores; ligands with minimal steric demands are
required.25−29,38−40 The comparatively low intensity and highly
blue-shifted nature of the T absorption features, as compared to
S
O, P, and TP, make it easy to discount T formation for
possible indiscriminate copper(II) byproducts when monitor-
ing oxygenation reactions by UV−vis spectroscopy. The optical
features for TnBu, TMe3, and TMe2 are significantly blue-shifted
from those previously reported, further complicating their
characterization.25−29 This blue-shifting is analogous with our
previous reports of O species ligated by LnBu, which acts as a
more strongly donating ligand, presumably raising the energy of
the accepting orbitals for the LMCT bands.22,23 Consistent
with these observations, the primary amine of LnBu is the most
blue-shifted of all T clusters known. Using a combination of
UV−vis, XAS, mass spectrometry, and O2 stoichiometry, the
formation of copper(III)-containing trinuclear T clusters by
direct oxygenation is shown to be viable.
With the few ligands known to produce T clusters, the
mechanism of T cluster formation has remained elusive.
Fukuzami and Itoh have demonstrated that dimeric O species
can serve as a precursor on the pathway to T cluster formation,
although their results do not indicate if the O species is the
direct precursor to T formation or if an unobserved
Figure 8. Space-filling models for the calculated structures trans-TMe3
(top left), trans-TnBu (top right), trans-OMe3 (bottom left), and trans-
OnBu (bottom right). The n-butyl groups of TnBu and OnBu are
modeled and illustrated as methyl groups. Color code: orange =
copper; red = oxygen; blue = nitrogen; gray = carbon; white =
hydrogen.
F
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intermediate is required for the transformation.26 In this work,
the addition of [Cu(I)LMe3]SbF6 to preformed OMe3 provides
insights into the generic T cluster formation mechanism.
Specifically, the 30 min reaction time for OMe3-to-TMe3
conversion is striking, given that the direct oxygenation of
[Cu(I)LMe3]+ at −125 °C is complete in 5 minOMe3 cannot
be on the direct TMe3 formation pathway. Though not observed
optically (Scheme 9), an intermediate species is required.
the trinuclear core: the less sterically demanding LnBu permits
faster reactivity due to the greater exposure of the oxide ligands
and the low barrier pathway for injection of an electron or
hydrogen atom into the cluster. This kinetic rate difference
highlights the highly compact core present in T clusters,
necessitating the use of sterically small ligands for its formation
in general. The contracted Cu−Cu vectors by ca. 0.1 Å in the T
cluster compared to the O clusters contributes additional steric
protection of the oxide ligand in TMe3 and TnBu. Our previous
report, with the reactivity of simple diamines in O being
enhanced by limiting the steric demands of the supporting
ligands, is consistent with the observations of steric effects in
the reactivity of T clusters.20 Also, selective reactivity of the O
species in O/T mixtures highlights that these clusters are not in
equilibrium with each other.
Scheme 9. Proposed T Formation Mechanism
4. CONCLUSION
The role of copper(III) in biology is reasonably discounted for
mononuclear copper systems due to their very positive
reduction potentials with biological ligation.42,43 However,
here faithful biological ligation of copper(I) directly yields two
different copper(III)-containing clusters with dioxygen. All told,
our results indicate that trinuclear copper oxide T clusters are
the thermodynamically preferred product in a homogeneous
solution with sufficiently non-sterically demanding amine or
imidazole ligands, although binuclear O clusters can also be
formed. These T species are also more thermally stable and less
reactive with substrates than their O counterparts. These
complexes represent the first example of copper(I) biological
ligation (histamine brace) that is kinetically competent to form
copper(III) species as the thermodynamic product directly
from O2, hinting at its potential importance in multinuclear
copper enzymes in biology.
−
S
Triflate (CF3SO3 ) counterions are known to bias P ⇄ O
equilibria toward the SP species more than the less coordinating
SbF6 anion.44−46 We observe that the addition of triflate
anions accelerates the OMe3-to-TMe3 conversion. A mechanistic
hypothesis consistent with these observations is that T
formation proceeds through the addition of a copper(I) species
to a peroxide-level intermediate, which we postulate as an P
species (Scheme 9).
The T species formation mechanism above (Scheme 9) is
consistent with the O and T cluster formations observed
experimentally. Cluster selectivity should be tunable, given that
the T species formation, but not the O species formation, is
copper(I) concentration dependent. From other studies, O
species are known to be favored enthalpically relative to their
−
S
45,47
S
5. EXPERIMENTAL DETAILS
isomeric P forms;
lower temperature oxygenations should
S
5.1. General Information. All chemicals were obtained from
commercial sources if not mentioned otherwise. THF solvent was of
HPLC grade and further purified by a Pure-Solv 400 solvent
purification system (Innovative Technology) before storage over
molecular sieves. 2-MeTHF (unstabilized, Aldrich) was degassed
under vacuum and stored under a N2 atmosphere, over 3 Å molecular
sieves. [Cu(MeCN)4]SbF6 was synthesized from Cu2O (Aldrich) and
hexafluoroantimonic acid (Aldrich) by a variation of a literature
method.48 Preparation and manipulation of air-sensitive materials were
carried out in a N2 drybox (MBraun). Low-temperature UV−vis
spectra were collected on a Varian Cary 50 Scan spectrophotometer
with fiber-optic leads to a custom-designed quartz immersion probe
(Hellma) of 0.1 or 1 cm optical path length in a custom-designed
sample cell (ChemGlass). Reactions at −125 and −95 °C were
maintained by N2(l)/pentane and N2(l)/acetone baths, respectively.
1H NMR spectra were collected in CDCl3 on an Inova 300 MHz,
Varian 400 MHz, or Mercury 400 MHz instrument. Reaction kinetics
were followed by either single-wavelength (310, 363, or 380 nm) or
multi-wavelength (200−1000 nm) monitoring. Kinetic analyses were
performed with SPECFIT 3.0.14 program.
All MS data were acquired on an LTQ Orbitrap XL hybrid mass
spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Analyte
solutions were drawn and infused by a manual syringe through a
commercial source set at position D. The spray potential was held at
3.4 kV, and the sheath gas was set to 100 for 100 psi N2. The Orbitrap
ion-transfer capillary was held at 275 °C. The resolution was set to
60 000 at m/z = 400 for all analyses. All data were analyzed using the
Qual Browser feature of the Xcalibur program (Thermo Fisher
Scientific).
bias P ⇄ O equilibrium positions toward the O species.
Consistent with these predictions, lowering the temperature
with [Cu(I)LnBu]SbF6 from −95 to −125 °C results in
increased relative formation of OnBu, and lowering the
concentration of [Cu(I)LMe3]SbF6 from 1 to 0.1 mM Cu at
−125 °C results in increased formation of OMe3. The
temperature and concentration dependence of T versus O
formation is consistent with a bifurcation point to these two
products through an unobserved peroxide-level dimer inter-
S
mediate, postulated as an P species.
It is difficult to decouple the inherent redox reactivity of the
O and T species from effects attributable to steric demands.
Isodesmic reactions from DFT calculations predict that, at
ligand parity, the trinuclear T cluster is a 200−300 mV stronger
oxidant than its related binuclear O analogue. Similar isodesmic
reactions involving a H-atom transfer between T and O clusters
suggest nearly equivalent H-atom affinities. This latter result
suggests comparable proton-coupled electron transfer (PCET)
reaction rates, which are not consistent with our experiments.
The Marcus self-exchange intrinsic barrier differences between
TMe3 and OMe3 are ca. 0.5 kcal mol−1 for H-atom exchange,
while the 106 difference in reaction rate with ascorbic acid, a H-
atom substrate, would indicate an expected difference of at least
4 kcal mol−1 at −125 °C. With the estimated intrinsic barrier
representing a small rate differentiation, steric demands are
implicated as the main component of the impressive reduction
rate difference. We postulate that this dramatic difference
results from the accessibility difference of the oxide ligands of
5.2. Preparation of Materials. Tetramethylpropylene diamine
was obtained from Aldrich. Ligands were stirred over CaH2 under N2
G
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Article
atmosphere before vacuum distillation unless stated otherwise.
Histamine free base was purchased from Matrix Scientific. 5,6-
Isopropylidene-L-ascorbic acid (Aldrich) was dried in a vacuum oven
before use. OnBu and OMe3 were prepared according to literature
methods.19
N2(l)/acetone frozen slurry (−95 °C). The vessel was purged with 1
atm of O2 using a fine needle for ca. 10 min to saturate the solution.
Next, 250 μL of 20 mM LMe2 and 250 μL of 20 mM [Cu(CH3CN)4]-
SbF6 were sequentially injected slowly into the solution (1 mM Cu),
leading to formation of TMe2 after 3 min of stirring.
5.3. Synthesis of Nτ-n-Butyl-histamine (LnBu). Nτ-n-Butyl-
histamine was synthesized from histamine free base according to the
common method of Nτ-alkylation through a cyclic urea intermediate.49
7,8-Dihydro-6H-imidazo[1,5-c]pyrimidin-5-one (2.9 g, 21 mmol, CAS
Registry Number 14509-66-1) and iodobutane (4.3 g, 23 mmol) were
stirred in 50 mL of DMF at 40 °C overnight. DMF solvent was
removed under vacuum. The residue was refluxed in 40 mL of
concentrated aqueous HCl overnight. A purple vapor indicating
gaseous iodine was observed evolving from the refluxing mixture. The
aqueous HCl was removed entirely by rotary evaporation, concurrent
with sublimation of additional iodine. Next, 20 mL of 4 M aqueous
NaOH was added before extraction three times with 30 mL of
dichloromethane. The organic extracts were combined, dried over
granular Na2SO4, and concentrated. The crude oil was stirred over
crushed CaH2 overnight before heated distillation under vacuum to
5.8. O2 Titration with LnBu. The optical immersion probe and
reaction vessel described above were charged with 5 mL of a 10 mM
solution of [Cu(CH3CN)4]SbF6 and LnBu in 2-MeTHF, sealed with a
septum, and equilibrated in a N2(l)/pentane frozen slurry (−125 °C)
under an atmosphere of N2. Next, 500 μL aliquots (0.1 equiv) of O2-
saturated THF (10 mM) were injected and allowed to maximize
formation. Each spectrum of the titrated species was allowed to
stabilize for at least 2 min before addition of more titrant. Fitting the
two linear regions of the absorbance (457 nm, Figure S10) changes
versus equivalents allows the Cu/O2 ratio to be the crossing point of
the two linear regions (0.33 equiv of O2) or Cu/O2 of 3.0.
5.9. O2 Titration with LMe3. The optical immersion probe and
reaction vessel described above were charged with 5 mL of a 10 mM
solution of [Cu(CH3CN)4]SbF6 and LMe3 in dried 2-MeTHF, sealed
with a septum, and equilibrated in a N2(l)/pentane frozen slurry
(−125 °C) under an atmosphere of N2. Next, 500 μL aliquots (0.1
equiv) of O2-saturated THF (10 mM) were injected and allowed to
maximize formation. Each spectrum of the titrated species was allowed
to stabilize for at least 2 min before addition of more titrant. Fitting the
two linear regions of the absorbance (510 nm, Figure S11) changes
versus equivalents allows the Cu/O2 ratio to be the crossing point of
the two linear regions (0.31 equiv of O2) or Cu/O2 of 3.2.
1
give a clear viscous oil. Yield: 0.447 g, 13%. H NMR (400 MHz,
CDCl3): δ 7.31 (s, 1H, aromatic H), δ 6.62 (s, 1H, aromatic H), δ 3.80
(t, 2H, −CH2−), δ 2.92 (t, 2H, −CH2−), δ 2.62 (t, 2H, −CH2−), δ
1.67 (quint., 2H, −CH2−), δ 1.25 (sext., 2H, −CH2−), δ 0.87 (t, 3H,
CH3). 13C NMR (100.6 MHz, CDCl3): δ 140.74, δ 136.62, δ 115.79,
δ 46.79, δ 42.06, δ 33.13, δ 32.57, δ 19.87, δ 13.64. HRMS (H+): m/z
= 168.1499 (C9H18N3, calcd 168.1495).
5.4. Synthesis of Nα,Nα,Nτ-Trimethyl-histamine (LMe3). Nτ-
Methyl-histamine dihydrochloride was synthesized from histamine-free
base through a cyclic urea intermediate.49 Nα-Dimethylation was
adopted from a known procedure.26 In general, Nτ-methyl-histamine
dihydrochloride (1.0 g, 5 mmol), paraformaldehyde (1.5 g, 50 mmol),
and sodium cyanoborohydride (3.1 g, 50 mmol) were stirred in 30 mL
of 0.2 M N4HOAc(aq) (pH 5.4) at 40 °C overnight. Solid sodium
hydroxide was added until pH ∼13. The aqueous solution was
extracted three times with 30 mL of dichloromethane. The organic
extracts were combined, dried over granular Na2SO4, and concen-
trated. The crude oil was stirred over crushed CaH2 overnight before
heated distillation under vacuum to give a clear viscous oil. Yield:
5.10. OMe3 to TMe3 Conversion. OMe3 was made according to
previously reported methods at 1 mM [Cu] at −125 °C in a N2(l)/
pentane frozen slurry.22 O2 was removed from the system by purging
with N2 for 10 min. Next, 250 μL (1 equiv to OMe3) of a 10 mM
solution of [CuLMe3(CH3CN)2]SbF6 was added, and the reaction was
monitored by UV−vis.
5.11. Geometric Predictions for TMe3 and TnBu. Geometry
optimizations of TMe3 and TnBu (butyl group was modeled as a methyl
group, TMe) were performed in C1 symmetry at the B3lyp/6-31g(d)
level of theory in the gas phase with Gaussian09.50−52 Optimized
calculation structures are tabulated in the Supporting Information. Of
the multiple cis/trans isomers tested, the lowest energy structure has a
trans relationship of the histamine ligands on the two Cu(II) metal
centers.
1
0.367 g, 52%. H NMR (400 MHz, CDCl3): δ 7.27 (s, 1H, aromatic
H), δ 6.60 (s, 1H, aromatic H), δ 3.57 (s, 3H, Nτ-methyl), δ 2.71−2.66
(mult., 2H, 2H, −CH2−), δ 2.58−2.52 (mult., 2H, 2H, −CH2−), δ
2.23 (s, 6H, Nα-methyl). 13C NMR (100.6 MHz, CDCl3): δ 141.30, δ
137.19, δ 116.63, δ 59.63, δ 45.57, δ 33.57, δ 26.90. HRMS (H+): m/z
= 154.1340 (C8H16N3, calcd 154.1339).
5.12. High-Resolution Mass Spectrometry of TnBu and TMe3
.
TnBu and TMe3 were formed as described above and stored at −125 °C
in a N2(l)/pentane frozen slurry prior to analysis. The solution was
injected quickly by syringe into the mass spectrometer. No special care
was undertaken to control syringe temperature. The resulting T
clusters were observed as monocations with two associated SbF6
anions (Figures S12−S15).
5.13. Titrations of TnBu, TMe3, and TMe2 with Decamethylfer-
rocene. TnBu, TMe3, and TMe2 were formed by the method described
above. Assuming a concentration [Cu] = 1 mM, 1/10 equiv of
decamethylferrocene (1e− reductant) in THF was added to
deoxygenated solutions of TnBu, TMe3, and TMe2 as formed above at
−95 °C. Each spectrum of the titrated species was allowed to stabilize
for at least 2 min before addition of more titrant. The resulting
titration spectra and absorbance versus equivalent plots are shown in
the Supporting Information. Titration data were used to established
typical formation yields of complexes TnBu, TMe3, and TMe2. Optical
data for one UV band and one visible band are illustrated (Figures S1−
S9) with good agreement between the two wavelengths for each
complex. Lower bound estimates of formation yields are 70%, 80%,
and 40% of complexes TnBu, TMe3, and TMe2, respectively.
5.5. Synthesis of Nα,Nα-Dimethyl-histamine (LMe2). Histamine
(2.0 g, 18 mmol), paraformaldehyde (2.7 g, 90 mmol), and sodium
cyanoborohydride (5.7 g, 90 mmol) were stirred in 60 mL of 0.2 M
NH4OAc(aq) (pH 5.4) at 40 °C overnight. Solid sodium hydroxide
was added until pH ∼13. The aqueous solution was extracted four
times with 30 mL of dichloromethane. The organic extracts were
combined, dried over granular Na2SO4, and concentrated. The crude
oil was purified by heated distillation under vacuum to give a clear
1
viscous oil. Yield: 0.763 g, 31%. H NMR (400 MHz, CDCl3): δ 7.47
(s, 1H, aromatic), δ 6.75 (s, 1H, aromatic H), δ 2.78−2.69 (mult., 2H,
−CH2−), δ 2.59−2.52 (mult., 2H, −CH2−), δ 2.27 (s, 6H, Nα-
methyl). 13C NMR (100.6 MHz, CDCl3): δ 134.52, δ 134.23, δ
119.23, δ 59.21, δ 45.31, δ 24.11. HRMS (H+): m/z = 140.1186
(C7H14N3, calcd 140.1182).
5.6. General Method of Formation of TnBu and TMe3. The
optical immersion probe and reaction vessel described above were
charged with 5 mL of 2-MeTHF, sealed with a septum, and
equilibrated in a N2(l)/acetone frozen slurry (−95 °C). The vessel
was purged with 1 atm of O2 using a fine needle for ca. 10 min to
saturate the solution. A 10 mM solution of [Cu(CH3CN)4]SbF6 and L
(LnBu, LMe3) in 500 μL of THF was injected slowly into the solution (1
mM Cu), leading to formation of TnBu and TMe3 after 3 min of stirring.
5.7. General Method of Formation of TMe2. The optical
immersion probe and reaction vessel described above were charged
with 5 mL of 2-MeTHF, sealed with a septum, and equilibrated in a
5.14. Mixture of TnBu and OnBu. The optical immersion probe and
reaction vessel described above were charged with 5 mL of 2-MeTHF,
sealed with a septum, and equilibrated in a N2(l)/pentane frozen slurry
(−125 °C). The vessel was purged with 1 atm of O2 using a fine
needle for ca. 10 min to saturate the solution. A 10 mM solution of
[Cu(CH3CN)4]SbF6 and LnBu in 500 μL of THF was injected slowly
into the solution, leading to formation of a mixture of TnBu and OnBu
H
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Journal of the American Chemical Society
Article
after 5 min of stirring. The mixture was fit by addition of pure optical
spectra for independently synthesized TnBu and OnBu (Figure S16).
5.15. Mixture of TMe3 and OMe3. The optical immersion probe
and reaction vessel described above were charged with 11 mL of
purified and distilled 2-MeTHF, sealed with a septum, and equilibrated
in a N2(l)/pentane frozen slurry (∼ −125 °C). The vessel was purged
with pure O2 using a fine needle for ca. 10 min to saturate the solution.
A 10 mM solution of [Cu(CH3CN)4]SbF6 and LMe3 in 110 μL of THF
was injected slowly into the solution, leading to formation of a mixture
of TMe3 and OMe3 after 5 min of stirring. The mixture wass fit by
addition of pure optical spectra for independently synthesized TMe3
and OMe3 (Figure S17).
Molecular Biology Program is supported by the DOE Office
of Biological and Environmental Research, and by the National
Institutes of Health, National Institute of General Medical
Sciences (including P41GM103393). The contents of this
publication are solely the responsibility of the authors and do
not necessarily represent the official views of NIGMS or NIH.
This work was supported by a subcontract with the University
of Utah (Agreement 10029173-S2) for which the Air Force
Office of Scientific Research (Grant FA9550-12-1-0481) is the
prime sponsor.
5.16. Reactions of O and T with Ascorbic Acid. OnBu and OMe3
were prepared by core capture strategies.22 TnBu and TMe3 were
prepared as illustrated above at −125 °C in a N2(l)/pentane frozen
slurry. 5,6-Isopropylidene-L-ascorbic acid was added, and the
decomposition was monitored by UV−vis. Kinetic rates are detailed
in the Supporting Information (Table S1).
5.17. Computational Isodesmic Reaction Energies. Geometry
optimizations were performed in C1 or Ci symmetry (when possible)
at the M06/TZVP level of theory using an SMD (water) solvation
model with Gaussian09 at multiple spin states if possible; only lowest
energy structure is tabulated in the Supporting Information.33−36
Single-point structures were calculated at the M06/TZVP level of
theory using an SMD (water) solvation model and a second-order
relativistic correction (DKH2) with Gaussian09.37 Optimized
structures of all calculated complexes are tabulated in the Supporting
Information.
5.18. Marcus Theory Calculations for TMe3, TnBu, OMe3, and
OnBu. Geometry optimizations of TMe3 and TnBu (butyl group was
modeled as a methyl group TMe) were performed in C1 symmetry at
the M06/TZVP level of theory using an SMD (water) solvation model
with Gaussian 09 with multiple spin states if possible; only lowest
energy structure is tabulated in the Supporting Information.33−36
Single-point structures were calculated at the M06/TZVP level of
theory using an SMD (water) solvation model and a second-order
relativistic correction (DKH2) with Gaussian 09.37 Associated OMe3
and OnBu (butyl group was modeled as a methyl group OMe) structures
were calculated in either C1 or Ci symmetry when viable. H-atoms
were placed at geometrically equivalent positions in the oxidized forms
as found in the optimized structures of the reduced form. Optimized
structures of all calculated complexes are tabulated in the Supporting
Information.
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ASSOCIATED CONTENT
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■
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DOI: 10.1021/jacs.6b05538
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