A dopaquinone model that mimics the water addition step of cofactor biogenesis in copper amine oxidases

Article abstract of DOI:10.1021/ja0455603

The consensus mechanism for biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper amine oxidases involves a key water addition to the dopaquinone intermediate. Although hydration of o-quinones seems straightforward and was implicated previously in aqueous autoxidation of catechols to give ultimately hydroxyquinones, a recent study (Mandal, S.; Lee, Y.; Purdy, M. M.; Sayre, L. M. J. Am. Chem. Soc. 2000, 122, 3574-3584) showed that the observed hydroxyquinones arise not from hydration, but from addition to the o-quinones of H₂O₂ generated during autoxidation of the catechols. In the enzyme case, hydration of dopaquinone is proposed to be mediated by the active site Cu(II). To establish precedent for this mechanism, we engineered a catechol tethered to a Cu(II)-coordinating unit, such that the corresponding o-quinone could be generated in situ by oxidation with periodate (to avoid generation of H₂O₂). Thus, coordination of 4-((2-(bis(2-pyridylmethyl)amino)ethylamino)methyl)-1,2-benzenediol (1) to Cu(II) and subsequent addition of periodate resulted in rapid formation of the TPQ-like corresponding hydroxyquinone. Hydroxyquinone formation was seen also using Zn(II) and Ni(II), but not in the absence of M(II). Under the same conditions, periodate oxidation of the simple catechol 4-fert-butylcatechol does not give hydroxyquinone in the presence or absence of Cu(II). M ^IIOH₂ pK_a data for the Cu(II), Zn(II), and Ni(II) complexes with the pendant tetradentate ligand in the masked (dimethyl ether) catechol form, and kinetic pH-rate profiles of the metal-dependent hydroxyquinone formation from periodate oxidation of catechol 1, suggested a rate-limiting addition step of the ligand-coordinated M^IIOH to the o-quinone intermediate. This study represents the first chemical demonstration of a true o-quinone hydration, which occurs in cofactor biogenesis in copper amine oxidases.

Full text of DOI:10.1021/ja0455603

Published on Web 03/11/2005
A Dopaquinone Model That Mimics the Water Addition Step of
Cofactor Biogenesis in Copper Amine Oxidases
Ke-Qing Ling and Lawrence M. Sayre*
Contribution from the Department of Chemistry, Case Western ReserVe UniVersity,
CleVeland, Ohio 44106
Received July 23, 2004; E-mail: lms3@case.edu
Abstract: The consensus mechanism for biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ)
cofactor in copper amine oxidases involves a key water addition to the dopaquinone intermediate. Although
hydration of o-quinones seems straightforward and was implicated previously in aqueous autoxidation of
catechols to give ultimately hydroxyquinones, a recent study (Mandal, S.; Lee, Y.; Purdy, M. M.; Sayre, L.
M. J. Am. Chem. Soc. 2000, 122, 3574-3584) showed that the observed hydroxyquinones arise not from
hydration, but from addition to the o-quinones of H2O2 generated during autoxidation of the catechols. In
the enzyme case, hydration of dopaquinone is proposed to be mediated by the active site Cu(II). To establish
precedent for this mechanism, we engineered a catechol tethered to a Cu(II)-coordinating unit, such that
the corresponding o-quinone could be generated in situ by oxidation with periodate (to avoid generation of
H2O2). Thus, coordination of 4-((2-(bis(2-pyridylmethyl)amino)ethylamino)methyl)-1,2-benzenediol (1) to
Cu(II) and subsequent addition of periodate resulted in rapid formation of the TPQ-like corresponding
hydroxyquinone. Hydroxyquinone formation was seen also using Zn(II) and Ni(II), but not in the absence
of M(II). Under the same conditions, periodate oxidation of the simple catechol 4-tert-butylcatechol does
not give hydroxyquinone in the presence or absence of Cu(II). MIIOH2 pKa data for the Cu(II), Zn(II), and
Ni(II) complexes with the pendant tetradentate ligand in the masked (dimethyl ether) catechol form, and
kinetic pH-rate profiles of the metal-dependent hydroxyquinone formation from periodate oxidation of
catechol 1, suggested a rate-limiting addition step of the ligand-coordinated MIIOH to the o-quinone
intermediate. This study represents the first chemical demonstration of a true o-quinone hydration, which
occurs in cofactor biogenesis in copper amine oxidases.
Introduction
site tyrosine-derived 2,4,5-trihydroxyphenylalanine quinone
(TPQ) cofactor to catalyze oxidative deamination of primary
Copper amine oxidases (CAOs) are a ubiquitous family of
copper-containing enzymes found in bacteria, yeast, plants, and
mammals.1 In microorganisms, CAOs permit the use of low
molecular weight amines as sources of carbon and nitrogen,
whereas in plants, CAOs are involved in developmental
processes and wound healing.2,3 In mammals, CAOs play
important roles in a variety of physiological processes including
detoxification, signaling, cell growth and maturation, cell
adhesion, lipid trafficking, drug binding, and glucose homeo-
stasis, and are also implicated in a number of human pathologi-
cal conditions such as diabetes, atherosclerosis, cardiovascular
diseases, and Alzheimer’s disease.4 Most CAOs utilize an active
amines to aldehydes and ammonia at the expense of reducing
oxygen to hydrogen peroxide.1,5
TPQ cofactor biogenesis was studied extensively in the 1990s
using both wild-type and active site mutant apoproteins of
different sources, showing it to be a posttranslational self-
processing reaction.6 Early biogenesis proposals6e-h,7 suggested
an initial monooxygenation of phenol to give catechol, which
subsequently was oxidized to an o-quinone intermediate. How-
ever, more recent proposals suggested a direct 4e- oxidation
(5) (a) Janes, S. M.; Mu, D.; Wemmer, D.; Smith, A. J.; Kaur, S.; Maltby, D.;
Burlingame, A. L.; Klinman, J. P. Science 1990, 248, 981-987. (b) Brown,
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J. AM. CHEM. SOC. 2005, 127, 4777-4784
4777

A R T I C L E S
Scheme 1
Ling and Sayre
Scheme 2 a
of the phenol to give o-quinone.8 The current consensus
mechanism for the overall 6e- oxidation biogenesis9 involves
interaction of the tyrosine phenol with the nearby trihistidinyl-
CuII,10 resulting in O2-dependent monooxygenation to generate
the dopaquinone (DPQ) intermediate and a residual trihistidinyl-
CuIIOH, followed by conjugate addition of the Cu(II)-bound
water to form the reduced triol form of TPQ,8 which then
undergoes autoxidation to TPQ (Scheme 1). However, since the
water delivered to C2 of the mature cofactor was shown to
derive from bulk solvent and not O2,6h the initially O2-derived
Cu(II)-bound water would have to exchange with bulk solvent
water before conjugate addition to the o-quinone took place.
Ligand exchange on Cu(II) is known to be rapid.
a Reagents and conditions: (a) (1) 2-picolyl chloride hydrochloride/
NaOH/THF/H2O/50 °C; (2) HCl/EtOH; (b) (1) 3,4-dihydroxybenzaldehyde/
HOAc/MeOH/NaBH4; (2) Boc2O/TEA/DCM; (3) HCl/EtOH; (c) (1) 3,4-
dimethoxybenzaldehyde/HOAc/MeOH/NaBH4; (2) Boc2O/TEA/DCM; (3)
HCl/EtOH; (d) (1) 2,4,5-trihydroxybenzaldehyde/HOAc/MeOH/NaBH4; (2)
Boc2O/TEA/DCM; (3) HCl/EtOH.
The chemically most challenging step in the proposed
biogenesis mechanism is the first Cu(II)-mediated monooxy-
genation, since most copper-mediated oxygenation reactions in
biology entail a Cu(I)-mediated activation of O2. In contrast,
the step involving conjugate addition of water to the o-quinone
seems straightforward, and was implicated earlier in the aqueous
autoxidation of dopamine to form the neurotoxin 6-hydroxy-
dopamine11 as well as in model TPQ cofactor biogenesis systems
that start with the catechol as a predecessor to the o-quinone.12
However, we recently demonstrated that starting directly with
o-quinones, water addition is very slow, even at elevated pH,
and that TPQ-like hydroxyquinones formed in the aqueous
autoxidation of catechols arise from conjugate addition to
o-quinone of H2O2 generated in the catechol autoxidation step.13
We therefore suggested that the o-quinone hydration step in
TPQ biogenesis must be a facilitated event, possibly by the
active site Cu(II).13 Although the latest TPQ biogenesis propos-
als typically depict the o-quinone being hydrated by the
trihistidinyl-CuIIOH species (which has evidently undergone
exchange),8,9 there is so far no direct chemical precedent for
such a facilitated o-quinone hydration.
not occur at physiological pH. We then have developed a novel
DPQ model with a pendant Cu(II)-chelating complex that readily
accomplishes Cu(II)-mediated quinone hydration at neutral pH.
To our knowledge, this is the first chemical demonstration of a
true o-quinone hydration, proposed in the consensus mechanism
of TPQ biogenesis.
Results and Discussion
Synthesis of Models. The target model in its catechol version
(1) was synthesized by reductive amination of 3,4-dihydroxy-
benzaldehyde with N,N-bis(2-pyridylmethyl)ethylenediamine
(BPEN) (Scheme 2). Direct isolation of 1 is inconvenient due
to its zwitterionic property, and pure product was obtained by
Boc protection and then deprotection of the purified tri-Boc-
protected ligand 1 under mild conditions (HCl/EtOH). To study
the Cu(II) coordination chemistry and to characterize the final
hydroxyquinone product, the redox-inactive dimethylcatechol
2 and the triol 3 were also synthesized by similar reductive
aminations with BPEN of 3,4-dimethoxybenzaldehyde and
2,4,5-trihydroxybenzaldehyde, respectively, using the same Boc
protection-deprotection approach. All three were obtained as
the corresponding tri-HCl salts.
In this paper, using a H2O2-free protocol, we have further
confirmed that hydration of a simple DPQ model quinone does
Acid-Base Properties and Coordination Chemistry of
Model Ligand 2 with Selected Metal Ions. Ligand 2 forms a
1:1 complex with Cu(II) as shown by UV-vis titration of
Cu(II) with the free base form of 2 (see the Experimental Section
and Figure S1 in the Supporting Information). The acid-base
properties of 2 and its coordination chemistry with Cu(II),
Zn(II), and Ni(II) were studied by potentiometric pH titration
(Figure 1A).14 The multistep protonation constants pK1, pK2,
and pK3 of ligand 2 were found to be 9.07, 5.00, and 3.08,
respectively. The fourth protonation occurs at strongly acidic
pH. This explains why ligand 2 was obtained as a tri- rather
than tetra-HCl salt. The stability constants (log Kf) for formation
of the 1:1 complexes between 2 and Cu(II), Zn(II), and Ni(II)
were all >10 (Table 1). Of particular interest are the acid disso-
ciation pKa values for the metal-bound water in the 1:1 com-
(8) (a) Ruggiero, C. E.; Smith, J. A.; Tanizawa, K.; Dooley, D. M. Biochemistry
1997, 36, 1953-1959. (b) Wilce, M. C. J.; Dooley, D. M.; Freeman, H.
C.; Guss, J. M.; Matsunami, H.; McIntire, W. S.; Ruggiero, C. E.; Tanizawa,
K.; Yamaguchi, H. Biochemistry 1997, 36, 16116-16133. (c) Ruggiero,
C. E.; Dooley, D. M. Biochemistry 1999, 38, 2892-2898. (d) Dooley, D.
M. J. Biol. Inorg. Chem. 1999, 4, 1-11.
(9) Brazeau, B. J.; Johnson, B. J.; Wilmot, C. M. Arch. Biochem. Biophys.
2004, 428, 22-31.
(10) (a) Chen, Z.-W.; Schwartz, B.; Williams, N. K.; Li, R.; Klinman, J. P.;
Mathews, F. S. Biochemistry 2000, 39, 9709-9717. (b) Dove, J. E.;
Schwartz, B.; Williams, N. K.; Klinman, J. P. Biochemistry 2000, 39, 3690-
3698. (c) Schwartz, B.; Dove, J. E.; Klinman, J. P. Biochemistry 2000, 39,
3699-3707. (d) Schwartz, B.; Olgin, A. K.; Klinman, J. P. Biochemistry
2001, 40, 2954-2963. (e) Kim, M.; Okajima, T.; Kishishita, S.; Yoshimura,
M.; Kawamori, A.; Tanizawa, K.; Yamaguchi, H. Nat. Struct. Biol. 2002,
9, 591-596.
(11) (a) Garcia-Moreno, M.; Rodriguez-Lopez, J. N.; Martinez-Ortiz, F.; Tudela,
J.; Varon, R.; Garcia-Canovas, F. Arch. Biochem. Biophys. 1991, 288, 427-
434. (b) Rodriguez-Lopez, J. N.; Varon, R.; Garcia-Canovas, F. Int. J.
Biochem. 1993, 1175-1182. (c) Senoh, S.; Creveling, C. R.; Udenfriend,
S.; Witkop, B. J. Am. Chem. Soc. 1959, 81, 6236-6240.
(12) Rinaldi, A. C.; Porcu, M. C.; Curreli, N.; Rescigno, A.; Finazziagro, A.;
Pedersen, J. Z.; Rinaldi, A.; Sanjust, E. Biochem. Biophys. Res. Commun.
1995, 214, 559-567.
(13) Mandal, S.; Lee, Y.; Purdy, M. M.; Sayre, L. M. J. Am. Chem. Soc. 2000,
122, 3574-3584.
(14) Martell, A. E.; Motekaitis, R. J. Determination and Use of Stability
Constants, 2nd ed.; VCH: New York, 1992.
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Water Addition Step of Cofactor Biogenesis in CAOs
A R T I C L E S
Figure 2. Periodate oxidation of 1 or 3 in 0.05 M pH 7.5 MOPSO buffer
at 25 °C: (A) UV-vis spectra recorded immediately upon mixing of a
solution of 1-Cu(II) or 3-Cu(II) (1 × 10-4 M) with NaIO4 (2 × 10-4 M);
(B) UV-vis spectra recorded immediately upon mixing of a solution of 1
or 3 (5 × 10-5 M) with NaIO4 (1 × 10-4 M), and then, in the case of 1,
recorded again upon addition of Cu(II) (5 × 10-5 M). In all cases, no further
spectral changes were observed after the first spectrum was recorded.
Figure 1. (A) Titration curves of 5.00 mM 2‚3HCl or a mixture of 5.00
mM 2‚3HCl and 5.00 mM M(II) with NaOH in 0.15 M KNO3 aqueous
solution in the presence of 5.00 mM HNO3. (B) Speciation diagram for
5.00 mM 2/5.00 mM Cu(II) in 0.15 M KNO3 as a function of pH at 25 °C.
Table 1. Equilibrium Constants of 1:1 Complexes of 2 with
Selected Metal Ionsa
Cu(II)
Zn(II)
Ni(II)
chols. Also, to avoid complications arising from production of
H2O2 via autoxidation events, an excess of periodate was main-
tained, ensuring immediate oxidation of not only the triol gen-
erated in the hydration reaction, but also any catechol arising
from reduction of o-quinone by o-quinone decomposition prod-
ucts.13 Formation of hydroxyquinone under such conditions can
thus be taken as a reliable indicator of “true” quinone hydration.
To validate the periodate protocol, we reevaluated the
behavior of 4-tert-butyl-1,2-benzoquinone (5) generated in situ
by periodate oxidation of catechol 4, which, under autoxidation
conditions, affords hydroxyquinone 6.13
log Kf
13.2
7.87
11.1
8.60
13.0
10.6
pKa of LMIIOH2
a Calculated from potentiometric pH titration data in 0.15 M aqueous
KNO3 at 25 °C.
plexes, which show a rank order consistent with the Lewis acid-
ity of these divalent metal ions. The speciation diagrams14 for
2-M(II) as a function of pH show domination of two consecu-
tive acid-base equilibria, MIIHL/MIIL and MIIL/MIIH-1L in
acidic and neutral-basic regions, respectively, as exemplified
by 2-Cu(II) (Figure 1B). The involvement of these metal
species was further confirmed by UV-vis monitoring of the
potentiometric pH titration of 2-Cu(II), and the λmax values of
these species were 672 nm for CuIIHL, 844 nm for CuIIL, and
837 nm for CuIIH-1L (Figure S2 in the Supporting Information).
Periodate Oxidation of 4-tert-Butylcatechol Does Not Give
Hydroxyquinone in pH 7.5 Buffer. To study quinone hydra-
tion, we have chosen to generate the required but only meta-
stable o-quinone species in situ by periodate oxidation of cate-
Incubation of 4 with 2 mol equiv of NaIO4 in 0.05 M pH 7.5
â-hydroxy-4-morpholinepropanesulfonic acid (MOPSO) buffer
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A R T I C L E S
Ling and Sayre
instantaneously generated quinone 5 (λmax ) 400 nm),13 which
then converted (t1/2 of ∼1 h) in an isosbestic fashion to a new
species with λmax ≈ 250 nm, without apparent formation of
any hydroxyquinone 6, as shown by time-dependent difference
UV-vis spectroscopy (Figure S3 in the Supporting Information).
Identical behavior was seen starting with authentic 5 under the
same conditions (data not shown). Bleaching of authentic 6 was
found to be fairly slow under the same conditions (3% over 1
h), and did not generate the 250 nm species (Figure S3),
suggesting that quinone hydration is not occurring under this
condition. The presence of 1 equiv of CuSO4 (free or coordi-
nated 1:1 to bis(2-pyridylethyl)amine) in this system was found
to alter the stability of various intermediates and products, but
we saw no evidence for even transient generation of 6
(Supporting Information). Thus, in an intermolecular sense,
Cu(II) appears unable to sufficiently promote o-quinone hydra-
tion in this simple model.
crystal structures show coordination of the TPQ enolate O4 to
Cu(II), are colorless.5e
Importantly, periodate oxidation of 1 in the absence of M(II)
instantaneously afforded a light yellow solution (λmax ) 330
and 407 nm) with no evidence for the copresence of even a
trace of hydroxyquinone 7 (compare to the spectrum for
periodate oxidation of 3 under the same conditions, Figure 2B).
In addition, subsequent addition of 1 equiv of Cu(II) (or Zn(II)
or Ni(II)) did not stimulate generation of 7 (Figures 2B and S7
in the Supporting Information). These results show that hydra-
tion of the o-quinone moiety of 8 can only be achieved when
it is generated at the point M(II) is prebound to the ligand
system, and that, in the absence of M(II), the transiently formed
8 rapidly converts to a species with λmax at 330 and 407 nm.
The fate of quinone 8 in the absence of metal ion is unclear,
as all attempts to determine the nature of the product(s) have
failed so far due to continuous decomposition of the product
mixture during isolation. In fact, a whole series of variously
N-substituted quinone models 10, generated by periodate
oxidation of the corresponding catechols in pH 7.5 MOPSO
buffer, all decompose very quickly in the absence of M(II) and
show a UV-vis spectral behavior (λmax ≈ 330 and 405 nm)
very similar to that of “decomposed” 8.16 On this basis and the
finding that quinone model 11 is much more stable than 8 under
the same conditions,16 we conclude that the rapid decomposition
of 8 and its relatives (10) is most likely initiated by an
intramolecular conjugate addition of the distal tertiary amino
group to the quinone ring, which apparently is more favorable
than the intermolecular water addition. We have observed
intramolecular conjugate addition reactions for other o-quinones
containing pendant OH, COOH, and NH2 nucleophiles.16
Behavior of the Catechol Ligand 1 in pH 7.5 Buffer. In
contrast to the unsuccessful hydration of quinone 5, when the
catechol ligand model 1 was first coordinated with 1 equiv of
Cu(II), and then subjected to periodate oxidation in 0.05 M pH
7.5 MOPSO buffer, the solution immediately turned red (λmax
) 500 nm), the expected color of the TPQ-like hydroxyquinone
anion 7-Cu(II). Formation of 7 was verified by spectral
comparison with authentic 7-Cu(II) generated from periodate
oxidation of 3-Cu(II) under the same conditions (Figure 2A),
and further confirmed by HPLC analysis after reduction to
authentic triol 3 (see the Experimental Section and Figure S6
in the Supporting Information). The yield of 7-Cu(II) was
almost quantitative (93% by UV-vis), and the reaction was so
fast that the o-quinone intermediate 8-Cu(II) could not be
detected by routine spectrophotometry.
Interestingly, the M(II)-mediated quinone hydration was
found to be significantly inhibited by phosphate buffer; e.g.,
periodate oxidation of 1-Cu(II) in 0.05 M pH 7.5 phosphate
buffer gave 7-Cu(II) in only 48% yield (by UV-vis), along
with a major side product that is identical with that formed when
1 equiv of Cu(II) is added to a preoxidized mixture of 1 in the
same buffer (Figure S8 in the Supporting Information).
The crystal structures of Cu(II) complexes with tripodal
tetradentate ligands are well-known,17 and the Cu(II) may adopt
geometries ranging from typical trigonal bipyramidal to distorted
square pyramidal depending on the nature of the ligands used.
Because the UV-vis spectrum of complex 2-Cu(II) in aqueous
solution exhibits a λmax at 844 nm rather than 650 nm, we
conclude that Cu(II) mainly adopts a trigonal bipyramidal rather
than a distorted square pyramidal geometry,17b with the tertiary
amine and a fifth water ligand occupying the two axial positions.
We believe that this axial water molecule plays a key role in
Under the same conditions, complexes 1-Zn(II) and
1-Ni(II) also underwent quinone hydration to give 7-Zn(II)
(UV-vis yield 92%) and 7-Ni(II) (UV-vis yield 82%), but
the red color was seen to form more slowly, with a transient
yellow color, presumably representing the intermediate o-qui-
none, being seen in the case of Ni(II). In contrast, reaction of
1-Co(II) was complicated by rapid deterioration of the hy-
droxyquinone product as verified by periodate oxidation of
3-Co(II) under the same conditions. The ∼500 nm absorbance
observed for all M(II) complexes of 7 suggests that the
hydroxyquinone anion is not coordinated to the metal center in
the product complex, even though the enolate O2 should be
capable of doing so geometrically. Such coordination would
be expected to induce a significant blue shift of the hydroxy-
quinone chromophore due to charge localization,15 as implicated
by the fact that inactive forms of copper amine oxidases, whose
(16) Ling, K. Q.; Sayre, L. M. Unpublished results.
(17) (a) Nagao, H.; Komeda, N.; Mukaida, M.; Suzuki, M.; Tanaka, K. Inorg.
Chem. 1996, 35, 6809-6815. (b) Weitzer, M.; Schatz, M.; Hampel, F.;
Heinemann, F. W.; Schindler, S. J. Chem. Soc., Dalton Trans. 2002, 686-
694.
(15) In fact, we have observed an 81 nm blue shift of the hydroxyquinone
chromophore (from 493 to 412 nm) in another synthetic hydroxyquinone
ligand model upon coordination with Cu(II).16
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Water Addition Step of Cofactor Biogenesis in CAOs
A R T I C L E S
Scheme 3
of hydroxyquinone 7 is shown in Scheme 3. Control experiments
demonstrated that oxidations of catechol and triol by periodate
were essentially instantaneous, even at 5 °C. Thus, the rate-
limiting step for formation of 7 in Figure 3 must correspond to
a reaction other than periodate oxidation of catechol or triol
intermediates. Since aromatization of unstable tautomer 9 to
triol 3 should also be very fast, we believe that the rate-limiting
step is quinone hydration after equilibrium deprotonation of the
Cu(II)-bound water (Scheme 3). According to this mechanism,
the pH-rate profiles should reflect the critical ionization
equilibrium.
To obtain kinetic data over a wide pH range, which is not a
function of the particular buffer used in each corresponding
pH range,18 we introduced a mixed “uniVersal” buffer solu-
tion composed of 0.1 M acetate, 0.05 M MES, and 0.05 M
HEPES. The pH-rate profiles for formation of 7-M(II) from
the 1-Cu(II), 1-Zn(II), and 1-Ni(II) complexes in this
universal buffer are shown in Figure 3 (inset). Rate data could
not be obtained at high pH (>8.5 for Ni(II), >8.0 for Zn(II)
and Cu(II)) either because of rapid decomposition of complexes
7-M(II) or apparent complexities in the solubility behavior of
the metal complexes (especially Cu(II)) at basic pH.19 In the
pH range shown, the pH-rate profiles for all three M(II)
complexes show a monotonic increase with increasing pH that
is consistent with a rate-limiting quinone hydration step associ-
ated with an ionization equilibrium. A least-squares fit of the
the observed quinone hydration (Scheme 3). The inhibition of
quinone hydration by phosphate can be interpreted in terms of
preferential coordination of phosphate (substituting for the water)
as the fifth metal ligand. We see no reason for phosphate to
inhibit the quinone hydration reaction if it were not for the strict
metal ion dependence of this process.
It should be noted that o-quinones such as 11 should serve
as reasonable models to mimic water addition through a general-
base catalysis (GBC) mechanism (a favorable six-membered
ring transition state is depicted in 12). The fact that none of
them display significant conversion to the corresponding hy-
droxyquinone16 suggests that the GBC mechanism is not a facile
process in solution compared to other possible decomposition
pathways of these highly unstable o-quinones.
Kinetics of M(II)-Mediated Quinone Hydration. In contrast
to the very rapid Cu(II)-mediated quinone hydration at neutral
pH, at lower pH (e.g., pH 4 acetate buffer), the intermediacy of
the o-quinone 8-Cu(II) in formation of the final product
7-Cu(II) can be seen by routine spectrophotometry (Figure 3).
A clean isosbestic conversion of 8-Cu(II) (λmax ) 380 nm) to
7-Cu(II) is observed. A reasonable mechanism for formation
+
Cu(II) data to kobsd ) kKapp/(Kapp + RH ) yielded a first-order
rate constant k of 1.28 ( 0.15 s-1, and an apparent ionization
constant pKapp of 7.93 ( 0.09, which is close to the titrated
pKa of 2-Cu(II) (Table 1). Though there are insufficient data
to derive kinetic constants in the cases of Zn(II) and Ni(II), the
pH-rate profiles are consistent with kinetic pKapp values that
match the titrated pKa values for 2-M(II).
For the enzyme, the rate of quinone hydration in TPQ
biogenesis is unknown because it is not the rate-limiting step.
The half-life of the overall biogenesis process for AGAO has
been reported to be about 30 s.8c Assuming that the hydration
step is about 10 times this fast, it would be comparable to the
half-life of Cu(II)-mediated hydration in our model system,
which can be estimated to be a few seconds at pH 7.
Conclusion. We have obtained a model (1) that allows access
to a DPQ-like intermediate (8), which simulates the Cu(II)-
mediated o-quinone hydration step proposed in the consensus
mechanism of TPQ biogenesis.8-10 The success of this model
compared to simpler models (e.g., 5) where o-quinone decom-
position predominates over hydration appears to reflect the
favorable intramolecular delivery of the Cu(II)-bound OH, a
better nucleophile than bulk solvent water, to the o-quinone C2.
Thus, the occurrence of DPQ hydration in the enzyme must
reflect in part the entropic advantage afforded by the active site
orienting the DPQ intermediate with respect to the trihistidinyl-
CuIIOH to facilitate the desired water addition.8 The active site
(18) As an example, the observed rate constant (0.345 s-1) for formation of
7-Cu(II) from periodate oxidation of 1-Cu(II) in 0.05 M pH 5.5 MES
buffer is nearly 10-fold faster than that (0.0404 s-1) of the same reaction
in 0.05 M pH 5.5 acetate buffer at 25 °C, showing a significant inhibitory
effect of acetate on the quinone hydration due to ligand exchange.
(19) Above pH 8.0, the rate for the Cu(II) complex decreased sharply in a non-
bell-shaped manner. Although there was no obvious precipitation, it seems
that the intermediate 8-Cu(II)OH may be self-associating in a way that is
equivalent to insolubilization. Interestingly, in the presence of the organic
cosolvent dioxane (slower rates), the rate was seen to increase monotonically
all the way up to pH 8.5.
Figure 3. Time-dependent (40 s interval) hydroxyquinone formation from
1-Cu(II) (1 × 10-4 M) in 0.05 M pH 4.00 acetate buffer upon periodate
(2 × 10-4 M) oxidation at 25 °C. Inset: pH-rate profiles of hydroxyquinone
formation from 1-Cu(II), 1-Zn(II), and 1-Ni(II) upon periodate oxidation
in mixed universal buffer (0.1 M acetate, 0.05 M MES, and 0.05 M HEPES)
at 10 °C.
9
J. AM. CHEM. SOC. VOL. 127, NO. 13, 2005 4781

A R T I C L E S
Ling and Sayre
organic layer was separated, washed with brine (2 × 50 mL), dried
over Na2SO4, and evaporated to give N-(2-aminoethyl)carbamic acid
tert-butyl ester as a colorless oil (15.2 g, 95% yield based on starting
Boc2O).
could possibly also ensure the success of the hydration step by
suppressing decomposition pathways.
It is curious that following delivery of the CuIIOH to C2 and
subsequent oxidation, the Cu(II) in our model does not coor-
dinate to the newly delivered oxygen (a partial oxyanion). The
same scenario holds for the case of the active enzyme, pre-
sumably due to a spatial reorientation of the TPQ ring. While
we do not suggest that our model simulates the proposed cofac-
tor reorientation, the affinity of the Cu(II) for the C2 oxyanion
is apparently not so high (it is a delocalized rather than localized
oxyanion), and this factor should contribute to the lack of coor-
dination observed both in the model and in the active enzyme.
A recent quantum mechanical study on TPQ biogenesis
suggested that the active site CuIIOH species might be acting
as a general-base catalyst to mediate DPQ hydration rather than
as the attacking nucleophile.20 A GBC mechanism would obviate
the issue of water exchange strictly required if the Cu(II)-bound
OH generated in the initial monooxygenation reaction is indeed
the attacking nucleophile. While we have not carried out studies
to exclude the operation of GBC in our model, direct nucleo-
philic attack takes advantage of a six-membered ring transition
state, and the corresponding eight-membered ring GBC mech-
anism seems less favorable in our case. At the same time, a
model described above (11) that could take advantage of a six-
membered ring tertiary amine-catalyzed GBC mechanism was
unsuccessful in achieving o-quinone hydration. While not ruling
out the GBC mechanism, our study demonstrates chemical
precedent for facilitation of o-quinone hydration by a trihis-
tidinyl-CuIIOH species. Independent of the issue of TPQ
biogenesis, to our knowledge, this facilitated hydration is the
first demonstration of formal conjugate addition of H2O (as
opposed to H2O2) to an o-quinone.
A mixture of the latter ester (2.4 g, 15 mmol), 2-picolyl chloride
hydrochloride (5.0 g, 30.5 mmol), and NaOH (2.4 g, 60 mmol) in 100
mL of THF and 100 mL of water was stirred at 50 °C for 5 days. The
organic layer was separated, and the aqueous layer was extracted with
CH2Cl2 (100 mL). To the combined organic layer were added Boc2O
(2.0 g, 9.2 mmol) and Et3N (20 mL), and the mixture was stirred at
room temperature overnight. The solvent was removed, and the residue
was separated by silica gel flash chromatography using hexanes-
EtOAc-MeOH/NH3 (7:3:0.5) as eluent to give N-(2-(bis(2-pyridyl-
methyl)amino)ethyl)carbamic acid tert-butyl ester (4.67 g, 13.6 mmol,
91%) as a light brown viscous oil: 1H NMR (CDCl3) δ 1.40 (s, 9H),
2.66 (t, 2H, J ) 5.8 Hz), 3.19 (q, 2H, J ) 5.8 Hz), 3.82 (s, 4H), 5.87
(br, 1H, NH), 7.11 (t, 2H, J ) 6.1 Hz), 7.38 (d, 2H, J ) 7.7 Hz), 7.60
(dt, 2H, J ) 1.0, 7.6 Hz), 8.85 (ddd, 2H, J ) 0.8, 1.6, 4.8 Hz); 13C
NMR (APT, CDCl3) δ 28.5 (-), 38.5 (+), 53.6 (+), 60.2 (+), 78.8
(+), 122.1 (-), 123.1 (-), 136.5 (-), 149.1 (-), 156.2 (+), 159.3
(+). The small amount of Boc-protected secondary amine (side product)
was not characterized. The Boc protection was used to facilitate
separation of the otherwise coeluting secondary and tertiary amines.
A solution of N-(2-(bis(2-pyridylmethyl)amino)ethyl)carbamic acid
tert-butyl ester (3.0 g, 8.8 mmol) in 40 mL of HCl/EtOH was stirred
at room temperature overnight. The mixture was evaporated to dryness
to give BPEN‚3HCl (3.0 g, 8.5 mmol, 97%) as a light brown amorphous
solid: 1H NMR (CD3OD) δ 3.02 (t, 2H, J ) 5.8 Hz), 3.35 (t, 2H, J )
5.8 Hz), 4.35 (s, 4H), 8.09 (ddd, 2H, J ) 1.1, 5.9, 7.5 Hz), 8.26 (d,
2H, J ) 7.9 Hz), 8.65 (dt, 2H, J ) 1.6, 7.9 Hz), 8.90 (dd, 2H, J ) 1.0,
5.9 Hz); 13C NMR (APT, CD3OD) δ 37.9 (+), 53.4 (+), 56.8 (+),
127.9 (-), 129.1 (-), 143.2 (-), 148.7 (-), 153.4 (+).
B. 4-(2-(Bis(2-pyridylmethyl)amino)ethylaminomethyl)-1,2-ben-
zenediol Trihydrochloride (1‚3HCl). To a solution of BPEN‚3HCl
(1.16 g, 3.3 mmol) in 50 mL of methanol was added solid NaOH (0.4
g, 10 mmol). The mixture was stirred at 50 °C until all the NaOH was
dissolved. The resulting suspension was evaporated to dryness, and
the residue was extracted with 50 mL of 2-propanol. Solid NaCl was
filtered off, and the filtrate was evaporated to dryness. The dry free
base of BPEN was mixed with 1 mL of HOAc and 3,4-dihydroxyben-
zaldehyde (0.51 g, 3.6 mmol) in 50 mL of 2-propanol. The mixture
was stirred at 50 °C for 10 min, and then evaporated to dryness. The
resulting Schiff base was further dried by two rounds of dissolving in
2-propanol (50 mL each) and subsequent evaporation. The dry Schiff
base was dissolved in 2 mL of HOAc and 50 mL of MeOH, to which
was added solid NaBH4 (0.5 g, 13.3 mmol) in small portions with
stirring. The mixture was further stirred at room temperature for 30
min, and then 5 mL of 12 N aqueous HCl was added. The mixture
was evaporated to dryness, leaving a white residue to which were added
100 mL of CH2Cl2, Boc2O (2.6 g, 12 mmol), and 20 mL of Et3N. The
resulting mixture was stirred at room temperature overnight, and the
solvent was removed, leaving a residue that was purified by silica gel
flash chromatography using hexanes-EtOAc-MeOH/NH3 (7:3:0.5,
v/v) as eluent to afford tri-Boc-protected ligand 1 as a mixture of two
amide rotamers (1.75 g, 2.6 mmol, 79%): colorless oil; 1H NMR
(CDCl3) δ 1.34 and 1.41 (2s, 9H total), 1.526 (s, 9H), 1.530 (s, 9H),
2.68 (m, 2H), 3.25 and 3.36 (2t, 2H total), 3.81 (s, 4H), 4.26 and 4.38
(2s, 2H total), 6.9-7.2 (5H), 7.48 (m, 2H), 7.65 (m, 2H), 8.51 (m,
2H).
Experimental Section
General Procedures. NMR spectra were obtained on Varian Gemini
200 and 300 instruments (13C NMR at 50 and 75 MHz, respectively),
with chemical shifts being referenced to the TMS or solvent peak. High-
resolution mass spectrometry (HRMS; FAB) spectra were obtained on
a Kratos MS-25A instrument. UV-vis spectra were obtained using a
Perkin-Elmer Lambda 20 spectrophotometer equipped with a temper-
ature-controlled multiple-cell compartment. 4-tert-Butyl-1,2-benzo-
quinone (5)13 and 2-hydroxy-5-tert-butyl-1,4-benzoquinone (6)21 were
prepared as shown previously. Bis(2-pyridylethyl)amine was prepared
according to a literature procedure.22 The NH3/MeOH used for
chromatography was prepared by saturation of dry MeOH with dry
NH3 followed by a 2-fold dilution with dry MeOH. The HCl/EtOH
solution used for deprotecting Boc-protected ligands was made by
saturation of absolute EtOH with dry HCl, followed by a 10-fold
dilution with absolute EtOH. The CO2-free Nanopure water used for
titration and kinetic studies was purified by a Milli-Q water purification
system from Millipore and was boiled before use.
Synthesis of Models. A. N,N-Bis(2-pyridylmethyl)ethanediamine
(BPEN) Trihydrochloride.23 A solution of Boc2O (21.8 g, 100 mmol)
in 90 mL of THF was added over 30 min to a well-stirred solution of
ethylenediamine (30 mL) in 80 mL of THF and 10 mL of water at 0
°C. The mixture was then stirred at room temperature for 2 days. The
solvent was removed under reduced pressure, and the residue was
partitioned between 200 mL of EtOAc and 100 mL of brine. The
The above tri-Boc-protected ligand (1.75 g) was deprotected by
dissolution in HCl/EtOH and heating at 50 °C for 5 min, followed by
evaporation of the solution to give 1‚3HCl (1.23 g, 2.6 mmol, 100%)
as a white amorphous solid: 1H NMR (CD3OD) δ 3.08 (t, 2H, J ) 7.8
Hz), 3.35 (t, 2H, J ) 7.8 Hz), 4.14 (s, 2H), 4.34 (s, 4H), 6.72 (d, 1H,
J ) 8.1 Hz), 6.87 (dd, 1H, J ) 2.2, 8.1 Hz), 7.01 (d, 1H, J ) 2.2 Hz),
8.05 (t, 2H, J ) 6.4 Hz), 8.21 (d, 2H, J ) 7.9 Hz), 8.65 (dt, 2H, J )
(20) Prabhakar, R.; Siegbahn, P. E. M. J. Am. Chem. Soc. 2004, 126, 3996-
4006.
(21) Lee, Y.; Sayre, L. M. J. Am. Chem. Soc. 1995, 117, 11823-11828.
(22) Brady, L. E.; Freifelder, M.; Stone, G. R. J. Org. Chem. 1961, 26, 4757-
4758.
9
4782 J. AM. CHEM. SOC. VOL. 127, NO. 13, 2005

Water Addition Step of Cofactor Biogenesis in CAOs
A R T I C L E S
1.4, 7.9 Hz), 8.90 (dd, 2H, J ) 1.1, 5.8 Hz); 13C NMR (APT, CD3OD)
δ 45.1 (+), 52.5 (+), 57.0 (+), 116.7 (-), 118.7 (-), 123.1 (+), 123.6
(-), 127.9 (-), 129.0 (-), 143.2 (-), 146.7 (+), 147.8 (+), 148.7
(-), 153.3 (+); HRMS (FAB) m/z calcd for C21H25N4O2 (MH+)
365.1978, found 365.1967 (rel intens 100).
chloride form. Similar observations have been reported in cases of other
dipicolylamine-type ligands,24 including BPEN.23 It is thus assumed
that the isolated models 1 and 3 are also in their trihydrochloride forms.
Coordination Chemistry of Ligand 3 with M(II). A. UV-Vis
Titration. A 0.0500 M standard solution of free base ligand 2 in 1:1
(v/v) aqueous MeOH was prepared by dissolving 25.1 mg of 2‚3HCl
in 1 mL of 0.150 M NaOH in 1:1 aqueous MeOH. A 3 mL quartz
cuvette was filled with 1.00 mM CuSO4 solution in 50% aqueous
MeOH (3.00 mL), and the UV-vis spectrum was recorded at 25 °C.
Small aliquots (6.0 µL each) of a standard solution of 2 were then
added to the cuvette, and the solution was well mixed before the
UV-vis spectrum was recorded after each addition. The results are
shown in Figure S1.
B. Potentiometric pH Titration. To an argon-protected solution
of ligand 2‚3HCl (5.00 mM) and HNO3 (5.00 mM) in 5.00 mL of 0.15
M aqueous KNO3 in the absence or presence of a metal ion (5.00 mM)
were added small aliquots (10.0 µL each) of standard 0.250 M aqueous
NaOH with stirring at 25 °C. The titration was monitored by an
Accumet model 910 pH meter equipped with a Fisher combination
glass electrode. The meter was calibrated to (0.01 using commercial
standard buffers before each run. Typical titration a(OH)-pH curves
are shown in Figure 1A, where a(OH) represents the number of
equivalents of added NaOH with respect to the total number of
equivalents of ligand or 1:1 ligand/metal complex in solution. To
calculate the various equilibrium constants, the collected data were
analyzed according to the program BEST,14 using a pKw value of 13.7325
and three metal-ligand species, MHL, ML, and MH-1L, which
represent a metal complex with a monoprotonated tridentate ligand, a
metal complex with a free base tetradentate ligand, and the metal-bound
water-deprotonated form of complex ML, respectively. Using the three
species above, the σ(pH) fit values defined in the program were less
than 0.03 in all cases, but omission of any species from the calculation
resulted in high σ values. The speciation diagrams for these titrations
were generated using the program SPE,14 as exemplified by that for
the 2-Cu(II) system (Figure 1B). To confirm the involvement of MHL,
ML, and MH-1L, the same pH titration of the 2-Cu(II) system was
repeated, with 3 mL aliquots being frequently removed for UV-vis
measurement and then transferred back to continue the titration. The
results are shown in Figure S2.
Oxidation of 4-tert-Butylcatechol (4) with Periodate in pH 7.50
MOPSO Buffer. To a 3 mL quartz cuvette were added 30.0 µL of
0.010 M catechol 4 or hydroxyquinone 6 in dioxane, 3.00 mL of 0.05
M pH 7.5 MOPSO buffer, and 6.0 µL of 0.1 M aqueous NaIO4. The
solution was quickly mixed and subjected to UV-vis monitoring at
25 °C (Figure S3). The difference spectra were generated using Origin
software (version 7.5) by subtracting the first spectrum from all other
spectra obtained thereafter.
In another experiment, a mixture of 30.0 µL of 0.010 M 4 or 6 in
dioxane and 3.0 µL of 0.100 M aqueous CuSO4 was diluted to 3.00
mL with 0.05 M pH 7.5 MOPSO buffer. The reaction was initiated by
addition of 6.0 µL of 0.1 M aqueous NaIO4 and then subjected to
UV-vis monitoring (Figure S4 in the Supporting Information).
In the third experiment, 4 and 6 (1 × 10-4 M) were both incubated
with a 1:1 complex (1 × 10-4 M) of Cu(II) with bis(2-pyridylethyl)-
amine and NaIO4 (2 × 10-4 M) under the same conditions. UV-vis
monitoring afforded spectra as shown in Figure S5 in the Supporting
Information.
C. N′-(3,4-Dimethoxybenzyl)-N,N-bis(2-pyridylmethyl)ethane-
diamine Trihydrochloride (2‚3HCl). The dry free base BPEN prepared
from 1.16 g of BPEN‚3HCl as for 1 above was dissolved in 50 mL of
2-propanol, and 3,4-dimethoxybenzaldehyde (0.61 g, 3.6 mmol) was
added. The mixture was stirred at 60 °C for 10 min before evaporation
to dryness. The residual water was removed azeotropically using
benzene (2 × 50 mL). The resulting dry Schiff base was dissolved in
50 mL of absolute EtOH, and solid NaBH4 (0.5 g, 13.3 mmol) was
then added in small portions with stirring. After being further stirred
for 30 min, the mixture was acidified with 12 N aqueous HCl to pH 1
and then evaporated to dryness. The residue was suspended in 100 mL
of CH2Cl2, and Boc2O (1.1 g, 5 mmol) and Et3N (20 mL) were added.
The mixture was stirred for 30 min and evaporated to dryness. The
residue was purified by silica gel flash chromatography using hexanes-
EtOAc-NH3/MeOH (6:1:0.7, v/v) as eluent to give the Boc-protected
form of amine 2. The latter compound was dissolved in 50 mL of HCl/
EtOH, and the mixture was heated at 50 °C for 10 min. Evaporation to
dryness gave 2‚3HCl (1.37 g, 2.72 mmol, 82%) as a pale solid: mp
209-211 °C dec; 1H NMR (CD3OD) δ 3.09 (t, 2H, J ) 4.6 Hz), 3.38
(t, 2H, J ) 4.6 Hz), 3.82 (s, 3H), 3.88 (s, 3H), 4.23 (s, 2H), 4.33 (s,
4H), 6.95 (d, 1H, J ) 8.0 Hz), 7.10 (dd, 1H, J ) 0.75, 8.0 Hz), 7.32
(d, 1H, J ) 0.75 Hz), 8.04 (t, 2H, J ) 6.6 Hz), 8.19 (d, 2H, J ) 8.0
Hz), 8.59 (t, 2H, J ) 7.7 Hz), 8.86 (d, 2H, J ) 5.7 Hz); 13C NMR
(APT, CD3OD) δ 45.4 (+), 52.6 (+), 52.7 (+), 56.5 (-), 56.8 (-),
57.0 (+), 112.8 (-), 115.2 (-), 124.5 (+), 124.6 (-), 127.9 (-), 129.0
(-), 143.2 (-), 148.5 (-), 150.7 (+), 151.5 (+), 153.5 (+); HRMS
(FAB) m/z calcd for C23H29N4O2 (MH+) 393.2291, found 393.2294
(rel intens 100).
D. 5-(2-(Bis(2-pyridylmethyl)amino)ethylaminomethyl)-1,2,4-ben-
zenetriol Trihydrochloride (3‚3HCl). A solution of BPEN‚3HCl (352
mg, 1 mmol) in 20 mL of MeOH was treated with solid NaOH (120
mg, 3 mmol) as for 1. The dry free base BPEN was dissolved in 20
mL of 2-propanol, and 1 mL of HOAc and 2,4,5-trihydroxybenzalde-
hyde (169 mg, 1.1 mmol) were added. The mixture was stirred at 50
°C for 10 min and evaporated to dryness. Trace water was removed
by evaporation of the residue twice with 2-propanol (2 × 10 mL). The
final Schiff base was dissolved in 2 mL of HOAc and 20 mL of MeOH,
and solid NaBH4 (250 mg, 6.6 mmol) was then added in small portions
with stirring. After the mixture was further stirred for 30 min, 2 mL of
12 N aqueous HCl was added, and the mixture was evaporated to
dryness. The residue was suspended in 50 mL of CH2Cl2, and then
Boc2O (1.31 g, 6 mmol) was added. The mixture was purged with argon
for 5 min and sealed with a septum. Degassed Et3N (5 mL) was
introduced by syringe, and the mixture was stirred at room temperature
overnight. Evaporation of the suspension, followed by silica gel flash
chromatography of the residue using hexanes-EtOAc-NH3/MeOH
(7:3:0.5, v/v) as eluent, afforded the tetra-Boc-protected form of triol
3. The latter compound was dissolved in 20 mL of HCl/EtOH, and the
mixture was heated at 50 °C for 5 min. Evaporation of the solution to
dryness gave 3‚3HCl (318 mg, 0.65 mmol, 65%) as a light brown
amorphous solid: 1H NMR (CD3OD) δ 3.05 (t, 2H, J ) 6.1 Hz), 3.30
(t, 2H, J ) 6.1 Hz), 4.12 (s, 2H), 4.32 (s, 4H), 6.38 (s, 1H), 6.81 (s,
1H), 8.04 (ddd, 2H, J ) 1.2, 5.9, 7.2 Hz), 8.17 (d, 2H, J ) 7.7 Hz),
8.59 (dt, 2H, J ) 1.6, 7.9 Hz), 8.86 (ddd, 2H, J ) 0.5, 1.5, 5.9 Hz);
13C NMR (APT, CD3OD) δ 44.6 (+), 47.6 (+), 52.3 (+), 57.0 (+),
104.4 (-), 108.2 (+), 119.4 (-), 127.9 (-), 128.9 (-), 139.4 (+),
143.1 (-), 148.6 (-), 148.9 (+), 151.0 (+), 153.4 (+); HRMS (FAB)
m/z calcd for C21H25N4O3 (MH+) 381.1927, found 381.1920 (rel intens
100).
Behavior of Catechol Ligand Model 1. A. Cu(II)-Mediated
Quinone Hydration in pH 7.5 MOPSO Buffer and Product
Characterization by HPLC. A mixture of 20.0 µL of 10.0 mM
(23) BPEN‚3HCl has been synthesized by a different route: Chiu, Y.-H.; Canary,
J. W. Inorg. Chem. 2003, 42, 5107-5116.
(24) See, for example: Sugimoto, H.; Miyake, H.; Tsukube, H. J. Chem. Soc.,
Dalton Trans. 2002, 4535-4540. Hartshorn, R. M.; Telfer, S. G. J. Chem.
Soc., Dalton Trans. 2000, 2801-2808.
A preliminary potentiometric pH titration (see below) showed that
the isolated 2 from HCl solution was the tri- rather than tetrahydro-
(25) Miranda, C.; Escarti, F.; Lamarque, L.; Yunta, M. J. R.; Navarro, P.; Garcia-
Espana, E.; Jimeno, M. L. J. Am. Chem. Soc. 2004, 126, 823-833.
9
J. AM. CHEM. SOC. VOL. 127, NO. 13, 2005 4783

A R T I C L E S
Ling and Sayre
aqueous 1‚3HCl and 2.0 µL of 0.100 M aqueous CuSO4 was diluted
with 0.05 M pH 7.5 MOPSO buffer to 1.00 mL, and then 4 µL of 0.1
M aqueous NaIO4 was added with stirring at 25 °C. The solution
immediately turned red, indicative of hydroxyquinone 7-Cu(II) arising
from oxidation of the initial quinone hydration product 3-Cu(II). After
being further stirred for 1 min, the mixture was acidified with 12 N
aqueous HCl. Solid NaBH4 (10 mg) was added in small portions with
maintenance of pH 1-2, and then 10 µL of 0.1 M aqueous diethyl-
enetriaminepentaacetic acid was added. After being further stirred for
2 min, the solution was filtered and subjected to HPLC analysis using
an Agilent SB-C18 9.4 × 250 mm column and aqueous CH3CN
containing 0.02% CF3COOH as mobile phase, with the composition
of CH3CN increasing from 0 to 35% over 30 min. The same procedure
was repeated with authentic triol 3‚3HCl, and the results are shown in
Figure S6. Authentic triol 3 was found to survive this oxidation-
reduction cycle without significant change as determined by HPLC and
UV-vis (not shown).
B. UV-Vis Monitoring of M(II)-Depedent Quinone Hydration
in pH 7.5 MOPSO Buffer. To a 3 mL quartz cuvette were added 3.0
µL of 0.100 M aqueous CuSO4 and 30.0 µL of 0.0100 M aqueous
1‚3HCl or 3‚3HCl, and the mixture was diluted to 3.00 mL using 0.05
M pH 7.5 MOPSO buffer. After further addition of 6.0 µL of 0.1 M
aqueous NaIO4, the solution was quickly mixed and subjected to
UV-vis scanning at 25 °C (Figure 2A). Similar results were obtained
using Zn(NO3)2 and NiSO4 (not shown). The yields of 7-M(II) were
determined by UV-vis against authentic 7-M(II) according to the 550
nm absorbance in all cases to avoid interference of side products from
decomposition of o-quinone that absorb at 500 nm (see below and
Figure S7).
C. Decomposition of o-Quinone 8 in pH 7.5 MOPSO Buffer and
Interaction of the Decomposition Product with M(II). To a 3 mL
quartz cuvette were added 15.0 µL of 0.0100 M aqueous 1‚3HCl or
3‚3HCl, 3.00 mL of 0.05 M pH 7.5 MOPSO buffer, and then 3.0 µL
of 0.1 M aqueous NaIO4. The solution was quickly mixed and subjected
to UV-vis scanning at 25 °C (Figure 2B). In another three runs of the
same reaction with 1‚3HCl, the final oxidation mixtures were mixed
with 1.5 µL of 0.100 M aqueous CuSO4, Zn(NO3)2, and NiSO4,
respectively, and the UV-vis spectra were recorded immediately
(Figures 2B and S7).
D. Cu(II)-Mediated Quinone Hydration in pH 7.5 Phosphate
Buffer. Using a procedure similar to that above, oxidations of 1‚3HCl
or 3‚3HCl (1 × 10-4 M) were conducted in 0.05 M pH 7.5 phosphate
buffer at 25 °C in the presence of CuSO4 (1 × 10-4 M). Upon initiation
of the reactions with periodate (2 × 10-4 M), the UV-vis spectra were
immediately recorded. In another run, the same oxidation of 1‚3HCl
was conducted without CuSO4. After the UV-vis spectrum was
recorded, 1 equiv of CuSO4 was added, and the UV-vis spectrum was
recorded again. The results are summarized in Figure S8.
E. Time-Dependent Formation of 7-Cu(II) in pH 4.00 Acetate
Buffer. To a 3 mL quartz cuvette were added 3.0 µL of 0.100 M
aqueous CuSO4 and 30.0 µL of 0.0100 M aqueous 1‚3HCl. The mixture
was diluted to 3.00 mL using 0.05 M pH 4.00 acetate buffer. After
further addition of 6.0 µL of 0.1 M aqueous NaIO4, the solution was
quickly mixed and subjected to UV-vis scanning at 25 °C. The result
is shown in Figure 3.
F. Kinetic Studies of Quinone Hydration as a Function of pH
Using Mixed Universal Buffers. Mixed buffer solutions composed
of 0.1 M acetate, 0.05 M 4-morpholineethanesulfonic acid (MES), and
0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
from pH 5.00 through pH 8.50 in 0.25 pH unit increments were prepared
and cooled to 10 °C. To a 3 mL quartz cuvette at 10 °C were added
3.0 µL of 0.100 M aqueous CuSO4 and 30.0 µL of 10.0 mM aqueous
1‚3HCl. The mixture was diluted to 3.00 mL using the cooled mixed
buffers. After further addition of 6 µL of 0.1 M aqueous NaIO4, the
solution was quickly mixed, and A550 was recorded over time at 10 °C
(see Figure S9 in the Supporting Information for an example). The
data were treated using Origin software (version 7.5) to yield the
observed first-order rate constants kobsd (correlation coefficients r were
always >0.999), which were plotted against pH to give the pH-rate
profiles as shown in Figure 3 (inset). A nonlinear least-squares fit to
the pH-rate profile for the 1-Cu(II) system over the pH range
5.00-8.00 was achieved (Origin 7.5) according to the equation kobsd
+
) kKapp/(Kapp + RH ), where Kapp represents the apparent ionization
constant of the Cu(II)-bound water, k represents the real rate constant
of the dissociated complex species 8-M-, and RH represents the proton
+
activity of the reaction system. The kinetic data for Zn(II) and Ni(II)
were obtained following the same procedure. The formation of
hydroxyquinone 7 was observed only at pH > 6.5 for Zn(II) and pH
> 7.25 for Ni(II). The results are also shown in Figure 3 (inset).
Acknowledgment. We thank the National Institutes of Health
for support of this work through Grant GM 48812.
Supporting Information Available: UV-vis titration of
Cu(II) with ligand 2, UV-vis monitoring of periodate oxida-
tions of catechol 4, HPLC diagrams and the diode array spectra
for characterization of hydroxyquinone 7, UV-vis spectra
showing decomposition of o-quinone 8 in the absence of M(II)
and interaction of the resulting solution with Cu(II), Zn(II),
and Ni(II), and typical time driven UV-vis monitoring of
M(II)-dependent quinone hydration with first-order kinetics plots
(PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
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4784 J. AM. CHEM. SOC. VOL. 127, NO. 13, 2005