Dioxygenation of flavonol catalyzed by Copper(II)complexes supported by carboxylate-containing ligands: Structural and functional models of quercetin 2,4-dioxygenase

Article abstract of DOI:10.1002/ejic.201601371

To obtain insights into the role of the carboxylate group of Glu73 in the active site of quercetin 2,4-dioxygenase (2,4-QD), the copper(Icomplexes [Cu^IILn(AcO)] [1 (Ln = L1), 2 (Ln = L2), 3 (Ln = L3), and 4 (Ln = L4)] supported by a series of carboxylate-containing ligands [L1H = 2-{[bis(pyridin-2-ylmethyl)amino]methyl}benzoic acid L2H = 3-{[bis(pyridin-2-ylmethyl)amino]methyl}benzoic acid, L3H = 2-({bis[2-(pyridin-2-yl)ethyl]amino}methyl)benzoic acid L4H = 3-({bis[2-(pyridin-2-yl)ethyl]amino}methyl)benzoic acid] as well as the ternary Cu^II flavonolate (fla-) complexes [Cu^IILn(fla)] [5 (Ln = L1), 6 (Ln = L2), 7 (Ln = L3), and 8 (Ln = L4)] were synthesized and characterized as structural and functional models for the active site of 2,4-QD. The ternary complexes [Cu^IILn(fla)] showed different reactivities in the dioxygenation of bound flavonolate to benzoic acid, salicylic acid, and N,N-dimethylbenzamide at 75-90 °C (single-turnover reactioin the order 5 >> 7 > 8 ≈ 6. A similar reactivity tendency was found in the catalytic dioxygenation of the substrate flavonol (multiturnover reactioby the binary complexes. The different reactivities of the copper complexes could be attributed to the different Lewis acidities of the copper(Iions induced by the different coordination environments of the ligands. The results will provide important insights into the pivotal catalytic role of the carboxylate group of Glu73 in 2,4-QD.

Full text of DOI:10.1002/ejic.201601371

DOI: 10.1002/ejic.201601371
Full Paper
Structure–Reactivity Relationships
Dioxygenation of Flavonol Catalyzed by Copper(II) Complexes
Supported by Carboxylate-Containing Ligands: Structural and
Functional Models of Quercetin 2,4-Dioxygenase
Ying-Ji Sun,*[a] Pei Li,[a] Qian-Qian Huang,[a] Jian-Jun Zhang,[a] and Shinobu Itoh*[b]
Abstract: To obtain insights into the role of the carboxylate of 2,4-QD. The ternary complexes [CuIILn(fla)] showed different
group of Glu73 in the active site of quercetin 2,4-dioxygenase reactivities in the dioxygenation of bound flavonolate to
(2,4-QD), the copper(II) complexes [CuIILn(AcO)] [1 (Ln = L1), 2 benzoic acid, salicylic acid, and N,N-dimethylbenzamide at 75–
(Ln = L2), 3 (Ln = L3), and 4 (Ln = L4)] supported by a series 90 °C (single-turnover reaction) in the order 5 >> 7 > 8 ≈ 6. A
of carboxylate-containing ligands [L1H = 2-{[bis(pyridin-2- similar reactivity tendency was found in the catalytic dioxygen-
ylmethyl)amino]methyl}benzoic acid; L2H = 3-{[bis(pyridin-2- ation of the substrate flavonol (multiturnover reaction) by the
ylmethyl)amino]methyl}benzoic acid,; L3H = 2-({bis[2-(pyridin-2- binary complexes. The different reactivities of the copper com-
yl)ethyl]amino}methyl)benzoic acid; L4H = 3-({bis[2-(pyridin-2- plexes could be attributed to the different Lewis acidities of the
yl)ethyl]amino}methyl)benzoic acid] as well as the ternary CuII copper(II) ions induced by the different coordination environ-
flavonolate (fla–) complexes [CuIILn(fla)] [5 (Ln = L1), 6 (Ln = ments of the ligands. The results will provide important insights
L2), 7 (Ln = L3), and 8 (Ln = L4)] were synthesized and charac- into the pivotal catalytic role of the carboxylate group of Glu73
terized as structural and functional models for the active site in 2,4-QD.
Introduction
histidine imidazole rings (His66, His68, and His112), one water
molecule, and a carboxylate group of Glu73 (Figure 1a); the
major conformer has a distorted tetrahedral geometry without
the Glu73 ligation (Figure 1b), and Glu73 acts as a hydrogen-
bond acceptor for the metal-bound water molecule (dotted
lines in Figure 1b).[1]
Quercetin 2,4-dioxygenase (2,4-QD) is a unique copper-contain-
ing dioxygenase containing a mononuclear type-II copper reac-
tion center.[1–3] The enzyme catalyzes the oxygenative ring-
opening reaction of quercetin (3′,4′,5,7-tetrahydroxyflavonol,
QUE) to the corresponding depside (phenolic carboxylic acid
esters) with the release of CO (Scheme 1).[4]
Scheme 1. Dioxygenation of QUE catalyzed by 2,4-QD.
Figure 1. Structures of the active site of the resting state of the (a) minor and
(b) major conformations as well as the (c) enzyme–substrate (ES) complex of
2,4-QD from A. japonicus.
The copper center in the active site of 2,4-QD from
Aspergillus japonicus involves two distinct coordination geome-
tries in a 3:7 ratio. The minor conformer has a distorted trigonal-
bipyramidal geometry, and the Cu center is bound to three
The crystal structure with a bound substrate [enzyme–sub-
strate (ES) complex] was also reported, and a flavonolate ion
(fla–, deprotonated substrate) is bound through the 3-hydroxy
group to the copper(II) ion, which exhibits a distorted square-
pyramidal geometry (Figure 1c).[5] The bound substrate is stabi-
lized by Glu73 through a hydrogen-bonding interaction (dotted
line in Figure 1c).[1,5,6] A mutational study has indicated that the
removal of Glu73 causes a loss of enzyme activity and, there-
fore, implies that Glu73 plays an important role in the catalytic
reaction.[1]
[a] School of Chemistry, Dalian University of Technology,
2 Linggong Road, Dalian 116024, China
E-mail: yingjis@dlut.edu.cn
[b] Department of Material and Life Science, Division of Advanced Science
and Biotechnology, Graduate School of Engineering, Osaka University,
2-1 Yamada-oka, Suita, Osaka 565-0871, Japan
E-mail: shinobu@mls.eng.osaka-u.ac.jp
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejic.201601371.
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Even though a large number of model compounds have reported in our previous paper.[14] Detailed inspections of their
been developed with N-chelating polyamine ligands,[7] little at- structures, physicochemical properties, and reactivities have en-
tention has been focused on the role of the active-site carboxyl- abled the exploration of the important factors that control the
ate group (Glu73) in 2,4-QD. In this regard, Speier and co-work- electron-donor ability of the carboxylate group as well as the
ers reported that the dioxygenation of the bound flavonolate pyridine donor groups and provided useful information for
substrate was promoted by the addition of excess carboxylate mechanistic studies of the enzymatic reactions.
(potassium salt), the role of which was attributed to the alterna-
tion of the binding mode of the bound flavonolate from bi-
dentate to monodentate.[8,9] To date, several ligands containing
a carboxylate group have been developed,[10–13] but the effects
of the carboxylate group in the ligands have yet to be ad-
dressed in detail.
Results and Discussion
Syntheses and Crystal Structures
The ligands LnH (n = 2–4; see Scheme 2) were prepared by a
To shed light on the roles of the carboxylate group in the procedure similar to that for the synthesis of L1H (see Support-
active site of 2,4-QD, we herein synthesized and characterized ing Information).[15] The treatment of the ligands with
a set of copper(II) complexes [CuIILn(AcO)] and their flavonolate CuII(OAc)2·H2O gave the corresponding mononuclear copper(II)
adducts [CuIILn(fla)] with the series of carboxyl-group-contain- complexes [CuIILn(OAc)] (1–4), the formation of which was con-
ing ligands LnH shown in Scheme 2. As a preliminary result, firmed by ESI-MS, IR spectroscopy, electron paramagnetic reso-
the L1–flavonolate complex [CuIIL1(fla)] (5) has already been nance (EPR) spectroscopy, and elemental analysis, as described
Scheme 2. Structures of ligands LnH.
Figure 2. ORTEP drawings of (a) [CuL1(AcO)]·CH3OH·H2O (1), (b) [CuL2(H2O)]2[ClO4]2·2H2O (2′), (c) [CuL3(AcO)]·2H2O (3), and (d) {[CuL4Cl]·H2O}n (4′); 50 %
probability thermal ellipsoids are shown for 1, and 30 % probability ellipsoids are shown for the others. The non-coordinating counteranions and the hydrogen
atoms attached to the carbon atoms are omitted for clarity.
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in the Experimental Section and the Supporting Information. meric complex [CuIIL4(Cl)]n (4′) was obtained for L4H, and the
For ligands L1H and L3H containing an o-carboxylate group, Cl– ions may have been incorporated from the solvent during
the mononuclear copper(II) complexes [CuIIL1(AcO)] (1) and crystallization in CHCl3. The crystal structures of 2′ and 4′ are
[CuIIL3(AcO)] (3) were obtained as blue single crystals, and their presented in Figure 2b and d, respectively. The crystallographic
structures are presented in Figure 2a and c. Although single data and selected bond lengths and angles of 1, 2′, 3, and 4′
crystals of the mononuclear copper(II) complex 2 have yet to be are summarized in Tables 1 and 2, respectively.
obtained, single crystals of the carboxylate-bridged dicopper(II)
The mononuclear copper(II) complex 1 (Figure 2a) exhibits a
complex [CuIIL2(H2O)]2 (2′) suitable for X-ray analysis were ob- distorted square-pyramidal structure (τ = 0.25)[16] with an N3O2
tained by counteranion exchange from AcO– ions in 2 to ClO4
donor set; the basal plane is constructed from the tert-alkyl-
–
ions in 2′. On the other hand, the carboxylate-bridged poly- amine nitrogen atom N(1), the pyridine nitrogen atoms N(2)
Table 1. Summary of the X-ray crystallographic data for 1, 2′, 3, and 4′.
1
2′
3
4′
Empirical formula
Mr
C23H27CuN3O6
505.02
C40H44Cl2Cu2N6O16
1062.79
C24H29CuN3O6
519.04
C22H24ClCuN3O3
477.43
Crystal system
Space group
a /Å
b /Å
c /Å
α /°
ꢀ /°
triclinic
triclinic
triclinic
monoclinic
P21/n
8.747(17)
14.695(3)
16.669(3)
90
90.34(3)
90
2142.5(7)
4
P1
P1
P1
¯
¯
¯
7.302(15)
8.682(17)
17.850(4)
103.65(3)
95.61(3)
97.00(3)
1082.0
7.948(16)
12.130(2)
12.135(2)
72.00(3)
80.53(3)
77.61(3)
1080.7
8.815(18)
10.978(2)
12.630(3)
75.83(3)
80.14(3)
80.72(3)
1158.4(4)
2
γ /°
V /Å3
Z
2
1
Dcalcd. /g cm–3
T /K
1.550
149.1
1.633
149.1
1.488
149.1
1.480
173.1
Crystal habit
Crystal color
Crystal size /mm
μ /mm–1
block
blue
0.36 × 0.32 × 0.15
1.056
block
blue
0.20 × 0.10 × 0.05
1.188
block
blue
0.27 × 0.23 × 0.12
0.989
block
blue
0.20 × 0.20 × 0.18
1.173
2θmax /°
50.00
50.00
50.00
50.00
Completeness to θ /%
Reflections collected
Independent reflections
Rint
99.6
8609
3631
0.0456
0.0444/0.1136
0.0457/0.1148
1.122
98.7
8272
3236
0.0937
0.0674/ 0.1752
0.0812/ 0.1860
0.927
99.7
9201
3854
0.0349
0.0404/0.1074
0.0415/0.1082
1.157
99.4
3763
3573
0.0000
0.0277/0.0759
0.0290/0.0765
1.094
R1[a]/wR2[b] [I > 2σ(I)]
R1[a]/wR2[b] (all data)
Goodness-of-fit (F2)
[a] R1 = Σ||Fo| – |Fc||/Σ|Fo|. [b] wR2 = [Σw(Fo – Fc2)2/Σw(Fo2)2]1/2
.
2
Table 2. Selected bond lengths [Å] and angles [°] for 1, 2′, 3, and 4′.[a]
Compound
1
2′
3
4′
Cu(1)–O(1)/(O1A)
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–O(3)/Cl(1)
τ
2.1512(18)
2.049(2)
1.997(2)
2.005(2)
1.9526(19)
0.25
1.932(4)
2.020(5)
2.002(4)
1.981(4)
2.306(4)
0.04
1.9666(17)
2.0232(13)
2.1412(16)
2.0164(16)
2.0318(17)
2.5562(7)
0.31
2.0912(18)
2.026(2)
2.222(2)
1.9886(17)
0.10
O(1)/(O1A)–Cu(1)–N(1)
O(1)/(O1A)–Cu(1)–N(2)
O(1)/(O1A)–Cu(1)–N(3)
N(1)–Cu(1)–N(2)
N(1)–Cu(1)–N(3)
N(2)–Cu(1)–N(3)
O(1)/(O1A)–Cu(1)–O(3)/Cl(1)
N(1)–Cu(1)–O(3)/Cl(1)
N(2)–Cu(1)–O(3)/Cl(1)
N(3)–Cu(1)–O(3)/Cl(1)
96.11(8)
102.04(8)
96.20(8)
83.30(9)
83.13(9)
158.29(9)
90.47(7)
173.35(7)
96.26(9)
95.27(9)
166.59(17)
98.06(17)
95.20(17)
81.89(18)
83.10(18)
163.91(18)
99.74(15)
93.67(16)
88.73(16)
98.00(16)
92.95(7)
166.98(7)
94.48(7)
91.45(8)
93.76(8)
97.45(8)
87.64(7)
172.97(7)
86.52(7)
93.18(7)
158.56(6)
90.92(6)
86.27(6)
86.76(7)
95.44(7)
176.97(6)
98.22(4)
103.20(5)
93.65(5)
87.89(5)
[a] Estimated standard deviations are given in parentheses.
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and N(3), and the carboxylate oxygen atom O(3) of the acetate
coligand (AcO–), and the axial position is occupied by the oxy-
gen atom O(1) of the carboxylate group of the anionic ligand
L1–.
The carboxylato-bridged dimeric complex [CuL2(H2O)]2-
(ClO4)2·2H2O (2′; Figure 2b) also shows a square-pyramidal ge-
ometry (τ = 0.04)[16] with an N3O2 donor set consisting of three
nitrogen atoms N(1), N(2), and N(3) from the ligand and one
oxygen atom O(1A) of the carboxylate group of another ligand
L2– in the basal plane and a water molecule O(3) at the axial
position. The coordination environment of the CuII center in 2′
is rather similar to that of the active site of the minor conforma-
tion of native 2,4-QD, which contains three nitrogen donor
atoms (His), one water molecule, and a directly coordinating
carboxylato group (see Figure 1a), even though the geometry
of the copper center is slightly different (square-pyramidal in 2′
vs. trigonal-bipyramidal in the minor conformation of 2,4-QD).
The Cu(1)–O(3) bond length [water oxygen atom, 2.306(4) Å] in
2′ is similar to the Cu–Owat bond length (2.2–2.4 Å) in native
2,4-QD from A. japonicus.[1]
Scheme 3. Interactions between the CuII center and the carboxylate group of
the ligand.
was treated with a solution of Cu(AcO)2 in dry MeOH/aceto-
nitrile (3:1) in a 1.1:1 molar ratio under N2 at room temperature.
The reaction mixture was stirred for 1 d, and the removal of the
solvent gave a yellow-green residue, which was then dissolved
in CH2Cl2. To this solution, the ligand LnH (1.1 equiv.) in dry
CH2Cl2 was added, and the mixture was stirred for another day
under N2 to give dark green powders of the ternary complexes
[CuIILn(fla)] (6–8). In these reactions, the acetate ions (AcO–) of
CuII(AcO)2 may act as a base to accept protons from both LnH
and flaH. Pure samples were obtained by slow diffusion of hex-
ane into CH2Cl2 solutions of the complexes under N2 at room
temperature. The formation of the ternary complexes was con-
firmed by ESI-MS, IR spectroscopy, EPR spectroscopy, and ele-
mental analysis, as described in the Experimental Section and
the Supporting Information.
Complex [CuL3(AcO)] (3; Figure 2c) exists as a mononuclear
copper(II) complex with a square-pyramidal (τ = 0.10)[16] geom-
etry with an N3O2 donor set consisting of two nitrogen atoms
N(1) and N(2) from L3–, one oxygen atom O(1) of the carboxyl-
ato group of the ligand, and one oxygen atom O(3) of the coor-
dinating AcO– counteranion in the basal plane and another pyr-
idine nitrogen atom N(3) of the ligand in the axial position.
Thus, the coordination mode of the three nitrogen atoms of the
ligand is changed from mer mode in 1 to fac mode in 3.
Redox Properties of the Complexes
Cyclic voltammetric measurements on [CuIILn(AcO)] (2 m
M in
The complex {[CuL4Cl]·H2O}n (4′) obtained by the recrystalli-
zation of the monomeric complex 4 from CHCl3 displays the
benzoate-bridged one-dimensional zigzag polymer-chain struc-
ture (Figure 2d). The metal center also shows a distorted
square-pyramidal geometry (τ = 0.31)[16] with an N3OCl donor
set involving the three nitrogen atoms N(1), N(2), and N(3) from
L4–, one oxygen atom O(1) of the carboxylate group of another
ligand in the basal plane, and Cl(1) [Cu–Cl(1): 2.5562(7) Å] at
the axial position. The coordination geometry in 4′ is similar to
that of 2′, although the Cu(1)–O(1) bond [benzoate oxygen
atom, 2.0232(13) Å] is somewhat longer than that in 2′
[1.932(4) Å].
N,N-dimethylformamide, DMF) were performed under N2 at
room temperature. The cyclic voltammogram of 4 is shown in
Figure 3a as a typical example, those of the others are pre-
sented in Figure S4, and the data are summarized in Table S1.
The binary complexes 1–4 each exhibit a quasireversible redox
couple at E1/2 = –0.448, –0.164, –0.269, and –0.086 V versus
saturated calomel electrode (SCE), respectively, and these cou-
ples can be assigned to a one-electron redox process between
CuII and CuI.
Although the CuII centers in all of the model complexes show
similar square-pyramidal coordination geometries, the positions
of the carboxylate groups are different (equatorial vs. axial) de-
pending on the position of the carboxylate group in the ligand
as well as the type of pyridylalkyl group (pyridylmethyl vs.
pyridylethyl). It should be noted that the core structures of our
model complexes supported by the o- and m-carboxylate li-
gands in solution are rather similar to those of the 3His1Glu
active site of the minor (coordinated carboxylate group of
Glu73) and major conformations (carboxylate group of Glu73
not coordinated) of 2,4-QD from A. japonicus, respectively, as
discussed below (Scheme 3).[1]
Figure 3. Cyclic voltammograms of the binary and ternary complexes (2 m
M
in DMF) under N2 at room temp.: (a) [CuIIL4(AcO)] (4) and (b) [CuIIL4(fla)] (8).
The E1/2 value of 1 is lower than that of 2. This can be ex-
Ternary complexes containing deprotonated flavonol (fla–) li- plained by the different interaction modes between the metal
gands [CuIILn(fla)] [6 (Ln = L2), 7 (Ln = L3), 8 (Ln = L4)] were center and the carboxylate group of the ligand. In particular,
obtained as follows {[CuIIL1(fla)] (5) has already been reported there is a strong coordinative interaction between the metal
in our previous paper}.[14] First, a solution of flaH in dry CH2Cl2 ion and the o-carboxylate group in 1, as is evident from the X-
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ray structure shown in Figure 2a, whereas the interaction be- (+0.502 V) > 6 (+0.517 V) ≈ 8 (+0.521 V), as was also observed
tween the metal ion and the m-carboxylate group may be experimentally (see below). The order of the oxidizability
weaker in 2, as illustrated in Scheme 3. The stronger coordina- among these ternary complexes can be attributed to the differ-
tive interaction of the o-carboxylate group in 1 makes the re- ences in the Lewis acidities of their metal centers. In particular,
duction of the metal ion more difficult owing to the strong the Lewis acidity of the copper(II) ion in 8 must be the strongest
electron donation, whereas the electron-donating effect of the among the four complexes owing to the weaker electron-donor
m-carboxylate group in 2 is weaker owing to the weaker inter- ability of the m-carboxylate group and the pyridylethyl groups
action, which makes the reduction of the metal ion easier. The in L4 as discussed above. Then, the strong Lewis ability of the
difference in E1/2 between 3 and 4 can be explained similarly.
On the other hand, the lower E1/2 value of 1 (–0.448 V) com-
pared with that of 3 (–0.269 V) can be explained by considering
the well-documented fact that the electron-donor ability of the
(2-pyridyl)methyl group is stronger than that of the (2-pyr-
idyl)ethyl group.[17] The lower E1/2 value of 2 (–0.164 V) com-
pared with that of 4 (–0.086 V) can be explained similarly.
Cyclic voltammetric measurements on the ternary complexes
5–8 were also performed under N2 at room temperature. The
cyclic voltammogram of 8 is shown in Figure 3b as a typical
example, those of the others are presented in Figure S4, and
the data are summarized in Table S2. The ternary complexes
also exhibit a quasireversible one-electron CuII/I redox couple at
E1/2 = –0.427, –0.136, –0.155, and +0.036 V versus SCE, respec-
tively. As for the binary complexes, the stronger coordinative
interaction of the o-carboxylate group in 5 and 7 makes the
reduction of the metal ion more difficult owing to the strong
electron donation (E1/2 = –0.427 and –0.155 V, respectively),
whereas the electron-donating effects of the m-carboxylate
groups in 6 and 8 may be weaker owing to a weaker interac-
tion, which would make the reduction of the metal ion occur
more readily (E1/2 = –0.136 and +0.036 V, respectively).
metal ion in 8 makes the coordinative interaction between the
copper(II) center and the fla– ligand stronger; thus, the oxid-
ation of fla– to fla· is harder. On the other hand, the weaker
Lewis acidity of the metal ion in 5, owing to the stronger elec-
tron-donor ability of the o-carboxylate group and the pyridyl-
methyl groups, renders the coordinative interaction between
the copper(II) center and the fla– ligand weaker and, thus, en-
hances the oxidation of fla– to fla·.
Dioxygenation of the Ternary Complexes [CuIILn(fla)]
(Single-Turnover Reactions)
The aerobic treatment of the ternary complexes [CuIILn(fla)] (5–
8) in DMF at 70 °C gave O-benzoylsalicylic acid (HObs; m/z =
243.2 [M + H]+) and CO[13,14] as the primary products, the
former of which was hydrolyzed to salicylic acid (69–77 %;
m/z = 137.0 [M – H]–) and benzoic acid (45–62 %; m/z = 121.2
[M – H]–) through the reaction with the small amount of water
present in the solvent over a prolonged reaction time. The
benzoic acid was further converted into the amide derivative
N,N-dimethylbenzamide (16–25 %; m/z = 150.1 [M + H]+) by
the reaction with the solvent molecule DMF (Scheme 4). These
products were identified by LC–MS (Figure S7) and 1H NMR
spectroscopy, as summarized in Table S3.[13,14] The oxygenative
ring-opening reaction of flavonol in the ternary complex is simi-
lar to that catalyzed by the native 2,4-QD; therefore, our ternary
ES model complexes [CuIILn(fla)] exhibit reactivity similar to that
of the native enzyme.
On the other hand, the lower E1/2 value of 5 (–0.427 V) com-
pared with that of 7 (–0.155 V) and the lower E1/2 value of 6
(–0.136 V) compared with that of 8 (+0.036 V) can be explained
by the fact that the electron-donor ability of the (2-pyridyl)-
methyl group is stronger than that of the (2-pyridyl)ethyl group,
as mentioned for the binary complexes.[17]
Interestingly, an irreversible anodic peak was observed at Epa
The oxygenative ring-opening reactions of the ternary com-
≈ +0.5 V versus SCE and may be due to the one-electron oxid- plexes [CuIILn(fla)] (5–8) were followed by monitoring the de-
ation of the coordinated fla– ligand to a flavonoxy radical (fla·). crease of the absorption band owing to the π→π* transition of
The order of Epa, 5 < 7 < 6 < 8, indicates that the fla– ligand is the coordinated flavonolate ligand in [CuIILn(fla)] at λ ≈ 420 nm
more readily oxidized in the order 5 (+0.480 V vs. SCE) > 7 in DMF at 75–90 °C. A typical example for the reaction of 6 is
Scheme 4. Dioxygenation of the ternary complexes [CuIILn(fla)].
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Figure 4. (a) Spectral changes observed during the dioxygenation reaction of 6 at 90 °C in DMF. Inset: time course of the absorption changes at λ = 420 nm.
Plots of vint versus (b) [CuIILn(fla)]0 and (c) [O2]0.
shown in Figure 4a, and those of others are presented in Fig- dimethylbenzamide (3.1–25 %; Scheme 5), as confirmed by LC–
1
ure S8. The initial rates (vint
/M
s–1) were determined from the MS and H NMR spectroscopy (Table S3 and Figure S7).[13,14]
initial linear portion of the plots of ΔA against time (insets of
Figures 4a and S8) and are summarized in Table S4 together
with the reaction conditions. As shown in Figure 4b and c, the
initial rates vint exhibit linear correlations with the concentra-
tions [CuIILn(fla)]0 and [O2]0, and the second-order rate con-
stants k2 were determined by dividing vint by both [CuIILn(fla)]0
and [O2]0, that is, vint = k2[CuIILn(fla)][O2].
Thus, the apparent second-order rate constants k2 were ob-
tained as 219 × 10–2
M
–1 s–1 for 5, 6.39 × 10–2
M
–1 s–1 for 6,
12.7 × 10–2
M
–1 s–1 for 7, and 7.80 × 10–2
M
–1 s–1 for 8 at 90 °C
Scheme 5. The dioxygenation of flavonol catalyzed by [CuIILn(AcO)] under
aerobic conditions.
(Table S4). The dioxygenation reactivity of the bound flavonol-
ate substrate of the ternary complexes [CuIILn(fla)] shows
notable differences in the order 5 >> 7 > 8 ≈ 6. The order of
the dioxygenation reactivity is the inverse of the order of the
oxidation peak potential Epa (fla–/fla·) for the ternary complexes
described above. As mentioned above, the oxidizability of the
fla– moiety in the ternary complexes is in the order 5 > 7 > 6
≈ 8 (Table S2), which is attributed to the differences in the
Lewis acidities of the copper(II) ions. That is, a stronger electron
donation to the copper(II) ion by the carboxylate and pyridyl-
methyl groups reduces the Lewis acidity of the metal ion; thus,
the electron density on the fla– moiety in the ternary complexes
follows the order 5 > 7 > 6 ≈ 8. The higher the electron density
on the fla– moiety, the easier it can be oxidized by O2. Conse-
quently, the O2 reactivity of the ternary complexes is controlled
by the strength of the interaction between the metal ion and
the carboxylate group, even though steric factors as well as the
coordination geometry around the metal center might also be
important for the control of the reactivity. These results suggest
that the key role of the carboxylate group in the enzymatic
system is to tune the redox potential of the bound substrate
by electron donation through the metal ion, that is, it is as
“electron conduit”, as shown in pink in Scheme 4.[1,6]
Then, the reactions were followed by monitoring the absorp-
tion changes of the substrate flavonol at λ = 343 nm (log ε =
4.24) in DMF at 85–100 °C under aerobic conditions. A typical
example for the reaction catalyzed by 1 is shown in Figure 5a.
The initial rates (vint /M
s–1) were determined from the initial
linear portions of the plots of ΔA against time (inset of Fig-
ure 5a), and the data are summarized in Table S4 together with
the reaction conditions. As shown in Figure 5b–d, the initial
rates vint exhibit linear correlations with the concentrations
[CuIILn(AcO)]0, [O2]0, and [flaH]0, and the third-order rate con-
stants k3 were determined by dividing vint by [CuIILn(fla)]0, [O2]0,
Dioxygenation of Flavonol Catalyzed by the Binary
Complexes [CuIILn(AcO)] (Multiturnover Reaction)
The dioxygenation of flavonol proceeded in the presence of
catalytic amounts of the binary complexes [CuIILn(AcO)]
(5.0 mol-% relative to the substrate flavonol) in DMF at 100 °C
under aerobic conditions to give the same products as those
for the single-turnover reaction mentioned above, that is,
benzoic acid (42–76 %), salicylic acid (64–79 %), and N,N-
Figure 5. (a) Spectral changes observed during the dioxygenation of flavonol
catalyzed by 1 at 100 °C in DMF. Inset: time course of the absorption changes
at λ = 343 nm. Plots of vint versus (b) [CuIILn(AcO)]0, (c) [flaH]0, and (d) [O2]0.
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and [flaH]0 according to the rate equation vint = k3[CuIILn- [CuIILn(fla)]. The formation of [CuIILn(fla)] was also confirmed by
(fla)][flaH][O2]. Thus, the apparent third-order rate constants k3 ESI-MS. Peak clusters for [CuIILn(fla)]H+ (Figures 6b and S10)
for the complexes [CuIILn(AcO)] (n = 1–4) were obtained as were observed, and the m/z values and the isotope distribution
10.9 × 103, 7.31 × 103, 9.00 × 103, and 4.90 × 103 –2 s–1, respec- patterns agreed well with the calculated values. These results
M
tively, at 100 °C (Table S4). The catalytic reactivities of our model indicate that the binding process between the fla– ion and the
complexes are higher than those of the 2,4-QD model com- binary complex is not the rate-limiting step in the catalytic reac-
plexes reported previously.[18,19]
tion.
It should be noted that the order of the catalytic activity of
the binary complexes 1 (L1) > 3 (L3) > 2 (L2) > 4 (L4) is almost
the same as that of the O2 reactivity of the ternary complexes
[5 (L1) > 7 (L3) > 8 (L4) ≈ 6 (L2)]. Thus, the rate-limiting step of
the catalytic oxygenation reaction is the oxidative ring-opening
process of the fla– moiety in the ternary complex, which is
formed during the catalytic cycle as discussed below.
Reaction Mechanism
To confirm the binding of flavonol (flaH) to the binary com-
plexes [CuIILn(AcO)] (1–4) in solution, optical spectroscopic ti-
trations were performed. The spectral changes observed upon
Figure 6. (a) Spectral changes observed upon the addition of flavonol (3 mM,
0.1 equiv. in 10 μL DMF) to a DMF solution of 1 (0.1 mM, 3 mL) under N2 at
room temperature. Inset: absorbance changes at λ = 423 nm. (b) ESI mass
spectrum of the final solution of the titration (line: experimental; column:
calculated).
the addition of flaH to a DMF solution of [CuIIL1(AcO)] (0.1 m
M)
under N2 at room temperature are shown in Figure 6a (for 2–4,
see Figure S9). The π→π* transition band at λ = 423 nm for the
coordinated flavonolate (fla–) substrate increased linearly with
When excess nitrotetrazolium blue chloride (NBT2+) was
increasing flaH concentration until the [CuIIL1(AcO)]/flaH molar added to a 1:1 mixture of 1 (0.5 m
M in DMF) and flavonol (flaH)
ratio reached 1:1, as shown in the inset of Figure 6a. Similar in an O2-saturated solution at room temperature, a new absorp-
results were obtained for complexes 2–4 (see Figure S9). These tion band arose at λ = 530 nm (Figure S11), which may be
results confirmed the strong 1:1 binding of the fla– ion to the due to the reduction of NBT2+ by O2·– radical species generated
binary complexes in solution to form the ternary complexes during the reaction.[20]
Scheme 6. Possible catalytic mechanism.
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For the reactivity of our present model system analogous to Experimental Section
that of Cu-2,4-QD,[5] a possible catalytic mechanism could be
General and Physical Methods: All reagents and solvents except
the ligand LnH were commercial products, which were used as re-
ceived unless otherwise noted. Air-sensitive reactions were per-
formed under N2 in a glovebox or by Schlenk techniques. H NMR
spectra were recorded with a Bruker 400WB spectrometer. FTIR
spectra were recorded with a Nicolet 6700 spectrophotometer. UV/
derived, as indicated in Scheme 6. The ligand-exchange reac-
tion of the acetate ligand in the binary complex [CuIILn(AcO)]
with the substrate (flaH) generates the ternary complex [CuII-
Ln(fla)] (path a). As shown in Figure 6, this process proceeds
rapidly. Then, inner-sphere electron transfer from the fla– ion to
1
the CuII center generates a CuI–fla· species A (path b). From this Vis spectra were recorded with an Agilent Technologies HP8453
diode-array spectrophotometer. ESI-MS and HPLC–MS data were re-
corded with a Thermo Fisher Scientific LTQ Orbitrap XL HPLC–ESI-
MS instrument with an on-line UV/Vis detector (λ = 210 nm). EPR
spectra were obtained with a Bruker A200 spectrometer fitted with
intermediate, there are two possible pathways. If O2 attacks the
fla· moiety in A (path c), concomitant electron transfer from
the CuI center to the generated fla–OO· moiety gives the alkyl
peroxide adduct B, from which the endoperoxide intermediate
C is generated by an intramolecular cyclization reaction
(path d). On the other hand, if O2 initially binds to the CuI site
in A, the CuII–superoxide adduct D may be generated (path e).
Then, the intramolecular radical coupling (path f) gives the
copper(II)–alkyl peroxide intermediate E, from which the same
a liquid-helium-cooled probe. The spectra of the complexes (2 m
M
in 0.5 mL of DMF) were recorded at –100 K, ca. 20 mW microwave
power, and ca. 9.40 GHz. The sample was placed in a nitric acid
washed quartz EPR tube and frozen in liquid nitrogen. Cyclic vol-
tammetry data were collected with a CHI620b system. All CV data
were obtained for samples in DMF with a complex concentration
endoperoxide intermediate C can be generated (path g). Once of 2 m
M and KClO4 (0.5 M) as the supporting electrolyte. The experi-
mental setup consisted of a glassy carbon working electrode, an
SCE reference electrode, and a platinum wire auxiliary electrode. All
potentials are reported versus SCE.
C is formed, the dissociation of CO provides intermediate F
(path h), which undergoes a ligand-exchange reaction with the
flaH present in the solution in a large excess (path i) to com-
plete the catalytic cycle. At this moment, no direct evidence is
available to determine the accurate reaction pathway (that is,
path c or path e). However, the reduction of NBT2+ mentioned
X-ray Crystallography: The single crystals were mounted on glass
fibers. The X-ray diffraction data were collected with a Rigaku
RAXIS-RAPID imaging-plate two-dimensional area detector using
above may support reaction path e, which involves a CuII–OO· graphite-monochromated Mo-Kα radiation (λ = 0.71069 Å) to
2θmax = 55°. The crystallographic calculations were performed by
direct methods with SHELXTL 97.[21] The non-hydrogen atoms and
hydrogen atoms were refined anisotropically and isotropically, re-
spectively. CCDC 1017425 (for 1), 1471869 (for 2′), 1471870 (for 3),
and 1471871 (for 4′) contain the supplementary crystallographic
data for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre.
species (superoxide-like species, intermediate D).
Conclusions
To obtain deeper insights into the role of the carboxylate group
of Glu73 in the active site of 2,4-QD, a series of CuII complexes
[CuIILn(AcO)] supported by carboxylate-containing (pyridyl-
Ligands LnH and Binary Complexes [CuLn(AcO)]: The synthetic
alkyl)amine ligands have been synthesized and characterized. procedures for the model ligands LnH are presented in the Support-
The CuII–flavonolate ternary complexes [CuIILn(fla)] have also
been prepared and characterized as structural and functional
models for the ES complex of 2,4-QD. The ternary complexes
exhibit reactivity similar to that of the enzyme (oxygenative O-
ing Information. The binary complexes were synthesized according
to the following procedure. A dry MeOH (5 mL) solution of
Cu(AcO)2·H2O (60.5 mg, 0.3 mmol) was added dropwise to an MeOH
solution (1 mL) containing LnH (0.3 mmol) at room temperature,
and the mixture was stirred for 30 min. The copper(II) complex was
heterocyclic ring-opening reaction) when treated in DMF at ca.
isolated as a blue powder through the addition of an excess of
90 °C under aerobic conditions. The reactivity is altered signifi-
diethyl ether to the final reaction solution and recrystallized from
cantly by the position of the carboxylate substituent of the li-
gand as well as the size of the chelate ring formed by the (pyr-
idylalkyl)amine moiety and the copper ion. A good correlation
MeOH and diethyl ether. Blue single crystals suitable for X-ray crys-
tallographic analysis were obtained by slow diffusion of diethyl
ether (for 1), hexane (for 2′), pentane (for 3), and toluene (for 4′)
between the reactivity and the oxidation peak potential of the into CH2Cl2 solutions of the complexes.
fla– moiety in the ternary complexes suggests that the Lewis
[CuL1(AcO)]·CH3OH·H2O (1): 70 mg, 46 %. ESI-MS (+): m/z = 477.1
acidity of the coordinating metal ion is crucial to the regulation
of the reactivity, that is, the lower the Lewis acidity of the metal
center, the higher the reactivity toward the oxygenation reac-
tion; the Lewis acidity can be controlled by the interaction be-
tween the copper(II) center and the carboxylate group as well
[CuIIL1(AcO) + Na]+. ESI-MS (+): m/z = 488.9/490.9 [CuIIL1(AcO) +
Cl]–, 513.0 [CuIIL1(AcO) + AcO]–. C23H29CuN3O7 (523.0383): calcd. C
52.82, H 5.59, N 8.03; found C 52.74, H 5.05, N 7.88. IR (KBr): ν =
˜
3406 (m), 3300 (m), 1610 (s), 1579 (s), 1562 (s), 1484 (w), 1443 (w),
1416 (s), 1377 (s), 767 (w), 757 (w) cm–1. IR (ethanol): ν = 3299 (m),
˜
as by the size of the chelate ring. The binary complexes can 1610 (s), 1580 (s), 1561 (s), 1485 (w), 1443 (w), 1416 (s), 1378 (s),
757 (m) cm–1
.
catalyze the oxygenative O-heterocyclic ring-opening reaction
under aerobic conditions, and the catalytic activities of the bi-
nary complexes are in the same order as that of the reactivity of
the ternary complexes; this suggests that the ternary complex
is involved as a key reactive intermediate in the catalytic reac-
tion. These results may provide important insights into the en-
zymatic mechanism of copper-containing dioxygenases.
[CuL2(AcO)]·CH3OH·2H2O (2): 93 mg, 59 %. ESI-MS (+): m/z = 477.2
[CuIIL2(AcO) + Na]+ (Figure S1a). C23H29CuN3O7 (523.0383): calcd. C
52.82, H 5.59, N 8.03; found C 52.68, H 5.03, N 8.00. IR (KBr): ν =
˜
3246 (m), 1610 (s), 1562 (s), 1481 (w), 1443 (s), 1372 (s), 1289 (w),
758 (m) cm–1. IR (ethanol): ν = 3334 (m), 1610 (s), 1562 (s), 1481
˜
(w), 1443 (s), 1373 (s), 1289 (w), 759 (m) cm–1
.
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[CuL3(AcO)]·2H2O (3): 98 mg, 63 %. ESI-MS (+): m/z = 505.4 [CuII-
L3(AcO) =
Na]+. ESI-MS (–): m/z [CuIIL3(AcO) +AcO]–. flavonol at λ = 343 nm. The concentration of O2 in the reaction
followed by monitoring the absorption changes of the substrate
+
C24H30CuN3O6.5 (528.0572): calcd. C 54.59, H 5.73, N 7.96; found C
solution was controlled by bubbling an O2/N2 mixture.
54.53, H 5.43, N 8.00. IR (KBr): ν = 3477 (m), 1667 (w), 1597 (s), 1568
Kinetic Measurements for [CuIILn(fla)]: After a DMF solution of
˜
(s), 1483 (w), 1444 (w), 1397 (s), 1354 (s), 1334 (w), 767 (m), 757 (m)
the ternary complex [CuIILn(fla)] (0.65–1.4 × 10–4
M in 3 mL of DMF)
cm–1. IR (ethanol): ν = 3353 (m), 1667 (w), 1596 (s), 1567 (s), 1483
˜
was kept at the desired temperature (75–90 °C) under N2 for several
minutes, O2 gas was introduced by gentle bubbling through a thin
Teflon tube. The time course of the reaction was followed by moni-
toring the absorption changes at λ ≈ 420 nm for the π→π* transi-
tion of the coordinated flavonolate ligand. The concentration of
O2 in the reaction solution was controlled by bubbling an O2/N2
mixture.
(w), 1444 (w), 1397 (s), 1353 (s), 1334 (m), 755 (m) cm–1
.
[CuL4(AcO)]·2H2O (4): 135 mg, 87 %. ESI-MS (+): m/z = 483.4 [CuII-
L4(AcO) + H]+. C24H30CuN3O6.5 (528.0572): calcd. C 54.59, H 5.73, N
7.96; found C 54.93, H 5.35, N 8.12. IR (KBr): ν = 3229 (m), 1608 (s),
˜
1558 (s), 1482 (w), 1445 (s), 1375 (s), 1287 (w), 758 (m) cm–1. IR
(ethanol): ν = 3235 (m), 1608 (s), 1562 (s), 1481 (w), 1443 (s), 1375
˜
(s), 1288 (w), 758 (m) cm–1. Blue single crystals of {[CuL4Cl]·H2O}n
(4′) suitable for X-ray crystallographic analysis were obtained during
the crystallization of 4 by slow diffusion of toluene into a CHCl3
solution of the complex. The Cl– anions originate from the solvent
CHCl3.
Products Analysis: For the catalytic reaction, the binary complex
[CuIILn(AcO)] (0.25 μmol in 50 μL DMF, 5 mol-%) was added to a
DMF solution of flavonol (1.2 mg, 5 μmol, in 5 mL of DMF), and the
mixture was stirred at 100 °C under dioxygen for 12 h. For the
oxidative degradation of the ternary model complexes, [CuIILn(fla)]
(ca. 1.6 mg, 2 μmol) was dissolved in DMF (2 mL), and the solution
was stirred at 70 °C under dioxygen for 8 h. After the reaction, the
mixture was concentrated by evaporation of the solvent, and the
remaining residue was dissolved in CH3OH (1.9 mL). o-Methyl-
Ternary Complexes [CuLn(fla)]: A dry CH2Cl2 (1.0 mL) solution of
flavonol (72.2 mg, 0.3 mmol) was added dropwise to a dry MeOH/
acetonitrile (3:1) solution (4.0 mL) of Cu(AcO)2·H2O (60.5 mg,
0.3 mmol) at room temperature under Ar. The mixture was stirred
for 1 d, the solvent was removed by evaporation, and the resulting
mixture was dissolved in CH2Cl2 (2.0 mL). A dry CH2Cl2 solution
(1.0 mL) containing ligand LnH (0.3 mmol) was added dropwise to
the above solution, and then the mixture was stirred under Ar for
another 1 d. The resulting ternary complexes [CuIILn(fla)] were iso-
lated as dark green powders by filtration. Pure microcrystalline sam-
ples were obtained by slow diffusion of hexane into dichloro-
methane solutions of the complexes at room temperature.
benzoic acid (10 m
solution) was added to the above solution as an internal standard.
Hypersil GOLD C18 column (Thermo Fisher Scientific
200 mm × 4.6 mm, 5 μm) was used for the HPLC analysis at room
M in 100 μL CH3OH, total 0.5 mM in 2.0 mL of
A
temperature with a mobile phase of CH3OH and 5 m
M NH4AcO
solution with a gradient at a constant flow rate of 0.5 mL min–1. The
yields of the products were calculated from the standard calibration
curve.
[CuIIL2(fla)]·3CH3OH (6): 68 mg, 31 %. ESI-MS (+): m/z = 633.3 [CuII-
L2(fla) + H]+. C38H39CuN3O8 (729.2776): calcd. C 62.58, H 5.39, N
Acknowledgments
5.76; found C 62.71, H 5.63, N 5.44. IR (KBr): ν = 3209 (m), 1608 (s),
˜
We gratefully acknowledge the financial support by the Na-
tional Natural Science Foundation of China (no. 21471025) and
1561 (s), 1481 (m), 1444 (m), 1415 (m), 1383 (m), 1351 (m), 1215
(m), 759 (m) cm–1. IR (ethanol): ν = 3233 (m), 1607 (s), 1562 /1541
˜
(s), 1481 (m), 1445 (m), 1416 (m), 1381 (m), 1351 (m), 1212 (m), 758 for a postdoctoral fellowship from the Japan Society for the
(w) cm–1
.
Promotion of Science (JSPS).
[CuIIL3(fla)]·4CH3OH (7): 123 mg, 52 %. ESI-MS (+): m/z = 661.1
[CuIIL3(fla) + H]+. C41H47CuN3O9 (789.2708): calcd. C 62.38, H 6.00,
Keywords: Dioxygen activation · Enzyme models ·
Carboxylate ligands · Copper
N 5.32; found C 62.14, H 5.65, N 5.30. IR (KBr): ν = 3393 (m), 1684
˜
(w), 1616 (s), 1542 (s), 1510 (m), 1495 (m), 1418 (s), 1214 (m), 752
(m) cm–1. IR (ethanol): ν = 3356 (m), 1615 (s), 1592 (s), 1541 (s),
˜
1510 (m), 1495 (m), 1440 (m), 1417 (s), 1357 (w), 1214 (m), 751 (m)
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.
[CuIIL4(fla)]·4CH3OH (8): 90 mg, 38 %. ESI-MS (+): m/z = 661.2 [CuII-
L4(fla) + H]+. C41H47CuN3O9 (789.2708): calcd. C 62.38, H 6.00, N
5.32; found C 62.19, H 5.78, N 5.30. IR (KBr): ν = 3399 (m), 1610 (s),
˜
1591 (s), 1542 (s), 1510 (m), 1495 (m), 1440 (w), 1420 (s), 1215 (m),
753 (m) cm–1. IR (ethanol): ν = 3325 (m), 1613 (s), 1590 (w), 1572
˜
(w), 1541 (s), 1509 (m), 1493 (vs), 1440 (m), 1418 (s), 1214 (m), 752
(w) cm–1
.
Kinetic Measurements for the Catalytic Reaction with [CuII-
Ln(AcO)]: The reactions of the substrate flavonol (flaH) with O2 cata-
lyzed by the complexes [CuIILn(AcO)] were performed in a 10 mm
path-length UV/Vis cell held in a Unisoku thermostated cell holder
USP-203 (the desired temperature can be set within 0.5 °C). After
the solution of substrate (0.72–1.4 × 10–4
kept at the desired temperature (85–100 °C) under N2 for several
minutes, the catalyst complex [CuIILn(AcO)] (2.0–5.0 × 10–6
in
M in 2.95 mL of DMF) was
M
50 μL DMF, 2.0–5.0 %) was injected into the solution with a micro-
syringe, and then O2 gas was introduced by gentle bubbling
through a thin Teflon tube. The time courses of the reactions were
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Received: November 13, 2016
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Full Paper
Structure–Reactivity Relation-
ships
Y.-J. Sun,* P. Li, Q.-Q. Huang,
J.-J. Zhang, S. Itoh* ......................... 1–11
Dioxygenation of Flavonol Cata-
lyzed by Copper(II) Complexes Sup-
ported by Carboxylate-Containing
Ligands: Structural and Functional
Models of Quercetin 2,4-Dioxygen-
ase
The CuII complexes [CuIIL(AcO)] and quercetin 2,4-dioxygenase (2,4-QD) to
[CuIIL(fla)] (fla– = flavonolate) sup- obtain insights into the role of the
ported by a series of carboxylate-con- carboxylate group of Glu73 in the ac-
taining ligands (L1–L4) are reported as tive site of 2,4-QD.
structural and functional models of
DOI: 10.1002/ejic.201601371
Eur. J. Inorg. Chem. 0000, 0–0
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11
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim