Nitric oxide-release study of a bio-inspired copper(i)-nitrito complex under chemical and biological conditions

Article abstract of DOI:10.1039/c8dt02281j

The selective and efficient nitrite reduction process is ubiquitous in biological systems. To understand copper-mediated nitrite reduction, we developed a bio-inspired model system to investigate the mechanism of copper-containing nitrite reductase. A well-characterized copper(i)-nitrate complex with amino functionalized 2-(diphenylphosphino)aniline ligands, [(Ph₂PC₆H₄(o-NH₂))₂Cu(ONO)], demonstrated the aniline protonation will cause NO release in an acidic environment. To further understand NO releasing ability, we also performed pH-dependency experiments and confocal imaging to release NO under physiological buffer conditions. According to titration and spectroscopic studies on the protonation reaction of complex [(Ph₂PC₆H₄(o-NH₂))₂Cu(ONO)], we proposed a mechanistic pathway for proton transfer and NO release. Furthermore, DFT calculations predicted that the release of NO takes place via aniline in both organic and aqueous media. These results highlight the importance of the proton-rich microenvironment around the copper(i)-nitrite core to induce nitrate reduction in a chemical and biological environment.

Full text of DOI:10.1039/c8dt02281j

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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
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a Zr-metal–organic
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Nitric Oxide Release Study of a Bio-inspired Copper(I)-
nitrito Complex on Chemical and Biological Conditions
Wan-Jung Chuanga,‡, Manmath Narwanea,‡, Hsing-Yin Chena, Chai-Lin Kaoa,b, Bin
Huangc, Kuang-Mei Hsud, Yun-Ming Wang*,c,d, Sodio C. N. Hsu*, a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The selective and efficient nitrite reduction process ubiquitous in a biological system. To understand the copper-mediated nitrite reduction,
we developed a bio-inspired model system to investigate the mechanism of copper-containing nitrite reductase. Structurally well-
characterized copper(I)-nitrate complex with amino functionalized 2-(diphenylphosphino)aniline ligands, [(Ph2PC6H4(o-NH2))2Cu(ONO)],
demonstrated the NO release and first protonation of aniline in an acidic environment. To further understand NO releasing ability we also
performed pH dependency experiments and confocal imaging method to release NO under physiological buffer condition. Based on
titration and spectroscopic studies of complex [(Ph2PC6H4(o-NH2))2Cu(ONO)], we proposed a mechanistic pathway for NO release and
first proton transfer site. Besides, DFT calculations predict that the release of NO takes place via aniline in both organic and aqueous media.
These results highlight the importance of proton-rich microenvironment around copper(I)-nitrite core to induce the nitrate reduction in a
chemical and biological environment.
releasing under acidic condition.23-25 Recently, another approach
towards these studies emphasized through the electron-
deficient β-diketiminate copper(II)-nitrito complexes, showed
Introduction
the activation of nitrite through the oxygen atom transfer
Copper containing nitrite reductase (Cu-NIRs) holds a blue-green
pathway or thiol reduction.26, 27 More recent studies also show
type 1 copper center that functions as a transfer of redox electron
that the electrocatalytic and proton coupled electron transfer
and type 2 copper center where nitrite binds and could catalyse
approach to reduction of nitrite to release NO.28
nitrite reduction of nitric oxide formation.1-13 Binding modes of
nitrite in type 2 Cu(I)/(II) redox process and microenvironment
Apart from nitrite binding mode and proton/electron transfer
around copper center, which is essential for catalytic copper redox
pathways for denitrification, Solomon et. al, demonstrated the
protein designs.14 In consideration of nitrite binding modes,
effect of pH using spectroscopic and DFT studies on the nitrite
copper(I)-nitro or -nitrito species both are postulated as the key
bound type 2 copper site in wild-type enzyme of Rhodobacter
intermediates for type 2 center of Cu-NIRs. Synthesis and reactivity
studies of the Cu-NIRs models such as copper(I)-nitro or -nitrito
sphaeroides. They found at high pH, there is no electron transfer
from reduced type 1 to the nitrite bound oxidized type 2 copper,
complexes lead to the detail understanding of the catalytic
while protonation triggers type 1 → type 2 electron transfer and
mechanism. Several Cu(I)-NO2 complexes possessing a η1-nitro (N-
generation of NO.11 Indeed, steady state kinetic turnover
bound) mode with biomimetic ancillary ligands intensively studied
experiments for nitrite reduction as function of pH indicated the
for their reactivity.15-21 Regarding copper(I)-nitrito (O-bound)
existence of two pKas, where the lower pKa (~5) has been invoked
complexes, there are few known examples containing η2-O,O-
as the protonation equilibrium of an Asp residue and the higher pKa
coordination.22-25 Moreover, only three of them have been
(~7) has been ascribed as the pKa of His residue.8 To understand
demonstrated that, the nitrite reduction activity accompany NO
the effect of pH and denitrification mechanism in Cu-NIRs, we used
biomimetic designing strategy and synthesized copper(I)-nitrito
model system.
A
copper(I)-nitrito complex [(Ph2PC6H4(o-
OMe))2Cu(ONO)] (1), was reported by our lab, represent as a rare
example of complexes exhibiting copper(I)-nitrito binding mode.24
Complex 1 confined as a hemilabile phosphine-ether type o-
(diphenylphos-phino)anisole (Ph2PC6H4(o-OMe)) ligand and could
mimic the reactivity of type 2 Cu-NIRs to produce NO gas under
acidic condition. We concerned in this regards, how does the
adjacent basic groups (e.g., Ph-NH2) will influence the nitrite
reduction ability of copper(I)-nitrito species? To address these
concern, we used a 2-(diphenylphos-phino)aniline (Ph2PC6H4(o-
NH2))29 as a ligand of choice and compared with known type 2
copper(I)-nitrito model complexes.23-25 The aniline group of this
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type of bidentate Ph2PC6H4(o-NH2) ligand can act as a proton exhibiting an asymmetric η2-O,O’-bonding mode (Fig. 1). The
acceptor, which may cause hydrogen bonding interaction asymmetric nitrito-O,O’ coordination (Cu-O) distance in 4 [2.226(3)
a
between the aniline hydrogen and copper(I)-nitrito unit. Herein, we and 2.257(3) Å] compared with the known copper(I) complexes
describe the synthesis of three copper(I) complexes containing the [(PPh3)2Cu(ONO)] [2.191(4) Å],22 suggests that the Cu(I)-Onitrito
Ph2PC6H4(o-NH2) ligands. Elucidate their reactivity and, interaction in 4 is rather weak. The exclusive bonding mode may be
furthermore, we explained how copper(I)-nitrito containing 2- derived from the more sterically hindered phosphine ligand in 4
(diphenylphosphino) aniline ligands, [(Ph2PC6H4(o-NH2))2Cu(ONO)] compared with other phosphine ligands.22-24
(4), releases the NO in chemical and biological conditions.
Result and Discussion
The reaction of bidentate P,N-donor ligand Ph2PC6H4(o-NH2) with
[Cu(CH3CN)4](BF4) (2:1 molar ratio) in CH2Cl2 under N2 gave the
copper(I) complex [Cu(κ2-Ph2PC6H4(o-NH2))2](BF4) (2), which was
previously isolated as a colourless solid.30-33 We also examined the
reaction of Ph2PC6H4(o-NH2) with CuCl to afford
a neutral
compound [CuCl(Ph2PC6H4(o-NH2))2] (3). Treatment of complexes 2
and 3 with NaNO2 gave a complex [(Ph2PC6H4(o-NH2))2Cu(ONO)] (4),
which exhibits an asymmetric η2-O,O’ bound nitrito ligation
(Scheme 1).
Fig. 1 ORTEP representation of complex 4 (50% ellipsoids, hydrogen atoms
are not shown for clarity).
H2N
PPh2
Ph2P
NH2
NaNO2
- NaCl
Cu
CuCl
Ph2P
Cl
H2N
We examined the NO-generating ability of 4 by using a reported
GC-TCD technique.19, 20 To a solution of 4 in CH2Cl2, excess of acetic
acid was added at room temperature, the color of the mixture
solution changed from yellow to green-blue, followed by the
evolution of NO gas (∼75% v/v; see Fig. S5). Comparing the NO
evolution results with those arising from studies of the known
copper(I)-nitrito complexes [(Ph2PC6H4(o-OMe))2Cu(ONO)] (1)24 and
[(2,6-(Ph2P(o-C6H4)CH=N)2C5H3N)Cu(ONO)]23 demonstrated that
complex 4 can also be a functional model for Cu-NIRs.
3
-
BF4
H2N
Cu
NH2
Ph2P
Ph2P
NH2
PPh2
NH2
O
NaNO2
[Cu(NCMe)4](BF4)
Ph2P
Cu
Ph2P
- NaBF4
N
O
NH2
2
4
Scheme 1 Synthesis of copper(I)-phosphine complexes.
All copper(I)-phosphine complexes 2-4 give significant π-π*
transitions bands due to the phenyl and aniline groups of phosphine
ligands and no absorption band observed in visible region (Fig. S1).
In case of complex 3 and 4, a shoulder peak appeared at 310 nm,
assigned as an absorption band for unbound aniline group.
Interestingly, complex 2 shows a different absorption peak at 275
nm that suggest the bound aniline groups to the copper center.
These observations are consistent with crystallographic data (Fig.
S4). The FT-IR spectra of 2-4 (KBr) displays the stretching
frequencies of v(N-H) in the range of 3200-3500 cm-1. Complex 4
displays νas and νs stretching of the copper-nitrito at 1265 and 1209
cm-1, respectively (Fig. S2). The 15N sensitive bands of 4 are at 1247
and 1169 cm-1 (Fig. S3), also assigned to νas and vs stretches,
respectively.15, 17 The 31P{1H} NMR spectra of 2-4 exhibit a broad
signal indicates the direct coordination of phosphorus atom of the
Ph2PC6H4(o-NH2) ligand to the copper(I) center. Additionally, the
resonance of NH2 occurred further downfield than the free
Ph2PC6H4(o-NH2) ligand in 1H NMR.
2
1
0
400
600
800
Wavelength (nm)
Fig. 2 Spectral changes observed during the anaerobic reaction of 4 with 300
eq. of TFA in CH2Cl2 at 273K. Inset: absorbance change over reaction time at
450 nm. (5 seconds interval)
The reaction of 4 with acetic acid is sufficiently slow that
intermediates might be detectable, but none were observed. We
think the protonation intermediate might be detected if a much
stronger acid were used. To examine this, we monitored the
reaction of 4 with trifluoroacetic acid (TFA), which is as stronger
acid than acetic acid. The reaction of 4 with TFA was monitored by
recording the UV-vis absorption spectra at 5-second intervals (Fig.
2). The 310 nm band of 4 was observed to be a shoulder at 273K.
Upon addition of excess TFA to a solution of 4 under N2, a decrease
in the peak at 310 nm and an increase in a peak at 266 nm were
The crystallographic results of 2 suggested that copper ion is
bound by two chelated Ph2PC6H4(o-NH2) ligands in a distorted
tetrahedral coordination geometry (Fig. S4), and consistent with
-
-
literature reports on ClO4 or PF6 salts.31, 32 Complex 3 affords a
four-coordination environment around the copper ion with axially
bound chloride (Fig. S4). Moreover, one of the aniline group of
Ph2PC6H4(o-NH2) in 3 is dangling while another one is bound, shows
a weaker Cu-N bonding interaction [Cu-N =2.251(2) Å]. In case of
complex 4, the structure consists of two Ph2PC6H4(o-NH2) ligands
with two dangling aniline groups and an O-bound nitrito ligand
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observed. A new intermediate with absorption peaks at 450 nm Cu(II) complex [Cu2(CF3COO)4(H2O)2] was isolated after NO
(less than 40 seconds) immediately disappeared and the absorption generation from reaction mixtures. These findings provide a novel
band at 325 nm, 410 nm, and 510 nm then rapidly increased. A insight for the bio-inspired Cu-NIRs models. The second
similar case on the protonation intermediated observed by stronger coordination sphere effects of aniline group on complex 4 would
acid could be found in literature.13 The spectrum of the final facilitate the elucidation of mechanism in type 2 copper center in
product is similar to the known Cu(II) complex [Cu(Ph2PC6H4(o- enzymes. As well, these results will also help us to understand the
NH2))2]2+ which was synthesized by following the literature interaction and function of amino group (aniline) residues in
procedure.30 Efforts on the isolation of final product microenvironment around copper(I)-nitrito core.
[Cu(Ph2PC6H4(o-NH2))2]2+ was failed, a known Cu(II) complex
[Cu2(CF3COO)4(H2O)2] was isolated after NO generation reaction
mixtures and demonstrated by crystallographic characterization.
Complex 4 contains two dangling aniline groups, which may
influence the nitrite reduction process. To address this issue, the
reactivity study of complexes 1 and 4 were performed (Table S1).
Both complexes 1 and 4 follow the first order kinetics at 273-243 K
and the rate of reaction almost directly proportional to the
concentration of TFA. Analysis of these results using the typical
Eyring approach gave the ꢀH‡ = 24.1
7.4 JK-1mol-1 for complex 4. Comparisons of the activation
parameters with known copper(I)-nitrito phosphine complex 1 (ꢀH‡
= 29.4
9.5 JK-1 mol-1) reveals that
±
2.8 kJ mo1-1 and ꢀS‡ = -196.7
±
±
7.6 kJ mo1-1 and ꢀS‡ = -171.1
±
complex 4 has smaller ꢀH‡ and larger ꢀS‡ values (Fig S7 and S8). The
smaller ꢀH‡ value and larger negative ꢀS‡ value for reactions of 4
means a nearly complete protonation (association) intermediate in
the nitrite reduction process.
Scheme 2 Proposed mechanism for nitrite reduction of complex 4 with TFA.
Density functional theory (DFT) calculations were carried out to
provide further insight into the nitrite reduction mechanism of
complex 4 on chemical and biological (aqueous) conditions. The
activation free energy of the HO−NO bond cleavage (i.e., from B to
C in Scheme 2) was estimated to be 22.4 and 19.6 kcal mol-1 in
dichloromethane and water, respectively. Our calculations indicate
the intermediate B as a closed shell singlet state. However, the
transition state of HO−NO bond breaking TSBC displays an open shell
singlet state that possesses biradical characteristics. The plots of
highest occupied molecular orbital (HOMO) and spin density at TSBC
are shown in Fig. 3. The distribution pattern of HOMO for α and β
electrons is dramatically dissimilar; the former mainly localized on
the leaving NO, whereas the latter spread over the Cu−P bonds and
OH ligand. The spin density plot also clearly shows the bi-radical
character of the bond breaking transition state. The natural
population analysis reveals that the atomic charge of copper
increases from 0.274(CH2Cl2)/0.307(H2O) at B to 1.025/0.655 at
TSbc. These results suggest that the HO−NO bond cleavage is
triggered by the electron transfer from Cu(I) to NO2H. In
comparison with complex 4, we also calculated the nitrite reduction
ability of complex 1. Interestingly, the activation free energy of
The possible protonation sites for complex 4 are either nitrogen
atom of aniline or the oxygen atom of nitrite. In order to
understand the preferred protonation site, control experiment in
complex 3 was chosen as this complex has dangling aniline arm
which may shed some light on the protonation mechanism.
Therefore, the titration of complex 3 with 0.5~4 eq. of TFA
performed anaerobically in CH2Cl2 and observed the UV-vis spectral
changes (Fig. S9). These results reveals, the protonation of complex
3, shows a decrease in a peak at 290 nm and no copper(II) species
formation observed. Moreover, after consumption of two
equivalents TFA by complex 3, anilines get protonated to become
anilinium ion which is supported by the FT-IR and NMR. In FT-IR, N-
H vibration peak after protonation shows peaks around 3050 cm-1
+
for the stretching modes of Ph-NH3 . Further, the symmetric
+
deformation peak for NH3 observed at 1558 cm-1 a weak band in
the FTIR spectrum (Fig. S10).34 Indeed, NMR titrations of complex 3
at room temperature also support the anilinium ion formation (Fig.
S11). Hence, the protonation should occur at the nitrogen of
aniline. UV-vis titration curve of complex 4 clearly illustrates the
consumption of only one equiv of TFA to reach the plateau (Fig.
S12). Extended titration experimentation to EPR for complex 4 also
shows the one equiv. of TFA gives a maxima signal of EPR (Fig. S13). HO−NO bond breaking for complex 1 was 17.4 kcal mol-1 in both
These phenomena may suggest that one equiv. TFA protonates the CH2Cl2 and aqueous solutions, somewhat lower than that for
amino group of complex 4 and triggers the nitrite reduction to
release the NO(g).
complex 4. The moderate activation energies of HO−NO bond
cleavage catalysed by Cu(I) in complex 1 and 4 are consistent with
the experimental observation that both complexes can release NO
in acidic conditions. However, as will be shown later, the complex 4
displays a superior NO releasing ability over complex 1 in neutral
and basic buffer environments, which seems to contradict with the
calculated activation energies of HO−NO bond cleavages. We thus
calculated the free energy changes of protonation of complex 1 and
4 by H3O+ in aqueous environment. It turned out that the
protonation on amine moiety of 4 was 3.4 kcal mol-1 lower than the
direct protonation on NO2- of 1. This result suggests that the amine
group on the second coordination sphere can act as an absorber to
capture proton from neutral and basic environments, which in turn
Based on UV-vis and EPR titration experimental observations in
acidic environments, a plausible mechanism of the nitrite reduction
of complex 4 was proposed and depicted in Scheme 2. The titration
experiments of 4 with TFA, is one of the supporting evidence for the
proposed mechanism. The shift of absorption bands towards the
shorter wavelength, support the existence of protonated aniliniun
intermediate A. From intermediate A, anilinium proton transferred
to nitrite group resulting into formation of intermediate B
(CuNO2H).
Further, intermediate
B
might trigger an
electron transfer process by releasing NO gas and become
rearranged to form a stable copper(II) species C. Finally, a known
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renders complex 4 capable of undergoing nitrite reduction and
releasing NO in physiological conditions.
To understand the application of NO release in actual biological
system, we performed in-vitro treatment of complexes 1 or 4 with
macrophages (RAW 264.7 cells) and investigated the NO release
studies using confocal microscopy. In this studies also, we used FA-
OMe as fluorescent material. In case of complex 1 no green
fluorescent signal were observed (Fig. 5a). However, in complex 4
strong green fluorescent signals observed with increase in
concertation as shown in Fig. 5(b). These results suggest that
microenvironment around copper(I)-nitrito core in complex 4 plays
a key role to release NO in pH 7.5 buffer conditions. From the
chemical and biological experimental data, we observed that amine
protons plays key role in the release of NO at basic or physiological
conditions. Usually, the nitrite reduction of copper(I)-nitrito species
will perform under acidic condition. As a result, these findings
demonstrated that nitrite reduction ability have great impact of
adjacent groups of complex 1 and 4 to release the NO(g).
Fig. 3 Representations of plots (a) highest occupied molecular orbitals and
(b) spin density for transition state TSbc.
To explore the potential of nitrite reduction and NO releasing
ability we extended the experimentations to physiological condition.
Complex 1 or 4 were tested under basic buffer condition with
different pH values and noted the fluorescence response for NO
release (see Fig. 4). In these experiments, FA-OMe (5-amino-2-(6-
hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester, green
fluorescence)35 used as a fluorescence detector to detect the nitric
oxide release from complexes 1 or 4. In this case, DETA NONOate
used as
a standard NO releasing source. The normalized
fluorescence response (F-F0) is directly proportional to the amount
of NO release and the linear correlation between NO release and
normalized fluorescence response.35 To determine the chemical
yield of NO, we recorded the normalized fluorescence response of
FA-OMe with varying equivalence of DETA NONOate (Fig S14 and
S15). The gradual decrease in the fluorescence intensity of DETA
NONOate indicates the decrease in the decomposition rate of
primary amine of DETA NONOate with increasing pH.36-38 For
complex 1 we didn’t observed a significant release of NO at
different basic pH values. However, complex 4 shows a similar trend
in fluorescence intensity like DETA NONOate which suggest the
importance of the amine group microenvironment closed to
copper(I)-nitrito center. Hence, the insight of pH dependency
experiments explains the influence of aniline group of complex 4 to
act as a NO donor source at physiological conditions.
(a)
50
40
30
20
10
0
(b)
Fig. 5 Confocal imaging of macrophage (RAW 264.7 cells) treated with
complexes 1 (a) or 4 (b) in 0, 1, and 5 µM at 37°C for 1 hr. Nuclei were
stained with DEPI in blue and FA-OMe were displayed in green fluorescence.
Conclusions
We successfully synthesized
a copper(I)-nitrito complex 4
containing Ph2PC6H4(o-NH2) ligands as a bio-inspired compound for
Cu-NIRs. The titration experiments and DFT studies helped to
understand the preferred protonation site and NO release from
complex 4. pH dependency, and confocal imaging results elaborates
the role of microenvironment around complexes 1 and 4, and how
aniline groups influenced the copper(I)-nitrito core to induce the
nitrite reduction under physiological as well in basic buffer
condition. Indeed, computational results also show the aniline
microenvironments in complex 4 acts as a proton absorber at
chemical and biological (aqueous) conditions. Overall, these results
6.5
7.5
8.5
9.5
10.5
11.5
pH
FA-OMe + 4
FA-OMe + NO
FA-OMe + 1
Fig. 4 Normalized fluorescence response of the FA-OMe (1 μM) with DETA
NONOate, 1, and 4 (5 μM) in different pH values (100 mM PBS buffer with
0.54% DMSO) and 25.0 ± 0.1 °C.
4 | J. Name., 2012, 00, 1-3
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will enhance our understanding of nitrite reduction process at A solution of 2 (0.5 g, 0.709 mmol) in MeOH (20 mL) was added to a
copper center of Cu-NIRs.
stirring MeOH (10 mL) solution of NaNO2 (0.049 g, 0.709 mmol) and
stirring for two hours. The solvent removed under vacuum. The
residue extracted with CH2Cl2 and layer with hexane. The resulting
white powder was recrystallized from CH2Cl2/ Et2O to yield the
product as colorless needles of [Cu(Ph2PC6H4(o-NH2))2(ONO)] (4).
Yield: 67.8% (0.319 g, 0.481mmol).1H NMR (DMSO-d6): δ 5.50(s, 4H,
-NH2), 6.57-7.55(m, 28H, Ph). 31P{1H} NMR (DMSO-d6): δ -12.93(s).
UV-vis absorption (CH2Cl2, λmax, nm) (ε/M-1cm-1) 309 (15165). ESI-
MS: 618.18 (100%) [M]+. Anal. Calcd for C36H32CuN3O2P2: C, 65.10;
H, 4.86; N, 6.33. Found: C, 64.79; H, 4.86; N, 6.31.
Experimental Section
General considerations
All manipulations carried out under an atmosphere of purified
dinitrogen with standard Schlenk techniques. Chemical reagents
purchased from Aldrich Chemical Co. Ltd., Lancaster Chemicals Ltd.,
or Fluka Ltd and Cayman Chemical. All the reagents were used
without further purification, apart from all solvents that were dried
over Na (Et2O) or CaH2 (CH2Cl2, CH3CN) and then thoroughly
degassed before use. The compounds [Cu(CH3CN)4](BF4),39
[(Ph2PC6H4(o-OMe))2Cu(ONO)] (1),24 Ph2PC6H4(o-OMe),40 and
Ph2PC6H4(o-NH2)29 were prepared as described in the literature.
Dulbecco’s Modified Eagle Medium (DMEM) and sodium pyruvate
purchased from Cellgro. Fetal bovine serum (FBS, 10%) purchased
from HyClone. Bright-field and fluorescence images recorded on an
Olympus IX71 fluorescence microscope equipped with a 100 W
mercury lamp, B-2A filters, and a color CCD camera system. IR
Measurement of NO Generated from 4.
A solution of 4 (4 mg, 0.006 mmol) in CH2Cl2 (2 mL) was prepared in
a small vial capped with a rubber septum. A solution of acetic acid
(10.3 μL) in CH2Cl2 (0.1 mL) was then introduced with a syringe at
room temperature. The solution changed immediately from
colorless to blue. Analysis of the headspace gas by a thermal
conductivity detector indicated that NO had been generated
(75.43±1.12% for 4). The NO generation data obtained by three
different experiments (see Table S1, Supporting Information). NO
concentration performed by calibrating curve response with known
concentrations of NO gas mixed with N2 (10, 20, 30, and 40 ppm of
NO in N2); molar quantities were calculated using the ideal gas
equation.
spectra recorded on
a
Perkin-Elmer System 2000 FT-IR
spectrometer. UV-vis spectra recorded on an Agilent 8453
spectrophotometer. The fluorescence spectra recorded on a Hitachi
F-7000 fluorescence spectrophotometer. 1H NMR, 13C NMR, and 31
P
NMR spectra were acquired on a JEOL JNM ECS 400 MHz. ESI mass
spectra were collected on a Waters ZQ 4000 mass spectrometer.
Elemental analyses performed on a Heraeus CHN-OS Rapid
Elemental Analyzer. Gas chromatography thermal conductivity
detector (GC-TCD) experiments were performed by using a Varian
CP-3800 gas chromatography, Porpak Q column (6 ft, 20 mL/min
flow rate, 30°C, nitrogen carrier gas), and TCD detector.
Kinetics. The kinetics studies of nitrite reduction of 4 in CH2Cl2 were
carried out by monitoring the intensity decrease of the 266 nm
band. The absorbance detected by an Agilent 8453
spectrophotometer. The reaction started with the addition of 100
equiv. of TFA, which degassed with N2 before use.
[Cu(κ2-Ph2PC6H4(o- NH2))2](BF4) (2)
pH Dependent NO release study.
The different pH values of buffer solution were prepared by 100
mM PBS buffer and adjust pH value by NaOH(aq). The stock solution:
FA-OMe was 2.5 mM in DMSO, complexes 4 and 1 were 0.5 mM in
DMSO, respectively and DETA NONOate was 0.25 mM in DMSO.
The FA-OMe stock solution diluted in different pH value of PBS
buffer, and added 4, 1 or NO solution, respectively for 4 hr at room
temperature. Then the reaction solution was recorded fluorescence
by the Hitachi F-7000 fluorescence spectrophotometer (λex = 460
nm; λem = 515 nm).
A solution of [Cu(CH3CN)4](BF4) (2.265 g, 0.0072mol) in CH3CN (30
mL) was added dropwise to a stirring CH3CN (20 mL) solution of
Ph2PC6H4(o-NH2) (4 g, 0.0144mol). The reaction mixture was stirred
for an hour and concentrated to about 10 mL, then slow diffusion of
Et2O into the solution and allowed to stay at room temperature for
one day to obtain colorless crystals of [Cu(κ2-Ph2PC6H4(o-
NH2))2](BF4) (2). Yield: 96% (4.88 g, 0.0069mol).1H NMR(DMSO-d6):
δ 5.68 (s, 4H,-NH2), 6.776-7.479 (m, 28H, Ph). 31P{1H}NMR (DMSO-
d6): δ -12.09(s). UV-vis absorption (CH2Cl2, λmax, nm) (ε/M-1cm-1)
260 (20531), ESI-MS: 618.11(100%) [M]+
.
Cell Culture and In Vitro Cell Imaging.
[CuCl(Ph2PC6H4(o-NH2))2] (3)
Raw 264.7 murine macrophages obtained from the American Type
Culture Collection (Manassas, VA). The cells cultured in Dulbecco’s
Modified Eagle Medium (DMEM) and supplemented with 10% fetal
bovine serum (FBS), 1% sodium pyruvate, and 1% MEM
nonessential amino acids at 37 °C under a humidified 5% CO2
atmosphere. For cell imaging studies, Raw 264.7 murine
macrophages passed and plated into poly-D-lysine coated plates
containing 2 mL of DMEM and incubated at 37 °C with 5% CO2. For
A solution of CuCl (0.196g, 1.98 mmol) in CH3CN (10 mL) was added
to a stirringCH3CN (15 mL) solution of Ph2PC6H4(o-NH2) (1.1 g, 3.97
mmol) and stirring for two hours. A white solid precipitate forms
immediately. The solvent removed under vacuum. The residue was
extracted withCH2Cl2, The resulting white powder was recrystallized
from CH2Cl2/ Et2O to yield the product as colorless needles of
1
[CuCl(Ph2PC6H4(o-NH2))2] (3). Yield: 82.5% (1.07 g, 1.64mmol). H
5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl
(FA-OMe)35
NMR (DMSO-d6): δ 3.32(s, 4H,-NH2), 6.42-7.39(m, 28H, Ph). 31P{1H}
NMR (DMSO-d6): δ -12.612(s). UV-vis absorption (CH2Cl2, λmax, nm)
(ε/M-1cm-1) 303 (14912). ESI-MS: 647.08 (100%) [M-Cl]+. Anal. Calcd
for C36H32ClCuN2P2: C, 66.16; H, 4.94; N, 4.29. Found: C, 66.12; H,
5.00; N, 4.29.
localization studies, Raw 264.7 murine macrophages were pre-
stimulated with complexes 1 or 4 in 0, 1, and 5 µM at 37°C for 1 h,
and then the cells were co-incubated with 10 μM of FA-OMe for 4
h. The cells washed three times with 1 mL of PBS prior to imaging
and then bathed in 2 mL of PBS during the imaging procedure.
[Cu(Ph2PC6H4(o-NH2))2(ONO)] (4)
DFT calculations.
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Geometry optimizations and vibrational frequency calculations 11.
were performed by using M06/6-31G* method at gas phase. To
obtain more reliable energies, M06/6-311+G** single point energy 12.
calculations were carried out at the M06/6-31G* optimized
geometries. The solvent effect (dichloromethane and water) taken 13.
into account in the energy calculations by SMD continuum solvation
model. The free energy corrections were made at the standard 14.
conditions of 1 atm and 298.15 K. Numerical integrations were
done using ultrafine grids. All calculations were accomplished by
S. Ghosh, A. Dey, Y. Sun, C. P. Scholes and E. I. Solomon, J.
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Gaussian 09 program.41
15.
X-ray crystal structure determinations.
16.
The crystal structures of complexes 2, 3, and 4 suitable for single
crystal X-ray diffraction analysis were grown by layering a CH2Cl2
solution with diethyl ether. All X-ray reflections were measured
with Mo Kα radiation (λ = 0.71073 Å) on a Nonius Kappa CCD
diffractometer. The data collection was executed using the SMART
program.42 Cell refinement and data reduction were made with the
SAINT program.43 The structure was determined using the
SHELXTL/PC program42 and refined using full-matrix least-squares.
All non-hydrogen atoms were refined anisotropically, whereas
hydrogen atoms placed at the calculated positions and included in
the final stage of refinements with fixed parameters.
17.
18.
19.
20.
21.
Acknowledgements
22.
23.
24.
25.
26.
27.
28.
We gratefully acknowledge financial support from the Ministry of
Science and Technology of Taiwan (MOST 106-2113-M-037-019)
and Kaohsiung Medical University “Aim for the Top University Grant,
grant No. KMU-TP105PR12”.
Conflicts of interest
There are no conflicts to declare.
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Table of contents entry:
Aniline groups create a microenvironment around copper(I)-nitrito core to induce the nitrite
reduction under physiological buffer condition.