Spectroscopic investigation of the noncovalent association of the nerve agent simulant diisopropyl methylphosphonate (DIMP) with zinc(II) porphyrins

Article abstract of DOI:10.1021/jp405976h

Organophosphonates pose a significant threat as chemical warfare agents, as well as environmental toxins in the form of pesticides. Thus, methodologies to sense and decontaminate these agents are of significant interest. Porphyrins and metalloporphyrins offer an excellent platform to develop chemical threat sensors and photochemical degradation systems. These highly conjugated planar molecules exhibit relatively long-lived singlet and triplet states with high quantum yields and also form self-associated complexes with a wide variety of molecules. A significant aspect of porphyrins is the ability to functionalize the peripheral ring system either directly to the pyrrole rings or to the bridging methine carbons. In this report, steady-state absorption and fluorescence are utilized to probe binding affinities of a series of symmetric and asymmetric zinc(II) metalloporphyrins for the nerve agent simulant diisopropyl methylphosphonate (DIMP) in hexane. The red shifts in the absorption and emission spectra observed for all of the metalloporphyrins probed are discussed in the frame of Gouterman's four orbital model and a common binding motif involving coordination between the metalloporphyrin and DIMP via interaction between the zinc metal center of the porphyrin and phosphoryl oxygen of DIMP (Zn-O=P) is proposed.

Full text of DOI:10.1021/jp405976h

Article
pubs.acs.org/JPCA
Spectroscopic Investigation of the Noncovalent Association of the
Nerve Agent Simulant Diisopropyl Methylphosphonate (DIMP) with
Zinc(II) Porphyrins
William A. Maza,† Carissa M. Vetromile,‡ Chungsik Kim,§ Xue Xu, X. Peter Zhang, and Randy W. Larsen*
Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States
S
* Supporting Information
ABSTRACT: Organophosphonates pose a significant threat as chemical
warfare agents, as well as environmental toxins in the form of pesticides. Thus,
methodologies to sense and decontaminate these agents are of significant
interest. Porphyrins and metalloporphyrins offer an excellent platform to
develop chemical threat sensors and photochemical degradation systems. These
highly conjugated planar molecules exhibit relatively long-lived singlet and
triplet states with high quantum yields and also form self-associated complexes
with a wide variety of molecules. A significant aspect of porphyrins is the ability
to functionalize the peripheral ring system either directly to the pyrrole rings or
to the bridging methine carbons. In this report, steady-state absorption and
fluorescence are utilized to probe binding affinities of a series of symmetric and
asymmetric zinc(II) metalloporphyrins for the nerve agent simulant diisopropyl
methylphosphonate (DIMP) in hexane. The red shifts in the absorption and
emission spectra observed for all of the metalloporphyrins probed are discussed
in the frame of Gouterman’s four orbital model and a common binding motif involving coordination between the
metalloporphyrin and DIMP via interaction between the zinc metal center of the porphyrin and phosphoryl oxygen of DIMP
(Zn−OP) is proposed.
postsynaptic membrane is not overstimulated. Inhibition of
AChE results in membrane overstimulation and involuntary
muscle contractions ultimately leading to severe heart muscle
arrhythmia.
INTRODUCTION
■
Weapons of mass destruction (WMD) continue to pose a
significant threat despite being banned completely or tightly
regulated/monitored. These weapon systems have remained a
critical threat due to the presence of large stockpiles of
chemical/biological weapons in countries that do not abide by
international treaties and the availability of materials that can be
utilized to construct primitive but effective biological/chemical
weapons. Since these weapon systems remain a dangerous
threat, methods to detect the use of these weapons as well as
methods for decontamination subsequent to the weapons use
are of critical importance.
Nerve agents (which affect the central nervous system by
inhibiting the acetylcholine esterase) are typically organo-
phosphonates based and include Tabun (ethyl N,N-dimethyl-
phosphoramidocyanidate), Sarin (O-isopropyl methylphospho-
nofluoridate), Soman (O-pinacolyl methylphosphonofluori-
date), and VX (methylphosphonothoic acid). Nerve agents
irreversibly inhibit the acteylcholine esterase (AChE) thus
preventing the hydrolysis of acetylcholine. Upon nerve
activation acetylcholine is released from the presynaptic
membrane associated with the cholinergic synapse and diffuses
across the synaptic cleft binding to nicotinic acetylcholine
receptors within the postsynaptic membrane. This results in an
action potential that initiates rapid Ca2+ influx triggering muscle
contraction. The AChE modulates the acetylcholine concen-
tration via hydrolytic degradation to ensure that the
Bacterial phosphotriesterases (PTE) have been shown to
catabolize a variety of organophosphate compounds.1,2 The
degradation mechanism has been proposed to occur via
hydrolysis of the fluorenyl group of Sarin or Soman (or
structurally equivalent substituent in other analogues).2,3 The
active site comprises a binuclear Zn2+ metal center coordinated
by four histidine residues and an aspartate residue and bridged
by a carbamylated lysine residue.4,5 Recently, the crystal
structure of PTE has been solved with the Sarin analogue/
simulant diisopropyl methylphosphonate (DIMP) and indicates
a mode of binding between DIMP and PTE via coordination of
the phosphoryl oxygen to the more solvent exposed Zn2+.3 A
similar mode of binding to PTE has been observed with the
simulant diethyl 4-methylbenzylphosphonate, though the
tetrahedral coordination geometry of this complex differs
from the distorted trigonal bipyramidal geometry of the DIMP-
PTE complex.5,6 The ability of Zn2+ cation to coordinate to the
phosphoryl oxygen provides a mechanism through which to
develop colorimetric/fluorescence-based sensors as well as to
Received: June 17, 2013
Revised: September 16, 2013
© XXXX American Chemical Society
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Figure 1. Structures of porphyrins examined for DIMP (top right) binding (M = Zn(II)). (Top left) 10-Monoborano-5,15-diphenylporphyrin,
MBDPP, (top middle) 5,10-diborano-15,20-diphenylporphyrin, DBDPP, (bottom left) 5,10-diphenylporphyrin, DPP, (bottom middle) 5,10,15,20-
tetraphenylporphyrin, TPP, (bottom right) 3,5-ditBu-IbuPhyrin, DtBIP.
facilitate decontamination of organophosphate toxins utilizing
Zn2+-based coordination complexes.
solutions were diluted in 1 cm2 quartz cuvettes to <10 μM in
hexane and capped to minimize volume reduction due to
solvent evaporation.
Colorimetric detection assays for the simulant, dimethyl
methylphosphonate (DMMP), have recently been proposed
based on a free-base tetraphenylporphyrin platform in polar
solvents.7−9 The attraction of free-base and metalloporphyrins
as sensor elements relies on their rich optical properties
including large molar extinction coefficients in the UV (on the
order of 1 × 105 M−1 s−1) and visible regions (on the order of 1
× 103−1 × 104 M−1 s−1) and relatively good quantum yields of
fluorescence for free-base and closed shell metalloporphyrins
(up to ∼0.20).10−13 These properties are a result of the
conjugated electronic structure of the porphyrin macrocycle
and, in the case of metalloporphyrins, the interactions between
the macrocycle and metal center.14−19 Zinc(II) metallopor-
phyrins are of particular interest for sensing organophospho-
nates considering that the nature of the closed shell metal
center allows for considerable fluorescence and a relatively
long-lived triplet excited state as well as the role of Zn2+ ions in
binding and hydrolysis of organophosphonates in phospho-
triesterase proteins.1−6,20−24 However, other metalloporphyrins
and corroles have also been shown to serve as sensor
elements.25,26
Steady-state UV−visible absorption spectra were collected
using a Shimadzu UV-2401PC spectrophotometer. Steady-state
emission spectra were collected using an ISS PC1 photon
counting spectrophotometer using 0.5 mm slits on the
excitation and emission monochromator. Samples were excited
either at an isosbestic point between DIMP-bound and
unbound porphyrin or, in the absence of a clear isosbestic
point, at the porphyrin Soret maxima. Since no appreciable
DIMP absorption was observed at the excitation or emission
wavelengths, primary and secondary inner-filter effects were
neglected and the emission spectra were normalized to the
number of photons absorbed at the excitation wavelength.
Affinity constants were obtained by plotting the changes in
absorption/emission as a function of DIMP concentration and
fitting the data to Benesi−Hildebrand27 expressions for
absorption (eq 1) or emission (eq 2):
Ka[DIMP]
1 + Ka[DIMP]
ΔAλ =
[P]oΔε
λ
(1)
Ka[DIMP]
1 + Ka[DIMP]
In the present study, the binding affinities of a variety of
meso-substituted Zn2+ metalloporphyrins for DIMP (figure 1)
are examined using steady-state absorption and fluorescence
spectroscopy in hexane. The effect of the porphyrin structure
on the binding affinity is considered and a preferred mode of
binding is postulated.
ΔF =
(F − F∞)
λ
o
(2)
where Ka is the association constant for a one-to-one binding
model, ΔAλ and ΔFλ are the changes in absorption and
fluorescence intensity, respectively, at wavelength λ, [P]o is the
total porphyrin concentration, Δελ is the change in molar
extinction at wavelength λ, and Fo and F∞ are the fluorescence
intensities in the absence of DIMP and at a saturating
concentration of DIMP, respectively.
Synthesis of Zinc(II)[5,10,15,20-tetraphenylporphyrin]
(ZnTPP). A solution of 5,10,15,20-tetraphenylporphyrin,28 TPP
(0.1 mmol), and Zn(OAc)2·4H2O (0.3 mmol) in 2:1
chloroform/methanol (15 mL) was heated to reflux for 2 h.
MATERIALS AND METHODS
■
All solvents were purchased from Sigma-Aldrich and used
without further purification. Diisopropyl methylphosphonate
(DIMP) was purchased from Alfa Aesar and used as received.
Porphyrin and DIMP stock solutions were freshly prepared in
hexane prior to photometric titrations. Porphyrin sample
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The reaction mixture was cooled to room temperature, and the
solvent was removed under reduced pressure. The desired
Zn(II)porphyrin was purified by flash chromatography using
chloroform as the eluent.
Synthesis of Zinc(II)[5,15-diphenylporphyrin]
(ZnDPP). A solution of 5,10-diphenylporphyrin,29 DPP (0.1
mmol) and Zn(OAc)2·4H2O (0.3 mmol) in HPLC grade
chloroform (15 mL) was heated to reflux for 2 h. After cooling
to room temperature, solvent was removed under reduced
pressure. The desired Zn(II)porphyrin was obtained as a violet
solid after purification by flash chromatography using chloro-
form as the eluent.
Synthesis of Zinc(II)[3,5-DitBu-IbuPhyrin] (ZnDtBIP). A
solution of ditBu-IbuPhyrin,30 DtBIP (0.1 mmol), and Zn-
(OAc)2·4H2O (0.3 mmol) in HPLC grade chloroform (15 mL)
in the presence of triethlyamine (TEA) was heated to reflux for
2 h. After cooling to room temperature, solvent was removed
under reduced pressure. The desired Zn(II)porphyrin was
purified by flash chromatography using chloroform as the
eluent.
Synthesis of Zinc(II)[5-(4′,4′,5′,5′-tetramethyl-
[1′,3′,2′]dioxaborolan-2′-yl)-10,20-diphenylporphyrin]
(ZnMBDPP). A solution of 10-monoborano-5,15-diphenylpor-
phyrin,31 MBDPP, (0.1 mmol) and Zn(OAc)2·4H2O (0.3
mmol) in the presence of triethylamine (TEA) in 10 mL
dichloroethane was heated to reflux under N2 for 2 h. The
mixture was then cooled to room temperature. After quenching
with NH4Cl (aq) and washed with brine (2 times), the organic
layer was dried with sodium sulfate (Na2SO4) before the
solvent was evaporated. The desired Zn(II)porphyrin was
isolated as a violet solid after purification by flash
chromatography using chloroform as the eluent.
Figure 2. Top panel: Steady-state absorption spectra of ZnTPP in
hexane as a function of DIMP concentration. Bottom panel:
Corresponding steady-state emission spectra (λexc = 418 nm).
Synthesis of Zinc(II)[5,15-di-(4′,4′,5′,5′-tetramethyl-
[1′,3′,2′]dioxaborolan-2′-yl)-10,20-diphenylporphyrin]
(ZnDBDPP). A solution of 5,10-diborano-15,20-diphenylpor-
phyrin,31 DBDPP (0.1 mmol), and Zn(OAc)2·4H2O (0.3
mmol) in 10 mL dichloroethane was heated to reflux under N2
for 2 h, The mixture was then cooled to room temperature.
After quenching with NH4Cl (aq) and washed with brine (2
times), the mixture was dried with sodium sulfate (Na2SO4)
before the solvent was evaporated. The desired Zn(II)-
porphyrin was isolated as a violet solid after purification by
flash chromatography using chloroform as the eluent.
RESULTS
■
In general, bathochromic shifts in the absorption and emission
spectra were observed for all of the Zn(II) porphyrins (i.e.,
ZnTPP, ZnDPP, ZnDBDPP, and ZnMBDPP) in the presence
of DIMP although the shift in the emission spectra of
ZnDBDPP was less pronounced than the others (see Figures
2−5). Specifically, the Soret band of ZnTPP shifts from 414 to
423 nm in the presence of ∼1 mM DIMP and the Q-bands
observed at 544 and 582 nm undergo bathochromic shifts to
558 and 596 nm, respectively (Figure 2). The emission bands
observed at 591 and 639 nm shift to 603 and 656 nm, whereas
the ratio of the intensities between the high energy emission
band and low energy emission band increases from 0.39 in the
absence of DIMP to 1.44 in the presence of ∼1 mM DIMP.
The ground state absorption and excited state emission spectra
in the presence of ∼1 mM DIMP resemble that of ZnTPP in
neat acetonitrile and approach the spectra previously reported
for ZnTPP in trimethylphosphate.32
Figure 3. Top panel: Steady-state absorption spectra of ZnMBDPP in
hexane as a function of DIMP concentration. Bottom panel:
Corresponding steady-state emission spectra (λexc = 411 nm).
For ZnDPP, a bathochromic shift was also observed in the
Soret shifting from 403 to 412 nm in the presence of ∼1 mM
DIMP with the Q-bands shifting to 545 and 582 nm from 532
C
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increases slightly from 0.35 to 0.47 in the presence of 1 mM
DIMP.
Spectral changes similar to those of ZnDPP were observed
for ZnMBDPP (Figure 3). The Soret band displayed a red shift
to 416 nm from 407 nm and the emission bands shift from 577
and 631 nm to 594 and 644 nm in the presence of the same
concentration of DIMP. However, despite the shift in emission
the high energy/low energy emission band intensity ratio
decreases slightly from 0.56 in the absence of DIMP to 0.44 in
the presence of 1 mM DIMP. The corresponding ZnDBDPP
(Figure 5) displays a red shift to 419 nm from 416 nm upon the
addition of DIMP. However, the Q-band centered at 543 nm
exhibited a slight hypsochromic shift to 539 nm while the 585
nm band shifted to 592 nm. The emission intensity significantly
decreased with increasing concentration of DIMP and the
emission bands were observed to red shift slightly from 600 and
647 nm to 604 and 650 nm.
The binding constants obtained from both absorption and
emission data are summarized in Table 1. It was found from the
Table 1. DIMP Binding Affinities (in Terms of Association,
Ka) for Zinc(II) Porphyrins in Hexane
absorbance
emission
porphyrin
Ka (104 M−1
)
Ka (104 M−1
)
ZnDtBIP
ZnDBDPP
ZnMBDPP
ZnDPP
1.50 0.03
0.41 0.02
2.09 0.04
2.51 0.03
2.17 0.07
0.58 0.05
0.28 0.02
1.4 0.20
1.94 0.09
0.37 0.04
Figure 4. Top panel: Steady-state absorption spectra of ZnDBDPP in
hexane as a function of DIMP concentration. Bottom panel:
Corresponding steady-state emission spectra (λexc = 419 nm).
ZnTPP
changes in the absorption spectra that all zinc(II) metal-
loporphyrins exhibited similar binding affinities with Ka values
between 1.5 × 104 and 2.5 × 104 M−1.The notable exception is
ZnDBDPP, which has a Ka of 4.1 × 103 M−1. The affinity
constants obtained from emission measurements, however,
were markedly different from those obtained by absorption. In
general the binding affinities were observed to decrease upon
generation of the porphyrin singlet excited state. The origin of
these differences is discussed below.
DISCUSSION
■
Steady-State Absorption. The optical spectrum of
metalloporphyrins provides important information regarding
the ligation, oxidation, and spin state of the central metal as well
as the polarity of the porphyrin’s solvent environment. Under
D
4h symmetry the lowest occupied MOs of the porphyrin are of
a1u and a2u symmetry with a degenerate set of orbitals with eg
symmetry comprising the lowest unoccupied molecular orbital
(LUMO). The porphyrin optical spectrum for these systems is
best described using a four orbital model, originally proposed
by Gouterman,14−24 that involves extensive configuration
interaction between the nearly degenerate a2u and a1u highest
occupied orbitals (HOMOs) and the degenerate set of eg*
LUMOs.14−24,33 The ground state of the D4h porphyrin π-
system has an electron configuration of ...(a2u)2(a1u)2 producing
1
Figure 5. Top panel: Steady-state absorption spectra of ZnDtBIP in
hexane as a function of DIMP concentration. Bottom panel:
Corresponding steady-state emission spectra (λexc = 424 nm).
an A1g state. The excited electron configurations ...
(a2u)2(a1u)1(eg )1, ...(a2u)2(a1u)1(eg )1, ...(a2u)1(a1u)2(eg )1, and ...
x
y
y
(a2u)1(a1u)2(eg )1 interact via the two electron repulsion term
x
and 567 nm. The emission bands display a shift from 571 and
623 nm to 587 and 638 nm upon addition of 1 mM DIMP. The
ratio of the high energy/low energy emission band intensities
H′ = e′2/rij, to give an energy level diagram where A1g′ is the
average energy splitting of the a2u and a1u orbitals and A1g″ is
the matrix element that couples the excited electron
D
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configurations (e.g., ⟨(a1ueg )|H|(a2ueg )⟩).20−24 Transitions
transitions upon the addition of DIMP. No significant
quenching of the fluorescence was observed with the exception
of ZnDBDPP. In fact, the red shift observed in the emission
spectra of ZnDtBIP was accompanied by an increase in
emission intensity with added DIMP. The changes in intensity
as a function of DIMP concentrations were fit to eq 2 to obtain
the corresponding association constants of the metalloporphyr-
ins for DIMP. These values are also summarized in Table 1.
Interestingly, the red shift corresponding to formation of the
metalloporphyrin:DIMP complex is accompanied by a change
in the Q(0,0)/Q(0,1) intensity ratio, though the direction and
degree of the change varied between metalloporphyrins. For
example, in the case of ZnTPP the Q(0,0) band increased in
intensity while a significant decrease in intensity was observed
for the Q(0,1) band. In the case of ZnDPP, both the Q(0,0)
and the Q(0,1) bands were observed to increase in intensity,
though disproportionately. Formation of the ZnDBDPP:DIMP
complex resulted in a decrease in intensities of both transitions,
though slightly disproportionately. ZnMBDPP displayed a
marked decrease in the Q(0,0) intensity with an almost
negligible increase in the Q(0,1) intensity upon association
with DIMP. DIMP complexation with ZnDtBIP resulted in a
narrowing of the Q(0,0) band and an intensity increase of both
Q(0,0) and Q(0,1) transitions.
x
x
between these states give rise to the B- and Q-band transitions
and accurately reflect the difference in molar extinction
coefficient.14−19,33 Introduction of a transition metal atom to
the porphyrin core results in shifts in the relative energies of the
a2u/a1u orbital energies, thus affecting the A1g′ matrix element
and the B- and Q-band positions.20−24 In addition, the 3d
orbitals of the metal can also interact directly with porphyrin
molecular orbitals of the same symmetry. Since the metal
2
2
2
orbitals transform as b1g (3dx −y ), a1g (3dz ), b2g (3dxy), eg (3dxz,
3dyz) in D4h, only the eg (3dxz, 3dyz) orbitals can mix with the
porphyrin eg* (π-orbitals). Occupancy of the metal 3dxz and
3dyz orbitals can then influence the electronic properties of the
metalloporphyrin.
Upon the addition of DIMP, the Soret and Q-band
absorption maxima of the Zn2+ complexes of porphyrins all
red-shifted. These shifts are similar to those previously
observed for the coordination of N, S, O, and P-containing
ligands to ZnTPP.34−36 Interestingly, ZnTPP, ZnDPP, and
ZnMBDPP each displayed a 9 nm red-shift in the Soret band.
The Soret band red-shift observed for ZnDBDPP and ZnDtBIP
is less pronounced, however, shifting only 3 and 6 nm,
respectively. Nappa and Valentine35 have proposed that the
observed red-shifts upon coordination of varying Lewis base
ligands (of S-, N-, O-, or P-containing donors) for ZnTPP are
less dependent on the strength of the interaction between the
zinc(II) and the ligand and more on the basicity of the ligand.
According to Gouterman, decreased electronegativity on the
central metal atom increases electron density of the pπ orbitals
of the porphyrin macrocycle and in turn increases the energy of
the a2u orbital, thereby decreasing the a2u → eg transition
energy.14 Nappa and Valentine further speculated that upon
coordination of a ligand of sufficient electron-donating
propensity, density in the pπ orbital of the porphyrin is
increased by conjugative effects via the zinc(II) metal center.35
Steady-State Emission. The observed emission spectra for
each zinc(II) porphyrin examined in the absence of DIMP
display two transitions between 550 and 750 nm, which
correspond to the Q(0,0) transition (typically between 550 and
620 nm) and the Q(0,1) transition (between 620 and 700 nm).
The emission Q-band maxima for the metalloporphyrins
probed are listed in Table 2. In general, bathochromic shifts
were noted in the maxima corresponding to the two Q-band
The intensity of transitions between vibronic manifolds of
two electronic states such as Q(0,0) and Q(0,1) of the S1 → S0
electronic transitions depends on the degree of overlap between
the nuclear wave functions corresponding to the two vibrational
states according to
F
= CiRe→g2⟨j|i⟩2
i→j
(3)
where Re→g is the electronic transition moment between the
porphyrin S1 excited state and S0 ground state, C is a constant
dependent on the population density of the excited state
vibrational manifold, i, and ⟨j|i⟩ is the nuclear overlap integral
between the excited state vibrational manifold, i, and ground
state vibrational manifold, j. The data suggests that binding of
DIMP to each metalloporphyrin affects the Q(0,0) and Q(0,1)
transition of the corresponding porphyrin differently by either
increasing or decreasing the ij overlap integral. The fact that the
relative spacing between the Q(0,0) and Q(0,1) transitions as
well as the change in Stokes shifts were essentially the same for
ZnTPP, ZnDPP, and ZnMBDPP in the absence and presence
of DIMP (Table 2) is indicative of a common binding motif,
which presumably involves the zinc(II) metal center. However,
the relative orientations between the DIMP and porphyrin
macrocycle may differ between the porphyrin species leading to
the changes observed in the emission intensities of the
transitions. Differences in the orientation and/or vibrational
overlap may arise from electronic (inductive or Coulombic)
effects related to the properties of the porphyrin substituents at
the meso-positions or steric factors. Distortion of the planarity
of the porphyrin macrocycle upon ligand binding may also
perturb the energies of the a2u, a1u, and/or eg orbitals. Crystal
structures of 5-coordinate complexes of ZnTPP and some O-
donating ligands, such as dimethylformamide and dimethyla-
cetamide, result in distortions of the porphyrin macrocycle
planarity while coordination of dimethylsulfoxide to the ZnTPP
macrocycle results in a relatively planar conformation.36−39
Plots of the ratio of the intensity of the Q(0,0) transition in
the absence of DIMP, Fo, to the intensity of the same transition
in the presence of DIMP, F, as a function of DIMP
Table 2. Absorbance and Emission Maxima for the Unbound
and DIMP Bound Metalloporphyrins
absorption
Q(0,0)
emission
Soret (B)
414 nm
Q(0,1)
Q(0,0)
Q(0,1)
ZnTPP
582 nm
596 nm
567 nm
582 nm
544 nm
558 nm
532 nm
545 nm
591 nm
603 nm
571 nm
587 nm
639 nm
656 nm
623 nm
638 nm
ZnTPP + DIMP 423 nm
ZnDPP
403 nm
412 nm
ZnDPP +
DIMP
ZnMBDPP
407 nm
416 nm
571 nm
584 nm
537 nm
549 nm
577 nm
594 nm
631 nm
644 nm
ZnMBDPP +
DIMP
ZnDBDPP
416 nm
419 nm
584 nm
591 nm
542 nm
539 nm
600 nm
604 nm
647 nm
650 nm
ZnDBDPP +
DIMP
ZnDtBIP
423 nm
429 nm
604 nm
606 nm
549 nm
560 nm
611 nm
613 nm
648 nm
666 nm
ZnDtBIP +
DIMP
E
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concentration were nonlinear displaying downward curvatures
in all cases. Downward curvature is indicative of the presence of
multiple emissive components in solution. It is likely that these
emissive species correspond to DIMP-bound and unbound
porphyrins in solution. If diffusional (collisional) quenching of
the zinc(II) porphyrin fluorescence is negligible, and the
resulting complex is emissive, a plot of Fo/F can be expressed as
F
F
1 + Ka[DIMP]
o
=
τ
o
Figure 6. Two different binding modes between Zn(II) porphyrins
and DIMP.
1 +
K [DIMP]
(τ ′)
a
(4)
where τo is the fluorescence lifetime of the unbound porphyrin,
τ′ is the fluorescence lifetime of the bound porphyrin, and Ka is
the association constant. Fits of the data to eq 4 are presented
in Table 3, and the Kas obtained agree very well with those
DIMP methyl group lies adjacent to the plane of the porphyrin
macrocycle and the second is a trans conformation in which the
DIMP methyl group is directed away from the porphyrin
macrocycle.
Table 3. Summary of Fluorescence Titration Data Fits to eq
4
Preliminary electrostatic surface potential calculations
performed with HyperChem using the PM3 method (see
Supporting Information) indicate (1) a positive electrostatic
surface is prominent over the phosphryl alkoxyl groups of
DIMP, (2) a large positive electrostatic surface over the face of
the parent macrocycle of zinc porphyrin, and (3) that the
surface potential is diminished in the ZnMBDPP, ZnDBDPP,
and ZnDtBIP macrocycles. It is proposed that the relative
orientation of the DIMP methyl group is gauche to the PO−
Zn interaction such that the DIMP alkoxyl groups are directed
away from the metalloporphyrin macrocycle thereby minimiz-
ing the Coulombic repulsion between the electrostatic surfaces
of the alkoxyl groups and the interior potential of the porphyrin
macrocycle.
porphyrin
Ka (104 M−1
)
τo (ns)
τ′ (ns)
ZnDtBIP
ZnDBDPP
ZnMBDPP
ZnDPP
0.55
0.29
1.44
1.91
0.45
1.14
0.78
1.30
1.05
1.82
2.42
0.32
0.82
0.63
0.34
ZnTPP
given in Table 1. The fluorescence lifetime value (∼1.9 ns)
extracted from the fit for unbound ZnTPP was found to agree
with that measured experimentally in benzene and methyl-
cyclohexane (∼1.5−1.8 ns).22,40,41
A marked difference in the binding affinities obtained by a
Benesi−Hildebrand treatment of the steady-state absorption
and steady-state emission data for the interaction between the
Zn(II) metalloporphyrins probed and DIMP was noted above.
The results obtained from fits of the emission data to eq 4
confirm the validity of the values obtained from eq 2. However,
the origin of the differences in affinities is not immediately
apparent. Previous studies involving ligand binding to ZnTPP
have attributed Lewis acid properties to the metalloporphyrin
and have noted that the binding affinity of ZnTPP with Lewis
base-type ligands correlate well with the strength of the base
(where deviations due to sterics were not considered).31,36,42,43
The decreased affinity constants obtained from the emission
data are, therefore, likely to be due to a decrease in the Lewis
acid character of the metal center in ZnTPP upon generation of
the excited state.44,45 In fact, Lampa-Pastirk et al.45 have noted
stepwise photodissociation of the axial ligands in Zn(II)-
substituted cytochrome c upon excitation at the zinc porphyrin
Soret band.
Proposed Binding Motif. A wide variety of oxygen-
containing ligands, including phosphine based liagnds, have
previously been shown to bind to ZnTPP in solvents of varying
polarity with reasonable affinity (i.e., down to Kd ≈ 1.4 mM for
dimethylsulfoxide).34,45−50 The primary mode of binding
between Zn based porphyrins and phosphonates is through
the phosphoryl oxygen and the porphyrin zinc metal center,
PO···Zn.26,45−50 A similar mode of interaction has been
observed in crystal structures of ZnTPP and O-donating ligands
such as dimethylsulfoxide, dimethylformamide, and dimethyla-
cetamide36−38 as well as open shell metalloporphyrins and
corroles.25 Two geometries are possible for the PO···Zn
binding mode with ((CH3)2CHO)2(CH3)PO as a ligand
(Figure 6). The first is a gauche conformation in which the
SUMMARY
■
Steady-state absorption and emission were used to probe the
binding and affinity constants for the nerve agent simulant
DIMP to a series of zinc(II) metalloporphyrins. In each case,
red shifts were observed in both the absorption and emission
spectra upon binding of DIMP. The magnitudes of the red shift
are attributed to inductive donation of electronic density from
the basic phosphoryl oxygen of DIMP into the porphyrin pπ
macrocycle via the zinc(II) metal center. It is proposed that the
binding of DIMP to the metalloporphyrins occurs via the
DIMP phosphoryl oxygen and the porphyrin zinc(II) metal
center where the DIMP alkoxyl groups are directed away from
the porphyrin macrocycle core. The resulting binding affinities
in hexane resembles that of some relatively strong N-donating
bases for ZnTPP in noncoordinating solvents and generally
diminish for the bulkier substituted porphyrins. The decrease in
affinity is attributed to a decrease in acidity of the porphyrin at
the porphyrin macrocycle core; possibly an effect of the nature
of the substituent.
ASSOCIATED CONTENT
■
S
* Supporting Information
Derivation of eq 4 as well as ZnPorphyrin-DIMP binding
curves and fits to eq 4. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(R.W.L.) E-mail: rwlarsen@usf.edu. Phone: 813 974 7925.
F
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The Journal of Physical Chemistry A
Article
(14) Gouterman, M. Study of the Effects of Substitution on the
Absorption Spectra of Porphin. J. Chem. Phys. 1959, 30, 1139−1161.
(15) Gouterman, M. Spectra of Porphyrins. J. Mol. Spectrosc. 1961, 6,
138−163.
(16) Gouterma, M.; Snyder, L. C.; Wagniere, G. H. Spectra of
Porphyrins. 2. 4 Orbital Model. J. Mol. Spectrosc. 1963, 11, 108−127.
(17) Liao, M. S.; Scheiner, S. Electronic Structure and Bonding in
Metal Porphyrins, (Metal = Fe, Co, Ni, Cu, Zn). J. Chem. Phys. 2002,
117, 205−219.
Present Addresses
†Department of Chemistry, Virginia Polytechnic Institute and
State University (Virginia Tech.), 306 Hahn Hall North,
Blacksburg, Virginia 24061, United States.
‡Department of Cell and Molecular Biology, University of
Hawaii Manoa, 651 Ilalo Street, BSB 222, Honolulu, Hawaii
96813, United States.
§Department of Chemistry, Kyushu University, Japan.
(18) Shelnutt, J. A.; Ortiz, V. Substituent Effects on the Electronic-
Structure of Metalloporphyrins: A Quantitative-Analysis in Terms of
4-Orbital-Model Parameters. J. Phys. Chem. 1985, 89, 4733−4739.
(19) Weiss, C.; Kobayash, H.; Gouterman, M. Spectra of Porphyrins.
3. Self-Consistent Molecular Orbital Calculations of Porphyrins and
Related Ring Systems. J. Mol. Spectrosc. 1965, 16, 415−450.
(20) Whitten, D. Photochemistry of Porphyrins and Their Metal
Complexes in Solution and Organized Media. Res. Chem. Intermed.
1978, 2, 107−138.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Department of Defense−
Defense Threat Reduction Agency (DoD−DTRA) through
HDTRA1-08-C-0035.
(21) Harriman, A. Luminescence of Porphyrins and Metalloporphyr-
ins. 1. Zinc(II), Nickel(II), and Manganese(II) Porphyrins. J. Chem.
Soc., Faraday Trans. 1 1980, 76, 1978−1985.
(22) Harriman, A. Luminescence of Porphyrins and Metalloporphyr-
ins. 3. Heavy-Atom Effects. J. Chem. Soc., Faraday Trans. 2 1981, 77,
1281−1291.
REFERENCES
■
(1) Caldwell, S. R.; Raushel, F. M. Detoxification of Organo-
phosphate Pesticides Using a Nylon Based Immobilized Phospho-
triesterase from Pseudomonas-Diminuta. Appl. Biochem. Biotechnol.
1991, 31, 59−73.
(23) Harriman, A.; Hosie, R. J. Luminescence of Porphyrins and
Metalloporphyrins. 4. Fluorescence of Substituted Tetraphenylpor-
phyrins. J. Chem. Soc., Faraday Trans. 2 1981, 77, 1695−1702.
(24) Krasnovsky, A. A.; Venediktov, E. A. Quenching of Singlet
Oxygen Luminescence by Porphyrins and Metalloporphyrins. Biofizika
1978, 23, 387−389.
(25) Kim, K.; Kim, I.; Maiti, N.; Kwon, S. J.; Bucella, D.; Egorova, O.
A.; Lee, Y. S.; Kwak, J.; Churchill, D. G. A Study of Nerve Agent
Model Organophosphonates Binding with Manganese-A2B-Corrole
and A2B2-Porphyrin Systems. Polyhedron 2009, 28, 2418−2430.
(26) Kim, K.; Tsay, O. G.; Atwood, D. A.; Churchill, D. G.
Destruction and Detection of Chemical Warfare Agents. Chem. Rev.
2011, 111, 5345−5403.
(27) Benesi, H. A.; Hildebrand, J. H. A Spectrophotometric
Investigation of the Interaction of Iodine with Aromatic Hydro-
carbons. J. Am. Chem. Soc. 1949, 71, 2703−2707.
(28) Adler, A. D.; Longo, F. R.; Kampas, F.; Kim, J. On the
Preparation of Metalloporphyrins. J. Inorg. Nucl. Chem. 1970, 32,
2443−2445.
(29) Frail, P. R.; Susumu, K.; Huynh, M.; Fong, J.; Kikkawa, J. M.;
Therien, M. J. Modulation of Dark Conductivity over a 1 × 10−12 to 1
× 10−5 S/cm Range through Ancillary Group Modification in
Amorphous Solids of Ethyne-Bridged (Porphinato)zinc(II)
Oligomers. Chem. Mater. 2007, 19, 6062−6064.
(30) Ruppel, J. V.; Jones, J. E.; Huff, C. A.; Kamble, R. M.; Chen, Y.;
Zhang, X. P. A Highly Effective Cobalt Catalysts for Olefin
Aziridination with Azides: Hydrogen Bonding guided Catalyst Design.
Org. Lett. 2008, 10, 1995−1998.
(31) Hyslop, A. G.; Kellett, M. A.; Iovine, P. M.; Therien, M. J.
Suzuki Porphyrins: New Synthons for the Fabrication of Porphyrin-
Containing Supramolecular Assemblies. J. Am. Chem. Soc. 1998, 120,
12676−12677.
(32) Humphry-Baker, R.; Kalyanasundaram, K. Influence of Axial
Ligation on the Fluorescence of Tetrakisphenylporphyrins. J. Photo-
chem. 1985, 31, 105−112.
(33) Owens, J. W.; Smith, R.; Robinson, R.; Robins, M. Photo-
physical Properties of Porphyrins, Phthalocyanines, and Benzochlorins.
Inorg. Chim. Acta 1998, 279, 226−231.
(34) Kolling, O. W. Soret Red Shift for Zinc Tetraphenylporphine in
the Presence of Uncharged Lewis-Bases. Inorg. Chem. 1979, 18, 1175−
1176.
(2) Dumas, D. P.; Caldwell, S. R.; Wild, J. R.; Raushel, F. M.
Purification and Properties of the Phosphotriesterase from Pseudomo-
nas-Diminuta. J. Biol. Chem. 1989, 264, 19659−19665.
(3) Benning, M. M.; Hong, S. B.; Raushel, F. M.; Holden, H. M. The
Binding of Substrate Analogs to Phosphotriesterase. J. Biol. Chem.
2000, 275, 30556−30560.
(4) Omburo, G. A.; Kuo, J. M.; Mullins, L. S.; Raushel, F. M.
Characterization of the Zinc-Binding Site of Bacterial Phosphotriester-
ase. J. Biol. Chem. 1992, 267, 13278−13283.
(5) Benning, M. M.; Kuo, J. M.; Raushel, F. M.; Holden, H. M.
Three-Dimensional Structure of the Binuclear Metal Center of
Phosphotriesterase. Biochemistry 1995, 34, 7973−7978.
(6) Vanhooke, J. L.; Benning, M. M.; Raushel, F. M.; Holden, H. M.
Three-Dimensional Structure of the Zinc-Containing Phosphotriester-
ase with the Bound Substrate Analog Diethyl 4-Methylbenzylphosph-
onate. Biochemistry 1996, 35, 6020−6025.
(7) Cao, H. T.; Nam, J.; Harmon, H. J.; Branson, D. H.
Spectrophotometric Detection of Organophosphate Diazinon by
Porphyrin Solution and Porphyrin-Dyed Cotton Fabric. Dyes Pigm.
2007, 74, 176−180.
(8) Huo, D. Q.; Yang, L. M.; Hou, C. J.; Fa, H. B.; Luo, X. G.; Lu, Y.;
Zheng, X. L.; Yang, J.; Yang, L. Molecular Interactions of
Monosulfonate Tetraphenylporphyrin (TPPS1) and Meso-Tetra(4-
sulfonatophenyl)porphyrin (TPPS) with Dimethyl Methylphospho-
nate (DMMP). Spectrochim. Acta, Part A 2009, 74, 336−343.
(9) Zhang, S. Y.; Yang, L. M.; Huo, D. Q.; Liu, X. J.; Li, J.; Fa, H. B.;
Zhang, L.; Hou, C. J. Molecular Interactions of Dimethyl
Methylphosphonate (DMMP) with Metalloporphyrins: Determina-
tion of the Binding Mechanism by Spectroscopic Methods.
Spectrochim. Acta, Part A 2012, 88, 137−143.
(10) Beer, P. D.; Cormode, D. P.; Davis, J. J. Zinc Metalloporphyrin-
Functionalised Nanoparticle Anion Sensors. Chem. Commun. 2004,
414−415.
(11) Di Natale, C.; Macagnano, A.; Repole, G.; Saggio, G.; D’Amico,
A.; Paolesse, R.; Boschi, T. The Exploitation of Metalloporphyrins as
Chemically Interactive Material in Chemical Sensors. Mater. Sci. Eng,
C. 1998, 5, 209−215.
(12) Gorski, L.; Malinowska, E.; Parzuchowski, P.; Zhang, W.;
Meyerhoff, M. E. Recognition of Anions Using Metalloporphyrin-
Based Ion-Selective Mebranes: State-of-the-Art. Electroanalysis 2003,
15, 1229−1235.
(13) Malinowska, E.; Niedziolka, J.; Meyerhoff, M. E. Potentiometric
and Spectroscopic Characterization of Anion Selective Electrodes
Based on Metal(III) Porphyrin Ionophores in Polyurethane
Membranes. Anal. Chim. Acta 2001, 432, 67−78.
(35) Nappa, M.; Valentine, J. S. Influence of Axial Ligands on
Metalloporphyrin Visible Absorption Spectra: Complexes of Tetra-
phenylporphyrinatozinc. J. Am. Chem. Soc. 1978, 100, 5075−5080.
G
dx.doi.org/10.1021/jp405976h | J. Phys. Chem. A XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry A
Article
(36) Vogel, G. C.; Stahlbush, J. R. Thermodynamic Study of the
Adduct Formation of Zinc Tetraphenylporphine with Several Neutral
Donors in Cyclohexane. Inorg. Chem. 1977, 16, 950−953.
(37) Vinodu, M.; Goldberg, I. A Dimethyl Sulfoxide/Chloroform
Clathrate of (Dimethyl Sulfoxide) (Tetraphenylporphyrinato)zinc(II).
Acta Crystallogr., Sect. E: Struct. Rep. Online 2004, 60, m579−m581.
(38) Lipstman, S.; Muniappan, S.; Goldberg, I. (Dimethylacetamide)-
(tetraphenylporphyrinato)zinc(II). Acta Crystallogr., Sect. E: Struct.
Rep. Online 2006, 62, m2330−2332.
(39) Lipstman, S.; Goldberg, I. Unsolvated (Dimethylformamide)-
(tetraphenylporphyrinato)zinc(II). Acta Crystallogr., Sect. E: Struct.
Rep. Online 2006, 62, m158−m160.
(40) Yu, H. Z.; Baskin, J. S.; Zewail, A. H. Ultrafast dynamics of
Porphyrins in the Condensed Phase. II. Zinc Tetraphenylporphyrin. J.
Phys. Chem. A 2002, 106, 9845−9854.
(41) Seybold, P. G.; Gouterman, M. Porphyrins. 13. Fluorescence
Spectra and Quantum Yields. J. Mol. Spectrosc. 1969, 31, 1−13.
(42) Vogel, G. C.; Searby, L. A. Lewis Acid-Base Interactions of Zinc
Alpha, Beta, Gamma, Delta-Tetraphenylporphine with Several Neutral
Donors. Inorg. Chem. 1973, 12, 936−939.
(43) Mizutani, T.; Kurahashi, T.; Murakami, T.; Matsumi, N.;
Ogoshi, H. Molecular Recognition of Carbohydrates by Zinc
Porphyrins: Lewis Acid Lewis Base Combinations as a Dominant
Factor for their Selectivity. J. Am. Chem. Soc. 1997, 119, 8991−9001.
(44) Lampa-Pastirk, S.; Lafuente, R. C.; Beck, W. F. Excited-State
Axial-Ligand Photodissociation and Nonpolar Protein-Matrix Reor-
ganization in Zinc(II)-Substituted Cytochrome C. J. Phys. Chem. B.
2004, 108, 12602−12607.
(45) Lin, C. L.; Fang, M. Y.; Cheng, S. H. Substituent and Axial
Ligand Effects on the Electrochemistry of Zinc Porphyrins. J.
Electroanal. Chem. 2002, 531, 155−162.
(46) Nardo, J. V.; Dawson, J. H. Spectroscopic Evidence for the
Coordination of Oxygen Donor Ligands to Tetraphenylporphyrinato-
zinc. Inorg. Chim. Acta 1986, 123, 9−13.
(47) Enakieva, Y. Y.; Bessmertnykh, A. G.; Gorbunova, Y. G.; Stern,
C.; Rousselin, Y.; Tsivadze, A. Y.; Guilard, R. Synthesis of meso-
Polyphosphorylporphyrins and Example of Self Assembling. Org. Lett.
2009, 11, 3842−3845.
(48) Chekmeneva, E.; Hunter, C. A.; Packer, M. J.; Turega, S. M.
Evidence of Partially Bound States in Cooperative Molecular
Recognition Interfaces. J. Am. Chem. Soc. 2008, 130, 17718−17725.
(49) Officer, D. L.; Lodato, F.; Jolley, K. W. Zinc-Porphyrin
Phosphonate Coordination: Structural Control through a Zinc
Phosphoryl−Oxygen Interaction. Inorg. Chem. 2007, 46, 4781−4783.
(50) Matano, Y.; Matsumoto, K.; Terasaka, Y.; Hotta, H.; Araki, Y.;
Ito, O.; Shiro, M.; Sasamori, T.; Tokitoh, N.; Imahori, H. Synthesis,
Structures and Properties of meso-Phosphorylporpyrins: Self-Oraniza-
tion through P-Oxo-Zinc Coordination. Eur. J. Chem. 2007, 13, 891−
901.
H
dx.doi.org/10.1021/jp405976h | J. Phys. Chem. A XXXX, XXX, XXX−XXX