Article
`
`pubs.acs.org/IC
`
`Lead(II) Complex Formation with L‑Cysteine in Aqueous Solution
`Farideh Jalilehvand,*,† Natalie S. Sisombath,† Adam C. Schell,† and Glenn A. Facey‡
`†
`Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada
`‡
`Department of Chemistry, University of Ottawa, 10 Marie Curie Private, Ottawa, Ontario K1N 6N5, Canada
`*S Supporting Information
`
`ABSTRACT: The lead(II) complexes
`formed with the
`multidentate chelator L-cysteine (H2Cys) in an alkaline
`aqueous solution were studied using 207Pb, 13C, and 1H
`NMR, Pb LIII-edge X-ray absorption, and UV−vis spectro-
`scopic techniques, complemented by electrospray ion mass
`spectrometry (ESI-MS). The H2Cys/PbII mole ratios were
`varied from 2.1 to 10.0 for two sets of solutions with CPbII =
`0.01 and 0.1 M, respectively, prepared at pH values (9.1−10.4)
`for which precipitates of lead(II) cysteine dissolved. At low
`H2Cys/PbII mole ratios (2.1−3.0), a mixture of the dithiolate
`[Pb(S,N-Cys)2]2− and [Pb(S,N,O-Cys)(S-HCys)]− complexes
`with average Pb−(N/O) and Pb−S distances of 2.42 ± 0.04
`and 2.64 ± 0.04 Å, respectively, was found to dominate. At
`high concentration of free cysteinate (>0.7 M), a significant amount converts to the trithiolate [Pb(S,N-Cys)(S-HCys)2]2−,
`including a minor amount of a PbS3-coordinated [Pb(S-HCys)3]− complex. The coordination mode was evaluated by fitting
`linear combinations of EXAFS oscillations to the experimental spectra and by examining the 207Pb NMR signals in the chemical
`Pb = 2006−2507 ppm, which became increasingly deshielded with increasing free cysteinate concentration. One-
`shift range δ
`pulse magic-angle-spinning (MAS) 207Pb NMR spectra of crystalline Pb(aet)2 (Haet = 2-aminoethanethiol or cysteamine) with
`
`PbS2N2 coordination were measured for comparison (δiso = 2105 ppm). The UV−vis spectra displayed absorption maxima at
`298−300 nm (S− → PbII charge transfer) for the dithiolate PbS2N(N/O) species; with increasing ligand excess, a shoulder
`appeared at ∼330 nm for the trithiolate PbS3N and PbS3 (minor) complexes. The results provide spectroscopic fingerprints for
`structural models for lead(II) coordination modes to proteins and enzymes.
`
`■ INTRODUCTION
`
`The efficiency of cysteine-rich proteins and peptides, e.g.,
`metallothioneins and phytochelatins,
`in removing harmful
`heavy metals from the cells and tissues1,2 has inspired the
`assessment of cysteine as an ecofriendly agent for extracting
`heavy metals from a contaminated environment. Cysteine
`+)COO−), as well as penicillamine
`(H2Cys = HSCH2CH(NH3
`(H2Pen) and glutathione (GSH), can liberate lead bound in
`contaminated soil,
`in iron/manganese oxides, and in lead
`phosphate/carbonate salts or in mine tailings by increasing its
`solubility and mobilization.3−5 Moreover, the ability of cysteine
`to capture heavy metals including lead from polluted water can
`be important
`in the development of new materials with
`treatment.6 A
`potential use in drainage and wastewater
`cysteine-based nanosized chelating agent
`that
`selectively
`removes PbII
`ions has recently been developed for
`the
`treatment of lead poisoning.7
`In recent years, cysteine has been introduced as an
`environmentally friendly source of
`sulfur
`for preparing
`nanocrystalline PbS, a widely used semiconductor. Such
`nanocrystals can be prepared by mixing Pb(NO3)2 or
`Pb(OAc)2 (OAc− = acetate) with cysteine to form a lead(II)
`cysteine precursor, followed by hydrothermal decomposition to
`PbS. Different morphologies, shapes, and sizes can be obtained
`
`depending on the metal-to-ligand mole ratio, concentration, or
`pH.8−11 It has been suggested that the precursor is polycrystal-
`line HSCH2CH(NH2)COOPbOH8 or has a polymeric
`[−SCH2CH(COOH)NHPb−]n structure,11 in well-aligned
`one-dimensional nanowires.12
`Corrie and co-workers have reported formation constants for
`several mononuclear lead(II) cysteine complexes in aqueous
`4−, Pb-
`2−, Pb(Cys)3
`solution,
`including Pb(Cys), Pb(Cys)2
`(HCys)+, Pb(Cys)(HCys)−, and Pb(Cys)2(OH)3−, however,
`with revised values, e.g., for the Pb(Cys) complex in their later
`reports.13−15 Bizri and co-workers also reported formation
`4− and
`constants for the above complexes, except for Pb(Cys)3
`Pb(Cys)2(OH)3−, but included a Pb(Cys)(OH)− complex; see
`Figure S-1a in the Supporting Information (SI).16 To explain
`the high stability of the Pb(Cys) complex, cysteinate was
`proposed to act as a tridentate ligand, binding simultaneously
`through the thiolate (−S−), carboxylate (−COO−), and amine
`(−NH2) groups.15−17 However, a subsequent study of the
`COO− stretching frequencies indicated that cysteinate in an
`alkaline solution exclusively binds to PbII via the amine and
`thiolate groups.18 Pardo et al. proposed formation constants for
`
`Received: October 22, 2014
`Published: February 19, 2015
`
`© 2015 American Chemical Society
`
`2160
`
`DOI: 10.1021/ic5025668
`Inorg. Chem. 2015, 54, 2160−2170
`
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`Nexus Ex. 1034
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`Inorganic Chemistry
`
`the Pb(Cys), Pb(HCys)+, Pb(HCys)2,
`a set consisting of
`2− complexes.19 Recently, Crea
`Pb(Cys)(HCys)−, and Pb(Cys)2
`et al. obtained formation constants for Pb(Cys), Pb(HCys)+,
`Pb(H2Cys)2+, Pb(Cys)(OH)−, and Pb(Cys)2
`2− to describe the
`stoichiometric composition of the lead(II) cysteine complexes
`formed at several ionic strengths (0 < I ≤ 1.0 M NaNO3) and
`temperatures; see Figure S-2a in the SI.20 The highest PbII
`concentration used in all studies above was 0.5 mM.
`In a high-field 1H and 13C NMR study, Kane-Maguire and
`Riley investigated the PbII binding to cysteine at both acidic
`(pD 1.9) and alkaline (pD 12.9) D2O solutions.21 The report
`includes proton-coupling constants for free cysteine (L), as well
`as mole fractions of its three rotamers: trans (t) and gauche (g
`and h), at different pD values in the range 1.80−12.92 (pD =
`pH reading + 0.4).21,22 Each rotamer was ascribed a preferred
`mode of binding: rotamer t to bidentate (S,N), h to tridentate
`(S, N, O), and g to bidentate (S,O). No significant lead(II)
`cysteine complex formation was observed in the acidic solutions
`(pD 1.9) with H2Cys/PbII mole ratios 0.5−6.0, which is
`consistent with the well-known ability of
`lead(II) to form
`nitrate complexes.23 At H2Cys/Pb(NO3)2 mole ratios ≥2.0
`(CPbII = 10 mM), only PbL2 complexes were proposed to form
`in alkaline media (pD 12.9), with cysteine mainly acting as a
`tridentate (S,N,O) or a bidentate (S,N) ligand. It was also
`suggested that when CPb(NO3)2 = CH2Cys = 10 mM (pD 12.9),
`PbL species with 63% Pb(S,N,O-Cys), 30% Pb(S,O-Cys), and
`7% Pb(S,N-Cys) coordination were formed in proportion to
`the mole fractions of h, g, and t
`rotamers,
`respectively.
`However, we could not prepare aqueous solutions with 1:1
`PbII/cysteine composition because the initially formed
`precipitate did not dissolve in alkaline media even at pH
`12.0, and stability constants for lead(II) hydrolysis indicate that
`precipitation of lead(II) hydroxide should start in such highly
`alkaline media;20 see Figures S-1b and S-2b in the SI.
`Reliable structural
`information to allow a better under-
`standing of the nature of the lead(II) complexes formed with
`cysteine is clearly needed. We used a combination of
`including UV−vis, 207Pb, 13C, and
`spectroscopic techniques,
`1H NMR, extended X-ray absorption fine structure (EXAFS),
`and electrospray ion mass spectrometry (ESI-MS) to study the
`coordination and bonding in lead(II) cysteine complexes
`formed in two sets of alkaline solutions with CPbII = 10 and
`100 mM for H2Cys/PbII mole ratios ≥2.1. To obtain such
`concentrations, the pH was raised (9.1−10.4) to dissolve the
`lead(II) cysteine precipitate that forms when adding lead(II) to
`cysteine solutions. Both the −SH and −NH3
`+ groups of
`cysteine deprotonate at about pH 8.5,24 thus increasing its
`ability to coordinate via the thiolate and amine groups.
`■ EXPERIMENTAL SECTION
`Sample Preparation.
`cysteamine (Haet,
`L-Cysteine,
`·3H2O, and sodium hydroxide
`H2NCH2CH2SH), PbO, Pb(ClO4)2
`were used as supplied (Sigma-Aldrich). All syntheses were carried out
`under a stream of argon gas. Deoxygenated water
`for sample
`preparation was prepared by bubbling argon gas through boiled
`distilled water. The pH values of the solutions were monitored with a
`Thermo Scientific Orion Star pH meter.
`Two sets of lead(II) cysteine solutions were prepared with different
`for CPbII ∼ 10 and 100 mM,
`H2Cys/Pb(ClO4)2 mole ratios
`respectively, at an alkaline pH at which the lead(II) cysteine
`precipitate dissolved; see Table 1. Lead(II) cysteine solutions A−G
`(CPbII ≈ 10 mM) and A*−F* (CPbII ≈ 100 mM) were freshly prepared
`·3H2O (0.05 mmol) to cysteine dissolved in
`by adding Pb(ClO4)2
`deoxygenated water (0.105−0.75 mmol, pH 2.0−2.4). For 207Pb NMR
`
`Article
`
`Table 1. Composition of Lead(II) Cysteine Solutions
`
`H2Cys/PbII mole
`ratio
`2.1
`3.0
`4.0
`5.0
`8.0
`10.0
`15.0
`15.0
`
`pH
`10.4
`9.1
`9.1
`9.1
`9.1
`9.1
`9.1
`8.9
`
`solution
`A
`B
`C
`D
`E
`F
`G
`G′
`
`CPbII
`(mM)
`10
`10
`10
`10
`10
`10
`10
`10
`
`solution
`A*
`B*
`C*
`D*
`E*
`F*
`
`CPbII
`(mM)
`100
`100
`100
`100
`100
`100
`
`and UV−vis measurements of solutions A−G, 50 mM stock solutions
`of enriched 207PbO (94.5%) from Cambridge Isotope Laboratories and
`PbO dissolved in 0.15 M HClO4 were prepared, respectively. Upon
`the dropwise addition of 6.0 M NaOH, an off-white precipitate
`formed, which momentarily dissolved at pH ∼7; after a few seconds, a
`cream-colored precipitate appeared. The addition of a 1.0 M sodium
`hydroxide solution continued until the solid dissolved above pH 8.5
`and gave a clear colorless solution. For solutions A (CPbII = 10 mM)
`and A* (CPbII = 100 mM), with the mole ratio H2Cys/PbII = 2.1, the
`solid dissolved completely at pH ∼10.4, and for solutions B and B*
`(H2Cys/PbII mole ratio = 3.0), it dissolved at pH 9.1. For consistency,
`the pH values of solutions with higher H2Cys/PbII mole ratios were
`also set at pH 9.1. The final volume for each solution was adjusted to
`5.0 mL. Solutions A−F were used for ESI-MS and 1H and 13C NMR
`(prepared in 99.9% deoxygenated D2O) measurements. For solutions
`in D2O, the pH-meter reading was 10.4 for solution A (pD = pH
`reading + 0.4)22 and 9.1 for B−F. 207Pb NMR spectra were measured
`for all solutions (10% v/v D2O), while Pb LIII-edge EXAFS spectra
`were measured for solutions A−E and A*−F*.
`Bis(2-aminoethanethiolato)lead(II) Solid, Pb(aet)2. A total of
`0.964 g (12.5 mmol) of Haet dissolved in 10 mL of deoxygenated
`water (pH 9.6) was added to a suspension of PbO (1.116 g, 5 mmol)
`in 50 mL of ethanol at 50 °C and refluxed for 3 h under an argon
`atmosphere, giving a pale-yellow solution, which was then filtered and
`cooled in a refrigerator. Colorless crystals formed after 48 h and were
`filtered, washed with ethanol, and dried under vacuum (turning
`yellow). Eleme anal. Calcd for Pb(SCH2CH2NH2)2: C, 13.36; H, 3.37;
`N, 7.79. Found: C, 13.41; H, 3.43; N, 7.81. The unit cell dimensions of
`the crystal were also verified, matching the literature values.25
`Methods. Details about instrumentation and related procedures for
`ESI-MS (Aglient 6520 Q-Tof), UV−vis (Cary 300), and 1H, 13C, and
`207Pb NMR spectroscopy (Bruker AMX 300 and Avance II 400 MHz),
`as well as EXAFS data collection and data analyses are provided
`elsewhere.26 UV−vis spectra of solutions A−G were measured using
`0.25, 0.5, and 0.75 nm data intervals, with a 1.5 absorbance Agilent
`rear-beam attenuator mesh filter in the reference position. ESI-MS
`spectra for solutions A, B, and F were measured in both positive- and
`negative-ion modes. 207Pb NMR spectra for solutions A−G enriched in
`207Pb were measured at room temperature using a Bruker AMX 300
`equipped with a 10 mm broad-band probe. For these solutions, the
`207Pb NMR chemical shift was externally calibrated relative to 1.0 M
`Pb(NO3)2 in D2O, resonating at −2961.2 ppm relative to Pb(CH3)4
`(δ = 0 ppm).27 Approximately 12800−51200 scans for 207Pb NMR,
`16−32 scans for 1H NMR, and 500−3000 scans for 13C NMR were
`coadded for the solutions. One-pulse magic-angle-spinning (MAS)
`207Pb NMR spectra for crystalline Pb(aet)2 were acquired with high-
`power proton decoupling on an AVANCE III 200 NMR spectrometer
`at room temperature (207Pb, 41.94 MHz). Ground crystals were
`packed in a 7 mm zirconia rotor, spinning at MAS rates of 5.8 and 5.5
`kHz using 800 and 895 scans, respectively, with a 5.0 s recycle delay.
`Chemical shifts were referenced relative to Pb(CH3)4, by setting the
`207Pb NMR peak of solid Pb(NO3)2 spinning at a 1.7 kHz rate at
`−3507.6 ppm (295.8 K).28,29 Static 207Pb NMR powder patterns were
`reconstructed by iteratively fitting the sideband manifold using the
`Solids Analysis package within Bruker’s TOPSPIN 3.2 software.
`
`2161
`
`DOI: 10.1021/ic5025668
`Inorg. Chem. 2015, 54, 2160−2170
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`Nexus Ex. 1034
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`Inorganic Chemistry
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`Article
`
`Figure 1. ESI-MS spectra measured in positive-ion mode for solutions A (left) and F (right) (CPbII = 10 mM) with H2Cys/PbII mole ratios 2.1 and
`10.0, respectively. The peak at 122.03 amu has 100% intensity. Selected peaks assigned to lead(II) species with distinct isotopic patterns for Pb are
`shown in the inset.
`
`Pb LIII-edge X-ray absorption spectroscopy (XAS) spectra were
`measured at ambient
`temperature at
`the Stanford Synchrotron
`Radiation Lightsource (SSRL) for freshly prepared solutions A−E
`and A* at BL 7-3 (500 mA; equipped with a rhodium-coated harmonic
`rejection mirror) and for solutions B*−F* at BL 2-3 (100 mA). A
`double-crystal monochromator with Si(220) was used at both
`beamlines. To remove higher harmonics, the monochromator was
`detuned to eliminate 50% of the maximum intensity of the incident
`beam (I0) at the end of the scan at BL 2-3. To avoid photoreduction of
`the samples at BL 7-3, the beam size was adjusted to 1 × 1 mm, and
`the intensity of
`the incident beam was reduced to 80% of
`the
`maximum of I0 at 13806 eV. A high beam intensity could result in
`precipitation of a small amount of black particles in the sample holder,
`especially in solutions containing excess
`ligand. For
`solutions
`containing CPbII = 10 mM, 12−13 scans were measured in both
`transmission and fluorescence modes, detecting X-ray fluorescence
`using a 30-channel germanium detector, while for
`the more
`concentrated solutions with CPbII = 100 mM between three and four
`scans were collected in transmission mode. For each sample,
`consecutive scans were averaged after comparison to ensure that no
`radiation damage had occurred. The energy scale was internally
`calibrated by assigning the first inflection point of a lead foil at 13035.0
`eV. The threshold energy E0 in the XAS spectra of the lead(II)
`cysteine solutions varied within a narrow range: 13034.0−13034.9 eV.
`Least-squares curve fitting of the EXAFS spectra was performed for
`solutions A, B, A*, B*, and F* over the k range = 2.7−11.7 Å−1, using
`the D-penicillaminatolead(II) (PbPen) crystal structure30 as the model
`in the FEFF 7.0 program.31,32 For each scattering path, the refined
`structural parameters were the bond distance (R), the Debye−Waller
`parameter (σ2), and in some cases the coordination number (N). The
`2) was fixed at 0.9 (obtained from
`amplitude reduction factor (S0
`EXAFS data analysis of solid PbPen),33 while ΔE0 was refined as a
`common value for all scattering paths. The accuracy of the Pb−(N/O)
`and Pb−S bond distances and the corresponding Debye−Waller
`parameters is within ±0.04 Å and ±0.002 Å2, respectively. Further
`technical details about EXAFS data collection and data analyses were
`provided previously.26
`Principal component analysis (PCA), introduced in the EXAFSPAK
`suite of programs,34 was applied on the raw k3-weighted experimental
`EXAFS spectra for solutions A−E and A*−F* over the k range of 2.7−
`11.7 Å−1. DATFIT, another program in the EXAFSPAK package, was
`the k3-weighted EXAFS spectra of
`lead(II) cysteine
`used to fit
`solutions A−E and A*−F* to a linear combination of EXAFS
`oscillations for species with PbS2N(N/O), PbS3N, and PbS3
`coordination to estimate the amount of such species in each lead(II)
`theoretical EXAFS
`the PbS3N model,
`cysteine solution. For
`oscillations were simulated by stepwise variation of the Pb−S and
`Pb−N parameters: Pb−S 2.67−2.70 Å [using σ2 = 0.0065 Å2 from
`
`EXAFS least-squares refinement of lead(II) glutathione solutions with
`excess ligand],33 Pb−N 2.40−2.43 Å (σ2 = 0.004, 0.006, and 0.008 Å2),
`2 = 0.9. The best fits were obtained for Pb−S = 2.68 Å (σ2 =
`and S0
`0.0065 Å2) and Pb−N = 2.40 Å (σ2 = 0.0080 Å2).
`■ RESULTS
`ESI-MS. ESI-MS spectra were measured in both positive-
`and negative-ion modes for the lead(II) cysteine solutions A, B,
`and F, with CPbII = 10 mM and H2Cys/PbII mole ratios 2.1, 3.0,
`and 10.0, respectively, as shown in Figures 1 and S-3 in the SI,
`to identify possible charged lead(II) cysteine complexes. The
`assignment of the mass ions, presented in Tables 2 and S-1 in
`
`Table 2. Assignment of Mass Ions Observed in ESI-MS
`Spectra (Positive-Ion Mode) for Lead(II) Cysteine Solutions
`A, B, and F (C
`PbII = 10 mM; H2Cys/PbII Mole Ratios 2.1, 3.0,
`and 10.0, Respectively)a
`
`m/z
`(amu)
`122.03
`144.01
`
`165.99
`252.97
`
`294.00
`
`308.99
`
`311.01
`
`assignment
`[H2Cys + H+]+
`[Na+ + H2Cys]+
`[2Na+ + H2Cys − H+]+
`[Pb(HCOO)]+
`[Pb(H2Cys) − H+ −
`H2S]+
`[3Na+ + 2(H2Cys) −
`2H+]+
`[PbC2H5N3O2]+
`
`m/z
`(amu)
`327.99
`349.97
`
`449.00
`451.99
`
`594.99
`
`635.97
`
`654.96
`676.95
`
`assignment
`[Pb(H2Cys) − H+]+
`[Na+ + Pb(H2Cys) −
`2H+]+
`− H+]+
`[Pb(H2Cys)2
`[4Na+ + 3(H2Cys) −
`3H+]+
`[5Na+ + 4(H2Cys) −
`4H+]+
`[3Na+ + Pb(H2Cys)3
`4H+]+
`− 3H+]+
`[Pb2(H2Cys)2
`−
`[Na+ + Pb2(H2Cys)2
`4H+]+
`
`−
`
`aH2Cys (C3H7NO2S); m /z 121.02.
`
`the SI, is facilitated by the distinct isotopic distribution pattern
`for lead(II) species due to the natural abundance of 52.4%
`208Pb, 22.1% 207Pb, 24.1% 206Pb, and 1.4% 204Pb.35 The ESI-MS
`spectra for solutions A and B were nearly identical, showing
`positive-ion mass peaks for species with metal-to-ligand mole
`ratios 1:1, 1:2, and 2:2. Such mass peaks were also previously
`detected for a 1:1 mixture of Pb(NO3)2 and cysteine in 50%
`methanol/water and considered to be independent of
`the
`reaction mixture stoichiometry (1:10 or 10:1).36 We could also
`− 4H+]+ at 635.97
`detect a 1:3 species [3Na+ + Pb(H2Cys)3
`
`2162
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`DOI: 10.1021/ic5025668
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`Nexus Ex. 1034
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`Article
`
`Figure 2. (left) UV−vis spectra of lead(II) cysteine solutions A−G with CPbII = 10 mM and H2Cys/PbII mole ratios 2.1−15.0 compared with that of
`a 10 mM cysteine solution (dots, pH 9.1). (right) UV−vis spectra of 10 mM lead(II) solutions containing H2Cys/PbII mole ratio 15.0 at pH 9.15
`(solution G) and at pH 8.95 (solution G′) and their difference (G′ − G) compared with that of a lead(II) glutathione solution with GSH/PbII mole
`ratio 10.0 (pH 8.5).33 Data interval = 0.5 nm.
`
`Figure 3. 1H and 13C NMR spectra of 0.1 M cysteine in D2O (pH 9.1) and alkaline lead(II) cysteine solutions (99.9% D2O) with CPbII = 10 mM and
`H2Cys/PbII mole ratios 2.1 (A), 3.0 (B), 4.0 (C), 5.0 (D), 8.0 (E), and 10.0 (F). See Table 1.
`
`amu in the spectrum of solution F. In negative-ion mode, only
`one mass peak corresponding to a lead(II) complex was
`− 3H+]− ion (446.99
`observed, assigned to the [Pb(H2Cys)2
`amu).
`Electronic Absorption Spectroscopy. Figure 2 (left)
`displays the UV−vis spectra for the lead(II) cysteine solutions
`A−G (CPbII = 10 mM). The absorption bands have been
`attributed to a combination of S− 3p → PbII 6p ligand-to-metal
`charge-transfer and PbII intraatomic transitions.37−40 The peak
`maximum for solution A, λ
`max = 298 nm (CH2Cys = 21 mM; pH
`10.4) shows a slight red shift to λ
`max = 300 nm as the ligand
`concentration increases in solution B with H2Cys/PbII mole
`ratio 3.0 at pH 9.1 (Figure S-4a in the SI).
`For solutions E−G with high free ligand concentration (50−
`120 mM), a growing shoulder appears around λ ∼ 330 nm,
`the peak at ∼300 nm reduces
`while the intensity of
`significantly. This shoulder
`is blue-shifted relative to the
`maximum absorption recorded at 335 nm for a lead(II)
`glutathione solution (pH 8.5) containing excess ligand (Figure
`2, right).33 The amplitude of this shoulder is pH-dependent, as
`shown in Figure 2 (right) for 10 mM lead(II) solutions
`
`containing H2Cys/PbII mole ratio 15.0 at pH 9.15 (solution G)
`and pH 8.95 (solution G′). The difference of these two spectra
`(G′ − G) shows that when the pH is lowered by 0.2 units, a
`max in the UV−vis
`peak at 335 nm emerges, very similar to λ
`spectrum of the lead(II) glutathione solution.33 There is no
`true isosbestic point around 312 nm, as shown in Figure S-4b in
`the SI by the systematic movement of crossing points of the
`absorption spectra of solutions B−G with that of solution A.
`1H and 13C NMR Spectroscopy. The 1H and 13C NMR
`spectra of a 0.1 M cysteine solution (pH 9.1) and the lead(II)
`cysteine solutions A−F (CPbII = 10 mM) prepared in D2O are
`shown in Figure 3, with the 1H NMR chemical shifts (δ
`H)
`shown in Table S-2 in the SI. For lead(II)-containing solutions,
`
`−Hc and C1−C3
`only one set of signals was observed for the Ha
`atoms in both PbII-bound and free cysteine because of fast
`ligand exchange on the NMR time scale. These average
`resonances were all
`shifted downfield relative to the
`corresponding peaks in free cysteine (see Table S-2 in the
`SI), with the largest shifts (Δδ) observed for solution A
`(H2Cys/PbII mole ratio = 2.1), which contains the least amount
`of free ligand. Satellites originating from 207Pb nuclei were not
`observed in the 13C NMR spectra.
`
`2163
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`Inorg. Chem. 2015, 54, 2160−2170
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`Nexus Ex. 1034
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`
`Figure 4. 207Pb NMR spectra of the alkaline aqueous lead(II) cysteine solutions A−G enriched in 207Pb (CPbII = 10 mM; H2Cys/Pb2+ mole ratios
`2.1−15.0) and A*−F* with 10% D2O (CPbII = 100 mM; H2Cys/Pb2+ mole ratios 2.1−10.0).
`
`207Pb NMR Spectroscopy. The chemical shift of the 207Pb
`nucleus spans over a wide range (∼17000 ppm). It is sensitive
`to the local structure and electronic environment, the nature of
`surrounding donor atoms,
`the bond covalency, and the
`coordination number and is affected by the temperature and
`concentration.27,28,41−43 We measured 207Pb NMR spectra for
`two sets of alkaline aqueous lead(II) cysteine solutions
`(containing 10% D2O), with increasing H2Cys/PbII mole ratios
`(Table 1). Calculated distribution diagrams based on different
`sets of stability constants indicate that the dominating lead(II)
`complexes would be either [Pb(Cys)2]2− (Figure S-2b in the
`SI),20 or a mixture of [Pb(Cys)2]2− and [Pb(Cys)(HCys)]−
`(Figure S-1b in the SI).16 Figure 4 presents the 207Pb NMR
`spectra for solutions A−G (CPbII = 10 mM; enriched in 207Pb)
`and A*−F* (CPbII = 100 mM), all with only an average NMR
`resonance. Solutions A and A*, both with the H2Cys/PbII mole
`ratio = 2.1 at pH 10.4, show sharp signals at 2006 and 2010
`respectively, which are ∼184−200 ppm deshielded
`ppm,
`relative to that of lead(II) penicillamine (3,3′-dimethylcysteine)
`solutions with similar composition (∼1806−1826 ppm).26 The
`sharpness of this signal results from fast ligand exchange (in the
`NMR time scale) between the lead(II) complexes in solution.
`As the ligand concentration increases in solutions B and B*
`(H2Cys/PbII = 3.0) and the pH changes to 9.1, the 207Pb
`resonance becomes broader and shifts ∼106 (B) and ∼209
`(B*) ppm downfield and more for
`the higher
`ligand
`concentration. Broad averaged signals indicate ligand exchange
`between several lead(II) species at intermediate rates. Solution
`F* containing CPbII = 100 mM and CH2Cys = 1.0 M shows the
`most deshielded 207Pb NMR resonance (2507 ppm), which still
`is ∼286 ppm upfield relative to that of the Pb(S-GSH)3
`complex (2793 ppm) with PbS3 coordination.33
`For comparison, we measured one-pulse MAS 207Pb NMR
`spectra of the crystalline bis(2-aminoethanethiolato)lead(II)
`complex, Pb(S,N-aet)2, at two different spin rates, 5.5 and 5.8
`kHz (see Figures 5 and S-5a in the SI), and observed an
`isotropic chemical shift of δ
`iso = 2105 ppm for this complex
`with PbS2N2 coordination.25 Reconstruction of a static 207Pb
`NMR powder pattern for spin rate 5.8 kHz resulted in the
`following principal components: δ
`11 = 3707.98 ppm, δ
`22 =
`33 = −223.12 ppm, leading to δ
`2831.04 ppm, δ
`iso = 1/3(δ
`11 +
`22 + δ
`δ
`33) = 2105.3 ppm (see Figure S-5b in the SI). The
`isotropic chemical shift is ∼600 ppm upfield relative to the only
`other 207Pb chemical shift
`that
`is reported for PbS2N2
`
`Figure 5. One-pulse proton-decoupled MAS 207Pb NMR spectra of
`crystalline Pb(S,N-aet)2, measured at two different spin rates (5.5 and
`5.8 kHz) at room temperature. The dashed vertical line shows the
`isotropic chemical shift δ
`iso = 2105 ppm (identified from overlapping
`spectra in Figure S-5a in the SI).
`
`coordination, δ
`iso = 2733 ppm for Pb(2,6-Me2C6H3S)2(py)2
`(py = pyridine). In that lead(II) complex, all
`ligands are
`monodentate (i.e., not forming a chelate ring) and pyridine is
`the N-donor ligand.44
`We also obtained the 207Pb NMR spectrum of an aqueous
`lead(II) cysteamine solution, prepared by dissolving crystalline
`(mononuclear) Pb(aet)2 in a solution containing the same
`number of moles of cysteamine, with a final
`lead(II)/
`cysteamine mole ratio of 1:3 (10% D2O; CPbII ∼ 76 mM; pH
`10.1). A signal at 2212 ppm was observed (Figure S-6 in the
`SI), probably from a mixture of mononuclear Pb(S,N-aet)2
`(PbS2N2 coordination), [Pb(S,N-aet)(S-Haet)(OH/OH2)]n (n
`= 0, 1; PbS2NO), and [Pb(S,N-aet)(S-Haet)2]+ (PbS3N)
`complexes. A minor amount of PbS3N species is a likely
`reason for the ∼100 ppm deshielding of the 207Pb NMR
`resonance for this solution, relative to the isotropic chemical
`shift of crystalline Pb(aet)2 (δ
`iso = 2105 ppm). Moreover,
`multinuclear species such as the [Pb2(aet)3]+ complex may
`form,45 where PbII ions can adopt PbS3N coordination through
`bridging thiolate groups. However, Li and Martell could only
`identify mononuclear [Pb(Haet)]2+, [Pb(aet)]+, and Pb(aet)-
`(OH) complexes in dilute solutions with CPbII = 1.0 mM (CHaet
`= 1.0−2.0 mM; pH 2−8).46
`Pb LIII-Edge XAS. The near-edge features in the XAS
`spectra are nearly identical for the lead(II) cysteine solutions, as
`shown in Figure S-7 in the SI, and are evidently not sensitive to
`the changes in lead(II) speciation as the ligand concentration
`
`2164
`
`DOI: 10.1021/ic5025668
`Inorg. Chem. 2015, 54, 2160−2170
`
`Nexus Ex. 1034
`Page 5 of 11
`
`

`

`Inorganic Chemistry
`
`the
`increases. When the extended regions are compared,
`EXAFS spectra and corresponding Fourier transforms are also
`quite similar for solutions A, B, and E in the CPbII = 10 mM
`series (Figure S-8 in the SI), while for solutions A*−F* (CPbII =
`100 mM), the amplitude of the EXAFS oscillation (and the
`Fourier transform) shows a gradual
`increase as the total
`concentration of cysteine increases from 0.21 to 1.0 M (Figure
`S-8 in the SI). The k3-weighted EXAFS spectrum of
`the
`lead(II) cysteine solution A* (CH2Cys = 210 mM; pH 10.4)
`closely overlaps with that of a lead(II) penicillamine aqueous
`solution (CPbII = 100 mM; H2Pen/PbII = 3.0; pH 9.6), in which
`the dithiolate [Pb(S,N,O-Pen)(S-HnPen)]2−n (n = 0−1) species
`dominates;26 see Figure S-9 in the SI.
`PCA of the raw k3-weighted EXAFS spectra of the six
`lead(II) cysteine aqueous solutions A*−F* and of the five
`solutions A−E displayed two major components with clear
`oscillatory patterns (Figure S-10,
`top,
`in the SI). Using
`TARGET in the EXAFSPAK package on the PCA output file
`showed that
`two of
`the PCA components in each series
`matched fairly well with the raw k3-weighted EXAFS
`oscillations obtained experimentally for 0.1 M lead(II) solutions
`containing penicillamine (CH2Pen = 0.3 M; pH 9.6) or N-
`acetylcysteine (CH2NAC = 1.0 M; pH 9.1). These solutions are
`dominated by PbS2NO and PbS3 species, respectively.26,47 In
`the next step, the DATFIT program in the EXAFSPAK suite
`was used to estimate the relative amounts of such PbS2N(N/
`O)- and PbS3-coordinated species in the lead(II) cysteine
`solutions A*−F*. This was achieved by fitting the k3-weighted
`EXAFS spectra of solutions A*−F* to a linear combination of
`EXAFS oscillations for the lead(II) penicillamine/N-acetylcys-
`teine solutions; however, a clear oscillatory residual was
`observed. That residual amplitude increased when fitting the
`EXAFS spectra of solutions with higher H2Cys/PbII mole
`ratios; see Figure S-11a in the SI,
`indicating a substantial
`additional contribution from another
`lead(II) species (or
`scattering path), probably a four-coordinated PbS3N complex.
`Note that coordinated O and N atoms cannot be distinguished
`by EXAFS spectroscopy, and the PbS2N(N/O) and PbS3N
`coordination modes used here are based on the interpretation
`of 207Pb NMR spectra (see the Discussion section).
`EXAFS oscillations were then theoretically simulated for a
`PbS3N model using the WinXAS48 and FEFF 7.0 programs,
`varying the Pb−S and Pb−N parameters stepwise within
`2 = 0.9 (see the Methods
`reasonable ranges and keeping S0
`section). The best fits with considerably smaller residuals were
`obtained when including the simulated EXAFS oscillation for
`this model in the linear combination, considering Pb−S 2.68 Å
`(σ2 = 0.0065 Å2) and Pb−N 2.40 Å (σ2 = 0.0080 Å2); see
`Figure S-11b in the SI. These fittings clearly show that di- and
`trithiolate lead(II) complexes with PbS2N(N/O) and PbS3N
`coordination give major contributions to the EXAFS spectra of
`the lead(II) cysteine solutions A*−F* (Table 3). Including the
`PbS3-coordinated model in the fitting only slightly improved
`the residual (Figure S-11c in the SI), indicating a minor and
`uncertain contribution (Table S-3 in the SI), especially because
`there are only two major oscillatory PCA components (Figure
`S-10 in the SI, top). Although the EXAFS spectra of solutions
`E* and F* nearly overlap (Figure S-8 in the SI),
`the
`percentages of PbS3N/PbS3 from their linear combination
`fitting results vary about ±10%. Similarly, the PbS3 contribution
`in the EXAFS spectra of solutions A−E is also minor and
`uncertain (Figure S-11e and Table S-3 in the SI). The fittings of
`
`Article
`
`Table 3. Results of Fitting the Raw k3-Weighted Pb LIII-Edge
`EXAFS Spectra of Lead(II) Cysteine Solutions A*−F* and
`A−E with Linear Combinations of EXAFS Oscillations for
`PbS2N(N/O) and PbS3N Models (See the Text; Figures S-
`11b and S-11d in the SI)a
`δ(207Pb)
`solution (H2Cys/PbII mole
`PbS3N
`PbS2NO + PbS2N2
`(%)
`ratio)
`(ppm)
`(%)
`11
`89
`A (2.1)
`2006
`20
`80
`B (3.0)
`2112
`26
`74
`D (5.0)
`2229
`27
`73
`E (8.0)
`2310
`A* (2.1)
`9
`91
`2010
`B* (3.0)
`23
`77
`2219
`C* (4.0)
`31
`69
`2316
`D* (5.0)
`37
`63
`2350
`E* (8.0)
`40
`60
`2418
`F* (10.0)
`49
`51
`2507
`aThe raw k3-weighted EXAFS spectrum of a 0.1 M lead(II)
`penicillamine solution (CH2Pen = 0.3 M; pH 9.6) was used for the
`PbS2N(N/O) model. The EXAFS oscillation for PbS3N was
`theoretically simulated (see the text). The estimated error limit of
`the relative amounts is ±10−15%. For the results when including the
`PbS3 contribution in the fitting, see Table S-3 in the SI.
`
`linear combinations of EXAFS oscillations for the PbS2N(N/
`O) and PbS3N models to the EXAFS spectra of the dilute
`lead(II) cysteine solutions A−E (CPbII = 10 mM) are shown in
`Figure S-11d in the SI, with results in Table 3.
`Least-squares curve fitting of the EXAFS spectra obtained for
`lead(II) cysteine solutions only provides an average of the bond
`distances around the PbII ions in the above species. Thus, a
`coordination model comprising the Pb−(N/O) and Pb−S
`paths was used to simulate the theoretical EXAFS oscillation,
`which was fitted to the extracted EXAFS spectra for the lead(II)
`cysteine solutions A, A* and B, B*, with H2Cys/PbII mole
`ratios 2.1 and 3.0, respectively, for which δ(207Pb) ∼ 2000−
`2220 ppm was observed. The curve-fitting results are displayed
`in Figure 6, with corresponding structural parameters in Table
`4. The average Pb-(N/O) and Pb−S bond distances were 2.42
`
`Figure 6. Least-squares curve fitting of k3-weighted Pb LIII-edge
`EXAFS spectra and corresponding Fourier transforms for lead(II)
`cysteine solutions A and B (CPbII = 10 mM) and A*−F* (CPbII = 100
`mM) with different H2Cys/PbII mole ratios (see Table 4).
`
`2165
`
`DOI: 10.1021/ic5025668
`Inorg. Chem. 2015, 54, 2160−2170
`
`Nexus Ex. 1034
`Page 6 of 11
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`

`

`Inorganic Chemistry
`± 0.04 and 2.64 ± 0.04 Å, respectively, with corresponding
`Debye−Waller parameters varying over the ranges σ2 = 0.014−
`0.024 and 0.0043−0.0061 Å2, respectively. Attempts to resolve
`two different Pb−(N/O) distances resulted in one quite short
`(2.33 Å) and another fairly long (2.50 Å) distances; see model
`III in Table S-4 in the SI. The shortest Pb−N distance found in
`crystal
`structures with PbS2N2 coordination is 2.401 Å
`(Cambridge Structural Database code: NOGQOQ), and the
`shortest recorded Pb−O distance in crystalline PbS2O2
`complexes is 2.349 Å (CSD