S1
© CSIRO 2017 Environ. Chem. 2017, 14, 243–253 doi:10.1071/EN17062_AC
Supplementary material
Thermodynamic stability of mercury(II) complexes formed with environmentally relevant low-molecular-mass thiols studied by competing ligand exchange and density functional theory
Van Liem-NguyenA, Ulf SkyllbergB, Kwangho NamA and Erik BjörnA,C
ADepartment of Chemistry, Umeå University, SE-901 87 Umeå, Sweden.
BDepartment of Forest Ecology and Management, Swedish University of Agricultural Sciences, SE-901
83 Umeå, Sweden.
CCorresponding author. Email: [email protected]
S2
S1. Calculation of stability constants for Hg(MAC)2 and HgIMAC using I- as competing
ligand. Components, reactions and a calculation scheme for determination of the stability constant
for the Hg(MAC)2 and HgIMAC complexes with I- as the competing ligand, using WinSGW
software. The pKa of the RSH and COOH groups for MAC are 10.2 and 3.5, respectively. A similar
set of corresponding equations were set-up when calculating constants for Hg(2-MPA)2.
[I-]total = [I-]free + [HgI+] + 2[HgI2] + 3[HgI3-] + 4[HgI4-] + [HgIMAC] (Sa)
[MAC]total= [MAC]free + 2[Hg(MAC)2] +[HgIMAC] + [HgMAC] (Sb)
[Hg2+]total= [Hg2+]free + [HgI+] + [HgI2] + [HgI3-] + [HgI4
-] + [HgIMAC] + [Hg(MAC)2] +[HgMAC] + [Hg(OH)n](Sc)
Hg2+ + I- ⇌ HgI+ Log K1= 13.42 (S3a)
Hg2+ + 2I- ⇌ HgI2 Log K2= 24.60 (S3b)
Hg2+ + 3I- ⇌ HgI3- Log K3=28.32 (S3c)
HgII + 4I- ⇌ HgI42- Log K4= 30.07 (S3d)
Hg2+ + MAC ⇌ HgMAC Log K = 32.5 (S4a’)
Hg2+ + 2MAC ⇌ Hg(MAC)2 Log β2 = ? (S1b’)
Hg2+ + I- +MAC ⇌ HgIMAC Log K = ? (S6)
Example of calculation
[I-]total = 20 µMa
[MAC]total = 16 µMa
[Hg2+]total = 4 µMa
[Hg(MAC)2] = 3.2 µMb
[HgIMAC] = (Peak Area (HgIMAC) / Peak Area (Hg(MAC)2)) ×[Hg(MAC)2] = 0.45 µMb
[HgInx] = [HgII]total - [Hg(MAC)2] - [HgIMAC]= 0.29 µMb
a Concentration of ligand used for complexation b Concentration of complexes determined by LC-ICPMS.
In the experiment, the concentration of I- was varied, and in the calculation scheme above it is
exemplified for 20 µM I-. Constants were recalculated to an ionic strength of 0 M and experimental
pH was 2.9. Based on the equations (Sa-Sc); reaction S3(a-d), (S4a’), (S1b’), (S6), and total
concentration of HgII and ligands, and measured concentration of Hg(MAC)2 and HgIMAC
complexes, the log K of Hg(MAC)2 and HgIMAC was determined to be 40.9 and 32.2, respectively.
S3
S2. Calculation of the stability constant for the Hg(Cys)2 complex using MAC as competing
ligand. Calculation scheme for determination of the stability constant for Hg(Cys)2 with MAC as
the competing ligand using WinSGW software. The pKa of the RSH group for MAC and Cys are
10.2 and 8.6, respectively and pKa of the COOH group for MAC and Cys are 3.4 and 2.0,
respectively. The same corresponding calculation scheme was used for the other Hg(SR)2
complexes using MAC or 2-MPA as the competing ligand.
[MAC]total= [MAC]free + 2[Hg(MAC)2] + [HgMAC] (Sd)
[Cys]total= [Cys]free + 2[Hg(Cys)2] + [HgCys] (Se)
[Hg2+]total= [Hg2+]free + [HgMAC] + [Hg(MAC)2] + [HgCys] + [Hg(Cys)2] +[Hg(OH)n] (Sf)
Hg2+ + MAC ⇌ HgMAC Log K = 32.5 (S4’)
Hg2+ + Cys ⇌ HgCys Log K = 29.0 (S4’’)
Hg2+ + 2MAC ⇌ Hg(MAC)2 Log β2= 40.9 (S1b’)
Hg2+ + 2Cys ⇌ Hg(Cys)2 Log β2=? (S1b’’)
Example of calculation
[MAC]total = 8 µMa
[Cys]total= 8 µMa
[Hg2+]total= 4 µMa
[Hg(MAC)2] = 2.27 µMb
[Hg(Cys)2] = 1.72 µMb a Concentration of ligand used for complexation b Concentration of complexes determined by HPLC-ICPMS
Constants were recalculated to an ionic strength of 0 M and experimental pH was 2.9. Based on
the equations (Sd-Sf); reactions (S4’), (S4’’), (S1b’), (S1b’’) and total concentration of the HgII
and ligands and measured complexes concentration, the log β2 of Hg(Cys)2 was determined to 37.5.
S4
1. Hydroxyl 2. Carboxyl 3. Carboxyl/carbonyl +secondary amine
4. Carboxyl/carbonyl +primary amine
(w/wo secondary amine)
5. Primary amine
Mercaptoethanol (ETH)
Mercaptoacetic acid (MAC)
N-acetyl-cysteine (NACCys)
Cysteine (Cys)
Cysteamine (Cyst)
Monothiolglycerol (Glyc)
3-Mercaptopropionic acid (3-MPA)
N-acetyl-penicilamine (NACPen)
Homocysteine (HCys)
2-Mercaptopropionic acid (2-MPA)
N-cysteinylglycine (CysGly)
Mercaptosuccinic acid (SUC)
γ-glutamylcysteine (GluCys)
Glutathione (GSH)
Penicilamine (Pen)
Fig. S1. The structure and abbreviation of investigated thiol ligands. The thiols are grouped according to
the presence of functional groups in addition to the thiol group.
S5
0
2
4
6
8
10
0 0.5 1 1.5 2 2.5 3 3.5 4
Peak
Are
a/T
l int
ensi
tyaHg(HCys)2 Hg(NACCys)2
0
2
4
6
8
10
12
0 0.5 1 1.5 2 2.5 3 3.5Time (h)
bHg(Cys)2 Hg(NACPen)2
Peak
Are
a/T
l int
ensi
ty
Fig. S2. The integrated peak area of Hg(SR)2 complexes to Thalium (204Tl+) signal ratios with
different reaction time for the Hg(SR)2 complex synthesis. (a) mixture of 1 µM of HgII and 2 µM
of each HCys and NACCys. (b) mixture of 1 µM of HgII and 2 µM of each Cys and NACPen. The
experiments were conducted at pH 3.0, and an ion strength of 0 M. A post column flow of a 10 ng
ml-1 Tl solution (flow rate of 100 µl min-1) was used to monitor and correct for signal drift of the
LC-ICPMS system over time.
S6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8
Time (day)
Hg(Cys)2 Hg(HCys)2 Hg(GSH)2 Hg(Glyc)2Pe
ak a
rea/
Tl si
gnal
Fig. S3. Peak area of Hg(SR)2 complexes to Thalium (204Tl+) signal ratios at different storage
time of Hg(SR)2 complex solutions. Samples contain 1 µM of HgII and 4 µM of individual LMM
thiols i.e. Cys, HCys, GSH and Glyc. A post column flow of a 10 ng ml-1 Tl solution (flow rate of
100 µl min-1) was used to monitor and correct for signal drift of the LC-ICPMS system over time.
S7
Hg-I-MAC
Hg(MAC)2
HgI2
HgI2
1
Hg-I-2MPA
HgI2
HgI2
0
5000
10000
15000
20000
0 200 400 600 800 1000
Sign
al (c
ps)
Time (s)
0
10000
20000
30000
0 200 400 600 800 1000 1200 1400
Time (s)
0
20000
40000
60000
0 200 400 600 800 1000 1200 1400
bHg I
0
5000
10000
15000
20000
0 200 400 600 800 1000
Sign
al (c
ps)
aHg I
c d
1
1
12
3
2
3
33
Hg(2MPA)2
Fig. S4. LC-ICPMS chromatograms showing 202Hg+ and 127I+ signals (counts per second, cps)
for (a) 4 µM of HgII and 50 µM of I-. A gradient elution was used with initially 3.5% of 1-propanol
during 12 min, then increased to 25% in a step gradient and kept for 4 min and then back to the
initial 3.5% concentration. (b) 4 µM of HgII and 50 µM of I-. A gradient elution was used with
initially 8.5% 1-propanol during 14 min, then increased to 25% in a step gradient and kept for 4
min and then back to the initial 8.5% concentration. (c) 4 µM of HgII, 16 µM of MAC and 100 µM
of I-. The elution gradient was the same as in (a). (d) 4 µM of HgII, 16 µM of 2MPA and 100 µM
of I-. The elution gradient was the same as in (b). The complexes of Hg(MAC)2, HgIMAC,
Hg(2MPA)2, and HgI(2-MPA) eluted at 600 s, 670 s, 780 s, and 1010 s, respestively. The HgI2
complex eluted at 790 s with the gradient of (a) and at 1070 s with the gradient of (b). The peaks
1, 2 correspond to the complexes of HgInn-2 (n=1, 3, 4) and the peak 3 to a system background
signal.
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m/z0
20
40
60
80
100
Rela
tive
Inte
nsity
468.8
466.9
465.9
470.9
464.9
530.6481.6
527.7553.9329.6 455.1367.8 369.8 496.7325.1
Hg(HCys)2
m/z m/z
698.8
696.9
695.9
694.8 699.8
734.9 796.7795.8
797.5751.0653.2
Hg(GluCys)2
496.8
494.8
493.8
492.8 498.8
597.6595.6499.7444.8 446.5 598.7581.8385.7 530.7
Hg(Pen)2
810.9
810.0
813.9809.0
850.8848.8
847.8 852.9908.5853.9757.8 806.9
Hg(GSH)2
m/z
498.8
497.0
496.1
495.1500.8
618.6616.6596.5
620.5593.6530.7440.8
Hg(SUC)2
390.8
388.8
387.8392.8
386.8
348.9312.8 393.8
410.8
408.9
407.9
412.8406.9
339.0414.9341.0335.0 493.8
Hg(3MPA)2
Hg(ETH)2
Hg(NACCys)2
524.8
522.8
521.9
520.8 526.8
527.8503.9500.8 609.4544.8482.9
0
20
40
60
80
100382.8
380.8
379.8
384.8
378.8
481.6
480.6479.6419.9325.7 324.7
Hg(MAC)2
352.9
350.9
349.9
354.9348.8
318.9400.8
382.9 418.9
328.0
Hg(Cyst)2
440.8
438.8
437.8
436.8
442.8
478.8357.7 444.7416.6 505.6
Hg(Cys)2
410.8
408.8
407.9
406.8 412.9
432.8 467.8339.8
Hg(2MPA)2
524.8Hg(CysGly)2
522.8
621.8571.8
460.8
580.7
579.5
578.2
582.8
583.7584.7 678.5 675.7575.4536.20
20
40
60
80
100Re
lativ
e In
tens
ity812.9
Hg(NACPen)2
Rela
tive
Inte
nsity
477.8
475.8
474.9
473.9479.8
414.9 450.9
512.7554.7
Hg(Glyc)2
521.8
520.8 526.8
m/z
Fig. S5. The mass spectra of 15 investigated Hg(SR)2 complexes achieved by direct infusion to
ESIMS, showing molecular mass and Hg isotope pattern of the Hg(SR)2 complexes. All Hg(SR)2
complex analyses were conducted in the negative ionization mode with the exception of
Hg(Cyst)2, which was analysed in positive mode. The concentration of HgII was 0.1 mM, the
molar ratio of RSH to HgII was 4 and pH was 3.0.
S9
Fig. S6. Measured concentration of the Hg(MAC)2 complex (2 µM of HgII and 8 µM of MAC)
at different added concentrations of the competing ligands EDTA, Cl-, and Br-. The experiments
were carried out at constant ionic strength of 0.5 M (NaClO4) and pH of 3.0.
S10
200 400 600 800 1200
1
23
45
6 78 9
1011 12
1314
15
3.5% 1-propanol
20% 1-propanol
Time (s)
Fig. S7. LC-ICPMS chromatograms of 202Hg+ signals Illustrating of retention time of the 15
investigated Hg(SR)2 complexes on the Kinetic Biphenyl LC column used in the LC-ICPMS
measurements. The retention time increased as, 1. Hg(Cyst)2, 2. Hg(CysGly)2, 3. Hg(Cys)2, 4.
Hg(HCys)2, 5. Hg(GSH)2, 6. Hg(GluCys)2, 7. Hg(Pen)2, 8. Hg(Glyc)2, 9. Hg(NACCys)2, 10.
Hg(ETH)2, 11. Hg(MAC)2, 12. Hg(SUC)2, 13. Hg(3MPA)2. 14. Hg(NACPen)2, 15. Hg(2MPA)2.
S11
Fig. S8. LC-ICPMS chromatogram of 202Hg+ signals for a mixture of 4 µM of HgII and 4 µM of
MAC with isocratic elution using 3.5% 1-propanol indicating the presence of the one-coordinated
HgMAC complex with a retention time of 700 s.
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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Time (min)
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100R
elat
ive
Abu
ndan
ceHg(MAC)2
Hg(Cys)2
NL:3.82E4TIC F: ITMS - c ESI SIM ms [378.00-385.00]
NL:4.07E2TIC F: ITMS - c ESI SIM ms [407.00- 414.00]
NL:6.17E3TIC F: ITMS - c ESI SIM ms [436.00- 443.00]
CysHgMAC
Fig. S9. LC-ESIMS chromatograms with selected ion monitoring (SIM) mode targeting m/z of
Hg(MAC)2, Hg(Cys)2, and CysHgMAC and their corresponding Hg isotope pattern 378-385 m/z,
436-443 m/z and 407-414 m/z, respectively. An elution gradient was used with initially 8% of
MeOH, 0.1%FA and 92% H2O, 0.1%FA from 3 to 10 min followed by a linear gradient to 90% of
MeOH, 0.1%FA which was kept for 3 min. From 13 to 16 min the concentration of MeOH, 0.1%FA
was reduced from 90% to 8% in a linear gradient and was kept for 9 min. The increased background
after 6 min is caused by the increase of MeOH in the mobile phase.
S13
0
20000
40000
60000
80000
0 200 400 600 800 1000 1200 1400 1600 1800
0
20000
40000
60000
80000
0 200 400 600 800 1000 1200 1400 1600 1800
0
50000
100000
150000
200000
250000
0 200 400 600 800 1000 1200 1400 1600 1800
Hg(Cys)2
Hg(MAC)2
Hg(MAC)2
Hg(Cys)2
a
b
c
Time (s)
Time (s)
Time (s)
Hg si
gnal
(cps
)Hg
sign
al (c
ps)
Hg si
gnal
(cps
)
Fig. S10. LC-ICPMS chromatograms showing 202Hg+ signals of (a) a mixture containing 4 µM
of HgII and 8 µM of each Cys and MAC, (b) a mixture containing 4 µM of HgII and 16 µM of
MAC, (c) a mixture containing 4 µM of HgII and 16 µM of Cys. The retention time of Hg(Cys)2
and Hg(MAC)2 is 85 s and 540 s, respectively with a mobile phase gradient of initially 3.5% of 1-
propanol for 12 min then a step gradient increase to 25% of 1-propanol. The appearance of a small
peak at 850 s is caused by the changed concentration of 1-propanol in the mobile phase.
S14
320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z
0
10
20
30
40
50
60
70
80
90
100R
elat
ive
Inte
nsity
411.8
409.8440.8
438.8
408.8
382.7
380.8 437.8
407.8379.8 436.7413.8
442.8512.5
384.9485.6 541.6483.7481.6378.8
456.6 538.8480.4 514.7508.5458.5 544.4 566.3 585.9415.7 523.7452.6 602.4355.6323.8 362.7 475.7353.8
Hg(MAC)2
CysHgMAC
Hg(Cys)2
Fig. S11. Full scan mass spectrum of a solution containing 0.1 mM of HgII and 0.2 mM of each Cys and
MAC by direct infusion to ESI-MS with a flow rate of 50 µl min-1. Observed signals indicate the presence
of Hg(MAC)2, Hg(Cys)2 and CysHgMAC complexes, matching their molecular mass and Hg isotope
pattern.
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Table S1. Thermodynamic stability constants for HgIn2-n complexes at different ionic strengths
The stability constants were corrected to different ion strength using free software Ionic Strength
Corrections for Stability Constants from IUPAC with Specific Interaction Theory (SIT) method[1]
Reaction Stability constant (log K1, log β2–β4) I=0.5* I=0 I=0.1 I=1
Hg2+ + I- = HgI+ 12.87 13.42 13.02 12.90 Hg2+ + 2I- = HgI2 23.82 24.60 24.00 23.92 Hg2+ + 3I- = HgI3
- 27.6 28.32 27.74 27.74 Hg2+ + 4I- = HgI4
2- 29.83 30.07 29.73 30.15 * Reference Martell et al. (2004)[2]
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Table S2. Computed complex formation free energies, bond lengths and angles of the Hg(SR)2
complexes determined at the B3LYP level of theory
All geometries were optimized at the gas phase with a mixed basis set containing Def2-TZVPP for Hg and
6-31++G(d,p) for all other atoms, respectively, and an initial geometry with all ligands fully extended
ΔG2a
(kcal/mol) Bond (Å) Angle (°)
Complexes Hg-Sb Hg-Oc S-Hg-S Hg-S-Cb O-Hg-Oc Hg(2-MPA)2 53.1 2.381 3.042 179.7 101.4 158.4 Hg(NACPen)2 56.4 2.364 175.6 105.0 Hg(NACCys)2 51.1 2.383 3.087 175.1 99.3 65.4 Hg(SUC)2 54.0 2.376 3.146 176.7 102.8 96.4 Hg(3-MPA)2 58.0 2.361 177.6 103.0 Hg(MAC)2 56.6 2.373 3.176 177.6 102.9 173.4 Hg(Glyc)2 55.2 2.361 177.7 103.0 Hg(GluCys)2 77.4 2.365 177.0 103.5 Hg(ETH)2 56.9 2.360 177.7 102.7 Hg(Pen)2 86.6 2.383 2.876 176.8 108.4 Hg(GSH)2 54.7 2.380 3.062 170.7 104.6 63.6 Hg(Cys)2 92.1 2.373 177.4 105.7 Hg(CysGly)2 91.7 2.375 176.6 106.0 Hg(HCys)2 87.3 2.372 176.7 106.1 Hg(Cyst)2 90.7 2.374 175.6 106.1
a The ΔG values are for the reaction 1a, i.e., Hg2+ + 2RSH = Hg(SR)2 + 2H+, in the gas phase at 298.15 K. b The Hg-S distances and Hg-S-C angles are the averages over the two Hg-S distances and Hg-S-C angles, respectively. c In the geometry optimisations, several complexes form an additional coordination between Hg and the ligand’s carbonyl or carboxyl oxygen (bidentate coordination). The Hg-O distances and O-Hg-O angles are the averages over the two Hg-O distances and O-Hg-O angles, respectively, except Hg(Pen)2, which only forms a single Hg-O interaction.
References
[1] IUPAC, Ionic Strength Corrections for Stability Constants using Specific Interaction Theory (SIT), version 1. 2004.
[2] A. Martell, R. Smith, R. Motekaitis, National Institute of Standard and Technology, NIST. Critically selected stability constants of metal complexes 2004 (PC-based Database: Gaithersburg, MD).
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