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: erik.bjorn@umu.se

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.

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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).