Reactions of hypobromous acid with dimethyl selenide, dimethyl diselenide and other organic selenium compounds: kinetics and product formation

Selenium (Se) is an essential micronutrient for many living organisms particularly due to its unique redox properties. We recently found that the sulfur (S) analog for dimethyl selenide (DMSe), i.e. dimethyl sulfide (DMS), reacts fast with the marine oxidant hypobromous acid (HOBr) which likely serves as a sink of marine DMS. Here we investigated the reactivity of HOBr with dimethyl selenide and dimethyl diselenide (DMDSe), which are the main volatile Se compounds biogenically produced in marine waters. In addition, the reactivity of HOBr with further organic Se compounds was tested, i.e., SeMet (as N-acetylated-SeMet), and selenocystine (SeCys2 as N-acetylated-SeCys2), as well as the phenyl-analogs of DMSe and DMDSe, respectively, diphenyl selenide (DPSe) and diphenyl diselenide (DPDSe). Apparent second-order rate constants at pH 8 for the reactions of HOBr with the studied Se compounds were (7.1 ± 0.7) × 107 M−1 s−1 for DMSe, (4.3 ± 0.4) × 107 M−1 s−1 for DMDSe, (2.8 ± 0.3) × 108 M−1 s−1 for SeMet, (3.8 ± 0.2) × 107 M−1 s−1 for SeCys2, (3.5 ± 0.1) × 107 M−1 s−1 for DPSe, and (8.0 ± 0.4) × 106 M−1 s−1 for DPDSe, indicating a very high reactivity of all selected Se compounds with HOBr. The reactivity between HOBr and DMSe is lower than for DMS and therefore this reaction is likely not relevant for marine DMSe abatement. However, the high reactivity of SeMet with HOBr suggests that SeMet may act as a relevant quencher of HOBr.


Text S3: Method for the production of N-acetylated-Selenomethionine and N-acetylated-Selenocystine
The method used to produce N-acetylated-selenomethionine (N-acetylated-SeMet) and N-acetylatedselenocystine (N-acetylated-SeCys 2 ) is described in McCurry et al. 2016. 16Here, we used 20 mL amber glass vials with screw caps.A solution of 390 mM BOC 2 O in methanol was produced in a 10 mL headspace amber crimp vial and stored in the refrigerator at 4 °C.5 mL methanol (CH 3 OH, LC/MS grade, Fisher Scientific, Loughborough, UK) was added to each vial, followed by the addition of BOC 2 O and the Se-amino acid to a total volume of 6 mL and a molar BOC 2 O:Se ratio of 10:1.Finally, 250 g sodium hydrogen carbonate (NaHCO 3 , analytical grade, Merck, Hohenbrunn, Germany) was added to the vial.
The vial was then placed in a beaker half-filled with ultrapure water and sonicated for 30 min in an ultrasonic bath (Sonorex Super 10 P, Bandelin electronic GmbH & Co., Berlin, Germany).The cap of the vial was slightly opened to avoid overpressure due to CO 2 formation.After sonication and settling of solid NaHCO 3 , 5 mL of the sonicated N-acetylated-SeMet solution was transferred to a 20 mL amber glass vial and mixed with 15 mL H 2 O.The sonicated N-acetylated-SeCys 2 solution was directly filtered (without dilution with water) through a 0.45 µm cellulose nitrate syringe filter (Whatman, Luer connection) and transferred to a 8 mL amber glass vial.
Stock solutions of N-acetylated-SeMet and N-acetylated-SeCys 2 were then quantified for total Se by ICP-MS/MS and the results were in good agreement with the calculated target concentrations (<3% deviation).In a separate experiment, the yield of N-acetylation of amines via BOC 2 O was tested by formation of chloramines (Text S4).

Text S4: Test for chloramine formation of N-acetylated-SeMet and N-acetylated-SeCys 2
Chlorination of N-acetylated Se amino acids was performed to examine the effectiveness of the derivatization.For fully derivatized amino acids, the N-acetylated amino group can no longer react with HOCl, wherefore, the added chlorine remains in solution and can be detected photometrically by the N,N-diethyl-p-phenylenediamine (DPD) method. 17If the amino acids are not or only partially derivatized, chlorine reacts with the amino group to the corresponding chloramines and can no longer be measured directly by DPD.However, upon addition of iodide, hypoiodous acid is formed, which reacts with DPD. 18Experiments were carried out with N-acetylated-SeMet and various doses of HOCl Tests for N-acetylated-SeCys 2 were performed similarly to the procedure described for SeMet but using a reaction volume of 3.5 mL and a final N-acetylated-SeCys 2 concentration of 4.9 µM.
No chloramine formation was observed in the experiment with N-acetylated-SeMet, as the signal before and after KI-addition is identical (Figure S1A).It is also visible that the difference between added HOCl and quantified total chlorine (via oxidized DPD) corresponds exactly to the concentration of Nacetylated-SeMet (with a slope of 1 regarding Δtotal chlorine/Δadded HOCl after the reaction of HOCl with Se in the N-acetylated-SeMet).However, for N-acetylated-SeCys 2 chloramine formation is observed which indicates an incomplete N-acetylation of the amino group (Figure S1B).Ca. 20 µM HOCl are consumed by N-acetylated-SeCys 2 .HOCl consumption can be explained by the three-stepoxidation of diselenides as seen for HOBr (see main text) and oxidation of non-N-acetylated amino groups.Quantified total chlorine after KI-addition is up to 3 µM (average 1.6 µM) higher compared to the first reading.However, the concentration of produced chloramine for different HOCl doses (representing the different data points in Figure S1) is inconsistent and the fraction of incomplete Nacetylated amino groups is difficult to predict (2.5 -58%, average: 33%).Despite the still available amino groups in solutions of N-acetylated-SeCys 2 , it can be excluded that the amino group will influence the kinetics of the reaction between N-acetylated-SeCys 2 and HOBr, because (i) the reactivity between HOBr and primary amines is around 10 6 M -1 s -1 at pH 8 19,20 which is 1-2 orders of magnitude lower than the observed reactivity between HOBr and N-acetylated-SeCys 2 and (ii) there is no HOBrreactivity difference reported for methionine and N-acetylated methionine, 19 which points to a limited influence of the amino group for the overall reactivity.
The apparent second-order rate constants for the reactions between resorcinol and HOBr/OBr -can be expressed by equation S1, with values indicated in Table S4 and graphic representation in Figure S2: where  k app (Res+HOBr) is the pH-dependent apparent second-order rate constant of the HOBrresorcinol reaction  k(Res+HOBr), k(Res -+HOBr), k(Res 2-+HOBr) and k(Res 2-+OBr -) are the species-specific secondorder rate constants for the reactions between protonated/deprotonated resorcinol species and HOBr (as indicated in Table S3)  αRes, αRes -and αRes 2-are the fractions of protonated and deprotonated resorcinol-species based on pK a values (Table S4) and the actual pH  αHOBr, αOBr -the protonated and deprotonated fractions of HOBr/OBr -based on pK a values (Table S4) and the actual pH FAC and total chlorine were quantified photometrically via oxidation of DPD at λ = 510 nm before and after KI addition, respectively.For Panel A, only values are shown after KI addition because of an insignificant difference to values before KI addition.
Table S3: Species-specific second-order rate constants for reactions between protonated/deprotonated HOBr and resorcinol species and pK a values for different resorcinol species and HOBr.

Reaction or acid-base equilibrium
Species     S4)

Text S6: Determination of limits of quantification for organic selenium compounds and associated reaction competitors
The limits of quantification (LOQ) for DMSe and DMDSe were calculated based on standard deviations of blanks relative to the calibration slope (Hubaux and Vos formula, equation S2), 24 while LOQs for DPSe, DPDSe, resorcinol and TMB were calculated based on the noise of the baseline relative to the signal of a standard (eq.S3).This method was not applied to N-acetylated-SeMet and N-acetylated-SeCys 2 due to an observed baseline drift.Instead, LOQs for these compounds were determined in an equivalent way compared to DMSe and DMDSe, but using a series of low-concentrated standard samples (eq.S4).where  SD standard is the standard deviation of the signal of a series of low-concentrated standards  S cal is the average signal for the target concentration

Text S7: Calculation of second-order rate constants for the reactions of organic selenium compounds with HOBr
Results from competition kinetics were analyzed by eqs.S5 and S6. 25 Selenium species and resorcinol (for DPDSe: TMB) compete with each other for their reaction with HOBr (competition kinetics).Based on the fraction of resorcinol and the Se organic compound that reacted with HOBr, a slope is derived: []0 [] []0 where  [Se] 0 is the initial concentration of the organic Se compound (before reaction)  [Se] is the residual concentration of the organic Se compound (after reaction)  [Res] 0 is the initial concentration of resorcinol (before reaction)  [Res] is residual concentration of resorcinol (after reaction)  k app (HOBr+Se) is the apparent second-order rate constant for the reaction between HOBr and the organic Se compound  k app (HOBr+Res) is the apparent second-order rate constant for the reaction between HOBr and resorcinol Multiplication of this slope with the apparent second-order rate constant for the reaction between resorcinol and HOBr results in the apparent second-order rate constant (k app (Se+HOBr)) of the reaction between the organic Se compound and HOBr (eq.S7): k app (HOBr+Se) was calculated for pH 8 based on the slopes indicated in Tables S5 and S6, and equation S7.S5) and the slopes represent the average slopes from all experiments.Average slope values are slightly different compared to Table S5 because in this figure the data points are fitted to a linear regression, representing the slopes in equations S5 -S7 and not calculated as an average as in Table S5.          ) and HOBr for four molar ratios.SeCys 2 elutes at 0.913 min.The first oxidation product appears at a RT of 1.1 min (HOBr:SeCys 2 ratio of 1:1), which becomes predominant at a molar HOBr:SeCys 2 ratio of 3:1.At a molar HOBr:SeCys 2 ratio of 3:1, there is only a small residual concentration of SeCys 2 and 2 other minor oxidation products are visible, i.e., at RT = 1.36 min, which is potentially Se(+IV), and at RT = 1.9 min.At the HOBr:SeCys 2 = 10:1 ratio, the main oxidation product appears at RT = 1.9 min followed by the one at RT = 1.36 min.Furthermore, a third product appears at RT = 3.8 min (potentially Se[+VI]).S8).Calculated Br 2 concentrations (based on equation S9) account for only 0.14% of HOBr (Table S8), which could increase the observed reactivity in artificial seawater medium compared to the phosphatebuffered medium by 40% at most, considering an upper reactivity limit of ≈2  10 10 M -1 s -1 for secondorder reactions (diffusion limit).This cannot explain the 6.5-fold higher reactivity of DMSe in buffered artificial seawater medium than phosphate-buffered medium.S9) at pH 8.The decrease of the ln of the relative residual concentration of HOBr is illustrated for two time periods of the same experiment: 0-60 s (blue fit) and 0-360 s (orange fit).The experimental conditions are indicated in Table S9.0.0088 Slope (i.e., k' app, pH8 BOC2O+HOBr ) 0-360s [s -1 ] 0.0041 Second-order rate constant (k'' app, pH8 BOC2O+HOBr ) 0-60s [M -1 s -1 ] 10.7 Second-order rate constant (k'' app, pH8 BOC2O+HOBr ) 0-360s [M -1 s -1 ] 5.0

Text S11: Stoichiometry for reactions between diselenide compounds and HOBr
The stoichiometries of the HOBr-target compound reactions were determined by experiments using an understoichiometric molar concentration of HOBr relative to the target compound concentration.

Figure S1 :
Figure S1: Total chlorine (i.e.free available chlorine (FAC = [HOCl] + [OCl -]) and chloramines) as a function of added HOCl for (A) N-acetylated-SeMet (10.0 µM) and (B) N-acetylated-SeCys 2 (4.9 µM).FAC and total chlorine were quantified photometrically via oxidation of DPD at λ = 510 nm before and after KI addition, respectively.For Panel A, only values are shown after KI addition because of an insignificant difference to values before KI addition.

Figure S2 :
Figure S2:Apparent second-order rate constant of the resorcinol-HOBr reaction as a function of pH (based on data "k app " from TableS4)

Figure S4 :
Figure S4: Competition kinetics plots of the ln of the relative residual concentrations of target organic Se compounds and competitors from kinetic experiments with HOBr performed in phosphate-buffered medium at pH 8.The plots include all data points from all replicates (TableS5) and the slopes represent the average slopes from all experiments.Average slope values are slightly different compared to TableS5because in this figure the data points are fitted to a linear regression, representing the slopes in equations S5 -S7 and not calculated as an average as in TableS5.(A) DMSe, (B) DMDSe, (C) DPSe, (D) DPDSe, (E) N-acetylated-SeMet, (F) N-acetylated-SeCys 2 .Conditions: pH 8, competitor: resorcinol (Res) or TMB as indicated at the X-axes.Buffer media: [PO 4 ] tot = 20 mM for experiments with DMSe, DMDSe, DPSe and N-acetylated-SeMet [PO 4 ] tot = 10 mM for experiments with DPDSe and N-acetylated-SeCys 2

2 Figure S6 :
Figure S4: Competition kinetics plots of the ln of the relative residual concentrations of target organic Se compounds and competitors from kinetic experiments with HOBr performed in phosphate-buffered medium at pH 8.The plots include all data points from all replicates (TableS5) and the slopes represent the average slopes from all experiments.Average slope values are slightly different compared to TableS5because in this figure the data points are fitted to a linear regression, representing the slopes in equations S5 -S7 and not calculated as an average as in TableS5.(A) DMSe, (B) DMDSe, (C) DPSe, (D) DPDSe, (E) N-acetylated-SeMet, (F) N-acetylated-SeCys 2 .Conditions: pH 8, competitor: resorcinol (Res) or TMB as indicated at the X-axes.Buffer media: [PO 4 ] tot = 20 mM for experiments with DMSe, DMDSe, DPSe and N-acetylated-SeMet [PO 4 ] tot = 10 mM for experiments with DPDSe and N-acetylated-SeCys 2

Figure S10 :
Figure S10: HR-MS mass spectra (between m/z 200 and 280) for solutions after the reaction between DPSe and HOBr (two molar ratios) allowing for the identification of DPSeO as the Se-containing oxidation product (detected with Na + -m/z 272.9788-andH + -m/z 250.9970-adducts and mass accuracy between -0.4 and 0.1 ppm).The space between the stippled lines indicates the m/z area of the Se isotopic pattern (excluding 74 Se, which is of low abundance) for the identified compounds.Experimental conditions: pH 8, [NaHCO 3 ] = 1 mM, [DPSe] = 6.25 µM, [HOBr] = 0 -62.5 µM.

Figure S13 :
Figure S13: LC-ICP-MS/MS chromatograms obtained for solutions after the reaction between DMDSe and HOBr (four molar ratios), which (i) confirm the formation of MSeIA (identified using a MSeIA standard) observed with HR-MS at molar HOBr:DMDSe ratios of 1:1 and 3:1; and (ii) show the reaction of MSeIA with HOBr and the formation of another Se compound that could not be identified with HR-MS.Neither Se(IV) nor Se(VI) were detected under any tested reaction conditions.

Figure S21 :
Figure S21: Kinetics of the HOBr-BOC 2 O reaction (pseudo-first order conditions, see TableS9) at pH 8.The decrease of the ln of the relative residual concentration of HOBr is illustrated for two time periods of the same experiment: 0-60 s (blue fit) and 0-360 s (orange fit).The experimental conditions are indicated in TableS9.

Table S1 :
Suggested marine biotic pathways for Selenium and Se compounds

Table S5 :
Slopes of competition kinetics experiments (reaction of the organic Se compound with HOBr in competition with the reaction of the competitor with HOBr).The slopes represent the ratios of the 2 nd order rate constants (Se-HOBr vs competitor-HOBr reactivity).The slope value for DMSe,seaw (26.0±2.8) is far beyond 10 and therefore not ideal for an exact quantification of kDMSe+HOBr in seawater medium.We used resorcinol and not sulfite (with a higher rate constant) because of sulfite oxidation by the DMSe oxidation product (i.e.DMSeO).Still, the decrease of resorcinol was large enough to enable a precise quantification by HPLC/UV.The coefficient of determination (R 2 ) for the two replicates was 0.95 and 0.96 tot = 20 mM for experiments with DMSe, DMDSe, DPSe and N-acetylated-SeMet [PO 4 ] tot = 10 mM for experiments with DPDSe and N-acetylated-SeCys 2

Table S7 :
Concentrations of halides, HOBr and H + in DMSe-HOBr experiments performed in buffered artificial seawater.

Table S8 :
Concentrations and mole fractions of BrCl, Br 2 O and Br 2 for used HOBr concentrations in buffered artificial seawater medium according to Table S7 at pH 8.

Table S9 :
Experimental conditions of the HOBr-BOC 2 O kinetic experiment and the determined apparent second-order rate constants at pH 8.