The species-specific acid-base and multinuclear magnetic resonance properties of selenocysteamine, selenocysteine, and their homodiselenides

Journal Pre-proofs Research paper The species-specific acid-base and multinuclear magnetic resonance properties of selenocysteamine, selenocysteine, and their homodiselenides Tamás Pálla, Arash Mirzahosseini, Béla Noszál PII: DOI: Reference:

S0009-2614(19)31057-7 https://doi.org/10.1016/j.cplett.2019.137076 CPLETT 137076

To appear in:

Chemical Physics Letters

Received Date: Revised Date: Accepted Date:

15 October 2019 22 December 2019 29 December 2019

Please cite this article as: T. Pálla, A. Mirzahosseini, B. Noszál, The species-specific acid-base and multinuclear magnetic resonance properties of selenocysteamine, selenocysteine, and their homodiselenides, Chemical Physics Letters (2019), doi: https://doi.org/10.1016/j.cplett.2019.137076

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TITLE PAGE The species-specific acid-base and multinuclear magnetic resonance properties of selenocysteamine, selenocysteine, and their homodiselenides

AUTHORS Tamás Pálla, Arash Mirzahosseini, Béla Noszál* Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary Research Group of Drugs of Abuse and Doping Agents, Hungarian Academy of Sciences, Hungary

*to whom correspondence should be addressed: Prof. Béla Noszál Department of Pharmaceutical Chemistry, Semmelweis University H-1092 Budapest, Hőgyes E. u. 9, Hungary Phone/Fax: +3612170891 e-mail: [email protected]

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GRAPHICAL ABSTRACT

The rationale of the study is shown together with the completely deprotonated molecular structure of the compounds studied. The NMR data of the most basic form of selenocysteine is shown as an example.

ABSTRACT Selenol-compounds were studied by 1H,

13C,

and

77Se

NMR spectroscopy as a function of pH, and

their acid-base properties were analysed by case-tailored evaluation methods. Macroscopic and microscopic protonation constants were determined, and the concomitant interactivity parameters were calculated. The species- and site-specific protonation constants are interpreted by means of inductive and shielding effects, also, in comparison with their sulfur containing analogues. The species-specific chemical shifts of these compounds show close correlation with the corresponding selenolate protonation constants. This relationship is observed and described for selenolates for the first time, and allows the prediction of selenolate oxidizabilities in important biomolecules.

2

KEY WORDS selenol; diselenide; pKa; microspeciation; NMR-pH titration; 77Se NMR

3

1. INTRODUCTION The inter-related acid-base and redox properties of selenol, selenolate and diselenide functional groups are important in the biological chemistry of selenocysteine and related compounds and the actions of enzymes

containing

such

moieties.

In

this

work

two

selenol/diselenide

pairs,

selenocysteine/selenocystine and selenocysteamine/selenocystamine were studied. The acid-base properties of the four compounds have been characterized thus far [1-5] using pH-potentiometric titration, complemented with UV [1] and 1H NMR-pH titrations [2] to determine the macroscopic protonation constants. These data are compiled in Table 1. As of these reports, however, two macroconstants of selenocystine are still unknown. Also, site-specific protonation constants are published only for selenocysteamine [3]. In general, a more comprehensive data set, describing the acid-base properties of these compounds would have to include all species-specific microconstants that characterize not only the molecule as a whole, but also, the site of protonation directly.

Table 1. Literature protonation constants of the studied compounds.

selenocysteine

selenocystine

selenocysteamine

logK1

9.96[1]; 10.68[4]

9.50[2]

logK2

5.24[1]; 5.43[4]

8.32[2]

logK3

2.01[1]; 2.21[4]

no data

not applicable

not applicable

logK4

not applicable

no data

not applicable

not applicable

10.99[3]; 11.45[4]; 10.8[5]

selenocystamine 10.77[2]

5.31[2]; 5.01[3]; 5.50[4];

9.65[2]

5.0[5]

These data refer to aqueous media and 298 K. The ionic strength was 0.1 mol/L in references [1,3] and 0.3 mol/L in references [2,4]. The ionic strength was not reported in reference [5].

The aim of this work was to elucidate the complete microspeciation scheme (i.e. the set of all microspecies and related microscopic protonation constants) of these four biologically important

4

compounds in terms of site-specific protonation constants (microconstants), and to quantify the extent of interactions between the various sites in terms of interactivity parameters. We have also determined the

77Se, 13C,

and 1H NMR chemical shifts for every microspecies. As a result, we observed and

quantified a correlation between the species-specific (or intrinsic) chemical shifts and the microscopic protonation constant values.

2. MATERIALS AND METHODS 2.1 Materials The chemicals (including selenocystamine dihydrochloride and seleno-L-cystine) were purchased from Sigma-Aldrich (Merck). Selenocystamine dihydrochloride was also purchased from a second source: AKos GmbH, so we could reproduce the results of selenocysteamine with two independent samples (see Results and Discussion). All these substances were used without further purification. Selenocysteamine and selenocysteine were synthesized in situ for the titration experiments, using sodium borohydride and dithiothreitol as reducing agents in three-fold molar excess.

2.2 NMR spectroscopy measurements NMR spectra were recorded on a Varian Unity Inova DDR spectrometer (599.9 MHz for 1H) with a 5 mm inverse-detection gradient (IDPFG) probehead at 298.15±0.1 K. The solvent in every case was H2O:D2O, 95:5 (V/V) containing approximately 10 mmol/L analyte (0.15 mol/L ionic strength) along with sodium 4,4-dimethyl-4-silapentane-1-sulfonate as chemical shift reference compound and in situ pH indicator molecules. The pH values were determined in tube by internal indicator molecules (ca. 1 mmol/L) optimized for NMR [6-7]: acetone oxime (pH range <2.8 and 11.6-13.1); sarcosine (pH range 1.0-3.5 and 8.7-11.6); acetate (pH range 3.6-5.6); imidazole (pH range 5.5-8.9). The sample volume was 550 μL. Water suppressed 1H spectra were recorded with presaturation sequence (number of transients = 16, number of points = 65536, acquisition time = 3.33 s, relaxation delay = 1.5 s). The 13C

NMR parameters were as follows: pulse and acquire sequence using 12.7 μs observe pulse, at most

the number of transients was 65536, number of points = 65536, spectral width = 37878.8 Hz, acquisition time = 0.865 s, relaxation delay = 1 s. The 77Se NMR parameters were as follows: pulse

5

and acquire sequence using 7.25 μs observe pulse, at most the number of transients was 65536, number of points = 262144, spectral width = 131578.9 Hz, acquisition time = 0.996 s, relaxation delay = 1 s. The

13C

peaks were referenced to the methyl peak of sodium 4,4-dimethyl-4-silapentane-1-

sulfonate used as an internal reference. The

77Se

peaks were referenced to the peak of dimethyl

selenide (1% solution in CDCl3) used as an external reference.

2.3 Data analysis For the analysis of NMR titration curves of proton chemical shifts (αCH 1H NMR signal for selenocysteine and selenocystine, and both CH2

1H

NMR signals for selenocysteamine and

selenocystamine) versus pH, the software Origin Pro 8 (OriginLab Corp., Northampton, MA, USA) was used. In all regression analyses the non-linear curve fitting option was used with the following function [8]:

log𝛽i ― i × pH

n

(1) 𝛿obs(pH) =

𝛿L + ∑i = 1𝛿HiL × 10 n

log𝛽i ― i × pH

1 + ∑i = 110

where δL is the chemical shift of an unprotonated ligand (L), 𝛿H𝑖L values stand for the chemical shifts of successively protonated ligands, where n is the maximum number of protons that can bind to L, and βi is the cumulative protonation macroconstant, as exemplified in equation (2); log henceforth refers to the base 10 logarithm. The standard deviations of logβ (and logK) values from the regression analyses were used to calculate the Gaussian propagation of uncertainty to the other parameters derived in the Results chapter.

3. RESULTS Figure 1 depicts the protonation schemes of selenocysteamine and selenocystamine, Figure 2 depicts the protonation schemes of selenocysteine and selenocystine. In the microspeciation schemes the protonation microspecies are shown with a pictogram, along with a one-letter symbol. The microspecies are interconnected with microscopic protonation constants (denoted with k). The

6

superscript of k indicates the protonating group while the subscript (if any) shows the site(s) already protonated. The microconstants characterize the protonation step at a submolecular level and assign the binding proton to a specific basic moiety within the molecule. Above the microspeciation schemes the step-wise protonation steps are shown, where the ligand is shown with the overall number of protons bound to it; the macroscopic protonation species are interconnected by the conventional, macroscopic protonation constants.

Figure 1 The protonation macroequilibrium (horizontal top lines) and microequilibrium schemes of selenocysteamine (A) and selenocystamine (B); N, and Se labels denote the amino, and selenolate groups, respectively; the major protonation pathway is shown in bold

7

Figure 2 The protonation macro- (horizontal top lines) and microequilibrium schemes of selenocysteine (A) and selenocystine (B); for the sake of simplicity not every protonation microspecies is shown with a structural formula, rather a schematic structure representing the basic moieties where N, Se, and O labels denote the amino, selenolate, and carboxylate groups, respectively; the major protonation pathway is shown in bold

Some examples of macro- and microconstants of selenocysteine are:

[HL ― ]

(2) 𝐾1 = [L2 ― ][H + ] [b]

(3) 𝑘N = [a][H + ]

[H2L]

𝐾2 = [HL ― ][H + ] [e]

𝑘Se N = [b][H + ]

[H2L]

𝐾1𝐾2 = 𝛽2 = [L2 ― ][H + ]2 [h]

𝑘ONSe = [e][H + ]

Concentrations of the various macrospecies comprise the sum of the concentration of those microspecies that contain the same number of protons. For example:

(4) [HL ― ] = [b] + [c] + [d] (5) [H2L] = [e] + [f] + [g]

8

Several microconstants were calculated using macroconstant(s) and the pair-interactivity parameter between the basic sites. The interactivity parameter shows to what extent the protonation of site “B” on a polyprotic molecule reduces the basicity of site “A”, and vice versa, irrespective of the protonation state of the other protonation sites (denoted here with “X” in equation (6)).

(6) logΔ𝐸A/B = log𝑘𝐴 ―log𝑘AB = log𝑘AX ―log𝑘ABX = log𝑘𝐵 ―log𝑘BA = log𝑘BX ―log𝑘BAX The interactivity parameter is generally considered to be the most invariant quantity in analogous moieties and also in various protonation states of the neighboring moiety in the same molecule [9]. This valuable parameter can therefore be used to elucidate the selenocysteine protonation microspeciation scheme by importing the amino-carboxylate interactivity parameter from cysteine. By introducing some parameters and constraints, all the microconstants can be determined with equations analogous to equation (6). Some microconstants of the major protonation pathway are practically identical with a certain macroconstant, provided the protonation in question is overwhelmingly predominant over that of its protonation isomers. For example, kN of selenocysteine highly predominates over kSe and kO, therefore kN is practically identical with K1. Similarly, 𝑘ONSe of N O selenocysteine overwhelmingly predominates over 𝑘Se NO and 𝑘SeO, therefore 𝑘NSe is practically identical

with K3. The imported parameters for the microspeciation scheme of selenocysteine are:

(7) log𝑘N = log𝐾1 (8) log𝑘Se N = log𝐾2 (9) log𝑘ONSe = log𝐾3 (10) log𝑘NSe = log𝑘N(of selenocystine) (11) logΔ𝐸N/O = logΔ𝐸N/O (of cysteine) (12) logΔ𝐸Se/O = logΔ𝐸S/O (of cysteine)

9

In other words, equations (7)-(9) hold true since the microconstants of the minor protonation pathways have insignificant contribution to the values of the macroconstants compared to the microconstants of the major protonation pathways. Equation (10) is valid from analogy to the case of cysteine-cystine [10] as the amino basicity in the disulfide form closely resembles the amino basicity in the protonated thiol form of cysteine. Equation (11) is actually valid for every α-amino acid, based on the same argument as above for interactivity parameters. Equation (12) is likely to be a good approximation since the determined log∆EN/Se interactivity parameter in selenocysteine (1.29±0.03) is in good agreement to the log∆EN/S interactivity parameter in cysteine (1.31±0.01). Selenocystine has two properties that allow the calculation of certain microconstants from macroconstants: symmetry and orders of magnitude difference between the amino and carboxylate basicity. Some microscopic parameters that could be obtained due to symmetry [8] in selenocystine are shown in equations (13)-(16).:

(13) log𝑘N = log𝐾1 ― log2 (14) log𝑘NN′ = log𝐾2 + log2 (15) log𝑘ONN′ = log𝐾3 ― log2 (16) log𝑘ONN′ ′O = log𝐾4 + log2 Selenocystine dimethyl ester is a derivative that mimics the species with two -COOH groups at any pH. It allows the calculation of constants for two minor microspecies:

(17) log𝑘NOO′ = log𝐾1 (of selenocystine dimethyl ester) ― log2 (18) log𝑘NOO′ ′N = log𝐾2 (of selenocystine dimethyl ester) + log2 The amino-carboxylate interactivity parameter for the same side of the diselenide bridge can be imported from selenocysteine:

10

(19) logΔ𝐸N/O = logΔ𝐸N/O (of selenocysteine) Thus, equations (13)-(18) harness the molecular mirror-symmetry and the use of model compounds that are the closest mimics of certain minor microspecies [11]. Equation (19) utilises the transferability of interactivity parameters. Note that the log∆EN/N’ interactivity parameter of selenocystine which can be calculated from logkN-logkNN’=logK1-logK2-log4 is in good agreement with the value derived from the macroconstants of selenocystine dimethyl ester: logK1 (of selenocystine dimethyl ester)-logK2 (of selenocystine dimethyl ester)-log4=0.32

compared to 0.37±0.03.

The fundamental parameters for the microspeciation of selenocysteamine are:

(20) log𝑘N = log𝐾1 (21) log𝑘Se N = log𝐾2 (22) logΔ𝐸N/Se = logΔ𝐸N/Se (of selenocysteine) The macroconstants of selenocysteamine and selenocystamine were determined by measuring the 1H NMR-pH titration curves followed by non-linear regression analysis using equation (1). The microconstants of selenocystamine could be calculated using equations analogous to equations (13)(14) due to molecular symmetry. The macro- and microconstants along with the interactivity parameters are compiled in Tables 2 and 3. The relative abundance of selenocysteine microspecies as a function of pH are depicted in Figure 3 as an example.

Table 2 Protonation constants of selenocysteamine and selenocystamine Selenocysteamine

Selenocystamine

Macroscopic Protonation Constants logK1

10.87±0.03

logK1

9.62±0.04

logK2

6.26±0.02

logK2

8.48±0.12

Microscopic Protonation Constants logkN logkSe

10.87±0.03

logkN

9.32±0.04

7.55±0.03

logkNN'

8.78±0.12

11

logkNSe

6.26±0.02

logkSeN

9.58±0.04

logΔEN/Se

1.29±0.03

Interactivity Parameters logΔEN/N'

0.54±0.13

The macroscopic and microscopic protonation constants and interactivity parameters (298.15 K, 0.15 mol/L ionic strength) in log units ± uncertainty in standard deviation or random error. The parameters of the major protonation pathways are shown in bold. For selenocystamine only the non-identical microconstants are depicted.

Table 3 Protonation constants of selenocysteine, selenocystine, selenocystine dimethyl ester, and the related interactivity parameters; assignments of the macro- and microconstants are in Figure 2

Macroscopic Protonation Constants Selenocysteine

Selenocystine

logK1

10.18±0.02

logK1

9.19±0.02

logK2

5.52±0.03

logK2

8.22±0.02

logK3

2.01±0.04

logK3

2.25±0.05

logK4

1.58±0.07

Selenocystine dimethyl ester logK1 6.74±0.12 logK2

5.82±0.32

Microscopic Protonation Constants Selenocysteine

Selenocystine

logkN

10.18±0.02

logkSeO

3.90±0.04

logkN

logkSe

6.81±0.04

logkO

N

logkO

5.02±0.05

logkOSe

logkNSe logkNO logkSe

N

8.89±0.02

logkNN'O

1.95±0.05

8.29±0.02

logkO

4.40±0.13

logkNO'N'

6.63±0.04

5.69±0.05

logkNN'

8.52±0.04

logkNO'O

2.44±0.14

5.52±0.03

logkNSe

O

2.01±0.04

logkNO'

3.84±0.05

logkNON'

7.96±0.12

3.13±0.05

logkNOSe

4.40±0.06

logkNO

2.51±0.13

logkNOO'

3.77±0.07

8.89±0.02

logkSeO

7.00±0.04

logkO

N'

8.33±0.12

logkOO'N

6.44±0.12

logkON

7.00±0.02

logkNN'OO'

1.88±0.07

logkOO'

4.33±0.14

logkNOO'N'

6.07±0.12

logΔEN/O

1.89±0.01

logΔEN/N'

0.37±0.03

logΔEN/O'

0.56±0.12

logΔEO/O'

0.07±0.09

N

Interactivity Parameters logΔEN/Se

1.29±0.03

logΔEN/O

1.89±0.01

logΔESe/O

1.12±0.03

The macroscopic and microscopic protonation constants and interactivity parameters (298.15 K, 0.15 mol/L ionic strength) in log units ± uncertainty in standard deviation or random error. The parameters of the major protonation pathways are shown in bold. For selenocystine only the non-identical microconstants are depicted.

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Figure 3 The pH-dependent relative abundance of selenocysteine microspecies.

The 13C and 77Se NMR spectra of the compounds were also recorded at pH values that correspond to plateaus on the titration curves. These data afforded the species-specific chemical shift values of the major microspecies, as the contribution of minor protonation microspecies to the mole fractionweighted observed chemical shifts is insignificant. The species-specific chemical shifts of the minor protonation microspecies were determined using Sudmeier-Reilly type equations [12]. These data are compiled in Tables 4 and 5. The relationships used to determine the intrinsic chemical shifts (for any nucleus) of selenocysteamine macrospecies and microspecies are elaborated below as an epitome. The chemical shifts for every selenocystamine, selenocysteine, and selenocystine microspecies were determined using analogous relationships.

13

Equation (23) shows how the protonation shift observed on nucleus Z, caused by the protonation of the amino site can be quantified in ppm. Superscripts pH=13.15 and pH=8.6 indicate that plateaus on the chemical shift-pH profiles are at these pH values, and only the amino site protonates in this pH range. Equation (24) is the analogous one for protonation shifts initiated by the protonation of the selenolate site. Note that ΔδNZ is nucleus-dependent: its value is different and distinctive not only for all types of nuclei, but also, for every atom in the molecule. The same specificity is true for ΔδSeZ. Subscript Z indicates the nucleus- and atom-specificity.

= 13.15 = 8.6 ― 𝛿pH (23) ∆𝛿NZ = 𝛿pH Z Z

pH = 8.6 = 1.60 ― 𝛿pH (24) ∆𝛿Se Z = 𝛿Z Z

Equations (25)-(28) show how the microspecies-, nucleus-, and atom-specific chemical shifts can be obtained.

= 13.15 (25) 𝛿aZ = 𝛿pH Z

(26) 𝛿bZ = 𝛿aZ + ∆𝛿NZ (27) 𝛿cZ = 𝛿aZ + ∆𝛿Se Z (28) 𝛿dZ = 𝛿aZ + ∆𝛿NZ + ∆𝛿Se Z Table 4 Observed and species-specific chemical shifts of selenocysteamine and selenocystamine on the ppm scale 77Se

lit[2]

77Se

1H (αCH

2)

1H (βCH

2)

13C (αCH

2)

13C (βCH

2)

Observed chemical shifts Secysteamine

pH=13.15

no data

-233.09

2.691

2.444

48.61

18.31

pH=8.6

-245.6

-214.16

3.079

2.583

46.67

10.91

pH=1.60

-81.6

-48.07

3.417

3.168

45.05

15.99

Protonation shifts ∆δN ∆δSe Microspecies

164.0

18.93

0.388

0.139

-1.94

-7.40

166.09

0.338

0.585

-1.62

5.08

Species-specific chemical shifts

14

a

-233.09

2.691

2.444

48.61

18.31

b

-214.16

3.079

2.583

46.67

10.91

c

-67.00

3.029

3.029

46.99

23.39

d

-48.07

3.417

3.168

45.05

15.99

Observed chemical shifts Secystamine

pH=13.15

234.3

261.82

2.941

3.016

43.69

35.06

pH=0.85

251.3

278.22

3.434

3.187

42.83

26.75

Protonation shifts 16.40

0.493

0.171

-0.86

-8.31

∆δN

17.0

18.93

0.388

0.139

-1.94

-7.4

∆δN' Microspecies

-2.53

0.105

0.032

1.08

-0.91

a

261.82

2.941

3.016

43.69

35.06

280.75

3.329

3.155

41.75

27.66

259.29

3.046

3.048

44.77

34.15

278.22

3.434

3.187

42.83

26.75

∆δNN'

Species-specific chemical shifts

b and c d

See text for the definition of the ∆δ values. Having no carboxyl group in selenocysteamine and selenocystamine, the α and β notation of carbons is done relative to the amino group. The amino protonation shifts (∆δN) of selenocystamine are imported from the parameters of selenocysteamine.

Table 5 Observed and species-specific chemical shifts of selenocysteine and selenocystine on the ppm scale 77Se

77Se

1H (αCH)

1H (βCH

2a)

1H (βCH

2b)

13C (αCH)

13C (βCH

-266.8

-233.09

Observed chemical shifts 3.152 2.828 2.434

62.55

22.18

pH=7.9

-268.5

-229.54

3.667

2.935

2.719

60.46

15.85

pH=3.8

no data

-84.06

4.076

3.242

3.190

58.37

20.12

pH=0

no data

-72.73

4.449

3.604

3.462

55.98

18.61 -6.33

pH=13.15 Se-cysteine

lit[2]

2)

Protonation shifts 3.55

0.515

0.108

0.285

-2.09

∆δSe

1.7

145.48

0.409

0.307

0.470

-2.09

4.27

∆δO

11.33

0.373

0.362

0.272

-2.39

-1.51

∆δN

Microspecies

Se-cystine

Species-specific chemical shifts

a

-233.09

3.152

2.828

2.434

62.55

22.18

b

-229.54

3.667

2.935

2.719

60.46

15.85

c

-87.61

3.561

3.135

2.904

60.46

26.45

d

-221.76

3.525

3.189

2.706

60.16

20.67

e

-84.06

4.076

3.242

3.190

58.37

20.12

f

-218.21

4.040

3.297

2.991

58.07

14.34

g

-76.28

3.934

3.497

3.176

58.07

24.94

h

-72.73

4.449

3.604

3.462

55.98

18.61

281.40

Observed chemical shifts 3.463 2.904 2.851

58.40

32.80

pH=13.15

254.9

15

pH=5.2

266.4

286.60

4.038

3.176

3.142

57.46

31.42

pH=0

268.2

291.92

4.440

3.289

3.217

55.00

29.41

Protonation shifts ∆δNN'

11.5

5.20

0.575

0.273

0.292

-0.94

-1.38

∆δOO'

1.8

5.32

0.402

0.113

0.074

-2.46

-2.01

∆δN

1.25

0.515

0.108

0.285

-2.09

-6.33

∆δN'

3.95

0.060

0.165

0.007

1.15

4.95

∆δO

10.14

0.373

0.362

0.272

-2.39

-1.51

∆δO'

-4.82

0.029

-0.249

-0.198

-0.07

-0.50

Microspecies a b and c d and e f g and j h and i k l and m n and o p

Species-specific chemical shifts 281.40

3.463

2.904

2.851

58.40

32.80

282.65

3.978

3.011

3.136

56.31

26.47

285.35

3.523

3.069

2.857

59.55

37.75

291.54

3.836

3.265

3.123

56.01

31.29

276.58

3.492

2.654

2.653

58.33

32.30

286.60

4.038

3.176

3.142

57.46

31.42

292.79

4.351

3.373

3.408

53.92

24.96

280.53

3.552

2.820

2.659

59.48

37.25

277.83

4.007

2.762

2.938

56.24

25.97

295.49

3.896

3.431

3.129

57.16

36.24

286.72

3.865

3.016

2.925

55.94

30.79

296.74

4.411

3.538

3.414

55.07

29.91

281.78

4.067

2.927

2.945

57.39

30.92

287.97

4.380

3.124

3.210

53.85

24.46

290.67

3.925

3.181

2.931

57.09

35.74

291.92

4.440

3.289

3.217

55.00

29.41

The amino and carboxylate protonation shifts

(∆δN

and

∆δO)

of selenocystine are imported from the

parameters of selenocysteine.

4. DISCUSSION Overall, the determined protonation constants in our work are in general agreement with those few that appeared in the literature (compiled in Table 1), except for the ones of selenocysteamine and selenocystamine. Our logK2 macroconstant of selenocysteamine differs by at least 1 log unit compared to the literature values (6.26 vs 5.0-5.5). Therefore, we have repeated this measurement numerous times making sure of the purity of the sample and comparing two different commercial sources of the substance. Our results confirm that the logK2 reported in Table 2 can be reproduced with 1H NMR-pH titrations reliably. Our selenocystamine macroconstants are also consistently lower than the literature values; therefore, we have repeated this measurement several times as well. Since our reported values for the selenocystamine protonation constants are consistent with the selenocysteamine protonation

16

constants (explanation below), we are confident that the data in Table 2 are accurate. In the literature the only microconstants are for selenocysteamine (using the Se-methyl model compound: logkN=10.99, logkSe=6.47, logkSeN=9.53, logkNSe=5.01), and the therein reported interactivity parameter is in agreement with ours (1.29 vs 1.46). Comparisons can be made between the protonation constants in Tables 2-3 and those of the thiolcontaing analogues [10], which reveal good agreement for the inherent amino and carboxylate basicities. It is noteworthy that the amino-selenolate interactivity parameter determined for selenocysteine is within the margin of error compared to the amino-thiolate interactivity parameter of cysteine (1.31). The species-specific protonation constants of the studied compounds also show that the kN of selenocystamine (9.32) and the kN and kO of selenocystine (8.89 and 4.40) are also within margin of error compared to 𝑘NSe and 𝑘OSe of selenocysteamine (9.58) and selenocysteine (8.89 and 3.90), i.e. the inductive effects of the diselenide moiety resembles the protonated selenolate, similarly to the case for disulfide and the protonated thiolate. It should be noted that the 𝑘NSe of selenocysteine was equated with the kN of selenocystine for our evaluation purposes considering this evidence, as described in the Results section. Our data also verify that the inherent selenolate basicity is 2.1-3.3 log units below that of the thiolate [15]: selenocysteamine (logkSe=7.55), selenocysteine (logkSe=6.81) vs cysteamine (logkS=9.67), cysteine (logkS=10.07). The 1H and

13C

chemical shift parameters (Tables 4-5) are congruent with the anticipated values,

taking into account the inductive effects in the various protonation microspecies. Our 77Se chemical shifts, however, exceed their literature counterparts [2] by a typical 30 ppm (as shown in Table 4 and 5), while the

77Se

protonation shifts (Δδ) are essentially identical with those in the literature. The

apparent disagreement between the 77Se chemical shifts lies in the non-standardised reference values, which used to be the case of 77Se NMR spectroscopy in its infancy. Our future projects will attempt to address this issue. The selenolate and amino protonation shifts present in selenocysteamine (18.93 and 166.09 for 77Se) and selenocysteine (3.55 and 145.48 for 77Se) are in good agreement with each other. It is noteworthy that the selenolate protonation shift is ‘wrong way’ on the Cβ, both

in

selenocysteamine and selenocysteine. While this effect is consistent and accurate, other ‘wrong way’ protonation shifts observed in the diselenides may be consequences of the inaccuracy of equating

17

amino and carboxylate protonation shifts of selenocysteamine and selenocysteine to those of selenocystamine and selenocystine. The species-specific chemical shifts of the various microspecies show linear correlation with the corresponding selenolate protonation microconstants with various degrees of correlation as presented in Figure 4 and Table 6. This is indicative of the well-established observation that electron density contributions are the main forces that influence acid-base, redox and chemical shift parameters [10,13-14].

Figure 4 Correlation of selenocysteine selenolate microconstants with the corresponding selenocysteine microspecies chemical shifts (left) and selenocystine microspecies chemical shifts (right); the chemical shift values are a result of three repeated measures

Table 6 Correlation data of selenolate microconstants with the corresponding selenocysteine microspecies chemical shifts (A) and the corresponding selenocystine microspecies chemical shifts (B) A 77Se

1H (αCH)

1H (βCH

2a)

1H (βCH

2b)

13C (αCH)

13C (βCH

2)

18

intercept

-192,5

5,7

4,1

4,0

50,0

-0,7

slope

-5,92

-0,37

-0,19

-0,23

1,84

3,38

-0,9961

-0,8436

-0,9989

0,9906

0,8866

0,0039

0,1564

0,0011

0,0094

0,1134

Pearson r -0,8514 p-value

0,1486

B 77Se

1H (αCH)

intercept

311,0

6,2

4,0

3,9

49,1

23,4

slope

-4,34

-0,41

-0,16

-0,16

1,36

1,38

-0,9945

-0,9481

-0,8939

0,8819

0,9679

0,0055

0,0519

0,1061

0,1181

0,0321

Pearson r -0,9967 p-value

0,0033

1H (βCH

2a)

1H (βCH

2b)

13C (αCH)

13C (βCH

2)

It is apparent from the correlation data in Figure 4 that the goodness of fit is limited by the small number of points that could be involved in the correlation analysis. Additional information from selenocysteine containing peptides could better corroborate the linear relationship between acid-base parameters and chemical shift values. While the value of correlation coefficient may be lower in some cases (e.g. 77Se chemical shifts of selenocysteine), it is the slope of correlation that characterizes the predictive power of chemical shifts in relation to the acid-base character of the selenolate (i.e. the 77Se nucleus chemical shifts are the most sensitive to changes in the selenolate basicity with the largest slope of -5.92). It is remarkable that the

77Se

chemical shifts of the diselenide microspecies show

excellent correlation with the basicity of the constituent selenolate. These correlations are highly promising data to estimate otherwise unavailable parameters. Namely, the chemical shift of the 77Se nucleus in selenocysteine-containing peptides can be a tool to predict the acid-base character of the corresponding selenolate, or the redox properties of the selenolate-diselenide system. The site-specific protonation constants and chemical shifts determined for selenocysteamine, selenocystamine, selenocysteine, and selenocystine constitute a significant dataset to interpret the behaviour of these biologically important compounds in their biochemical and analytical reactions. Since selenolate basicities are related to their redox and chelating properties, the correlation between the acid-base and NMR parameters provide sound means at the molecular level to predict

19

oxidizabilities, a key parameter to understand and influence oxidative stress-related biochemical reactions.

ACKNOWLEDGEMENT This research was supported by the FIKP-2018 New National Excellence Program of the Ministry of Human Capacities of Hungary.

CONFLICT OF INTEREST The authors report no conflict of interest.

REFERENCES 1. Huber, R. and R. Criddle, Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs. Archives of biochemistry and biophysics, 1967. 122(1): p. 164-173. 2. Tan, K.-S., A.P. Arnold, and D.L. Rabenstein, Selenium-77 nuclear magnetic resonance studies of selenols, diselenides, and selenenyl sulfides. Canadian journal of chemistry, 1988. 66(1): p. 54-60. 3. Tanaka, H., H. Sakurai, and A. Yokoyama, Acid Dissociation of Selenocysteamine (2Aminoethaneselenol). Chemical and Pharmaceutical Bulletin, 1970. 18(5): p. 1015-1020. 4. Arnold, A.P., K.S. Tan, and D.L. Rabenstein, Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 23. Complexation of methylmercury by selenohydrylcontaining amino acids and related molecules. Inorganic Chemistry, 1986. 25(14): p. 24332437. 5. Sugiura, Y., Y. Tamai, and H. Tanaka, Selenium protection against mercury toxicity: high binding affinity of methylmercury by selenium-containing ligands in comparison with sulfurcontaining ligands. Bioinorganic chemistry, 1978. 9(2): p. 167-180. 6. Szakács Z, Hägele G, Tyka R. 1H/31P NMR pH indicator series to eliminate the glass electrode in NMR spectroscopic pKa determinations. Anal Chim Acta, 2004; 522:247–258.

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7. Orgován G, Noszál B. Electrodeless, accurate pH determination in highly basic media using a new set of 1H NMR pH indicators. J Pharm Biomed Anal, 2011; 54:958-964. 8. Szakács Z, Noszál B. Protonation microequilibrium treatment of polybasic compounds with any possible symmetry. J Math Chem, 1999; 26:139–155. 9. M.A. Santos, M.A. Esteves, M.C. Vaz, J.J.R.F. Frausto da Silva, B. Noszál, E. Farkas, Microscopic acid–base equilibria of a synthetic hydroxamate siderophore analog, piperazine1,4-bis(N-methylacetohydroxamic acid), J. Chem. Soc. Perkin Trans. 2 10 (1997) 1977–1983. 10. Mirzahosseini A, Noszál B. The species- and site-specific acid-base properties of biological thiols and their homodisulfides. J Pharm Biomed Anal, 2014; 95C:184-192. 11. Ebert L. Zur Abschätzung der Zwitterionenmenge in Ampholytlösungen. Zeitschrift für Physikalische Chemie, 1926; 121:385-400. 12. Sudmeier JL, Reilly CN (1964) Nuclear magnetic resonance studies of protonation of polyamine and aminocarboxylate compounds in aqueous solution. Anal Chem 36:1698–1706. 13. Szajewski RP, Whitesides GM. Rate constants and equilibrium constants for thiol-disulfide interchange reactions involving oxidized glutathione. J Am Chem Soc, 1980; 102:2011-2026. 14. Keire DA, Strauss E, Guo W, Noszál B, Rabenstein DL. Kinetics and equilibria of thiol/disulfide interchange reactions of selected biological thiols and related molecules with oxidized glutathione. J Org Chem, 1992; 57:123-127. 15. Pleasants JC, Guo W, Rabenstein DL. A comparative study of the kinetics of selenol/diselenide and thiol/disulfide exchange reactions. J Am Chem Soc, 1989, 111:65536558.

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TITLE PAGE The species-specific acid-base and multinuclear magnetic resonance properties of selenocysteamine, selenocysteine, and their homodiselenides

AUTHORS Tamás Pálla, Arash Mirzahosseini, Béla Noszál* Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary Research Group of Drugs of Abuse and Doping Agents, Hungarian Academy of Sciences, Hungary

*to whom correspondence should be addressed: Prof. Béla Noszál Department of Pharmaceutical Chemistry, Semmelweis University H-1092 Budapest, Hőgyes E. u. 9, Hungary Phone/Fax: +3612170891 e-mail: [email protected]

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HIGHLIGHTS



Selenol-compounds were studied by 1H, 13C, and 77Se NMR spectroscopy



Species- and site-specific protonation constants were determined



Chemical shifts show close correlation with the selenolate protonation constants

23

Ga

24

25

Tamás Pálla: Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization

Arash Mirzahosseini: Conceptualization, Methodology, Validation, Formal analysis, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Supervision, Project administration, Funding acquisition

Béla Noszál: Conceptualization, Methodology, Resources, Writing - Review & Editing, ASupervision, Project administration, Funding acquisition

26