The species- and site-specific acid-base properties of penicillamine and its homodisulfide

Article abstract of DOI:10.1016/j.cplett.2014.07.026

Penicillamine, penicillamine disulfide and 4 related compounds were studied by ¹H NMR-pH titrations and case-tailored evaluation methods. The resulting acid-base properties are quantified in terms of 14 macroscopic and 28 microscopic protonation constants and the concomitant 7 interactivity parameters. The species- and site-specific basicities are interpreted by means of inductive and shielding effects through various intra- and intermolecular comparisons. The thiolate basicities determined this way are key parameters and exclusive means for the prediction of thiolate oxidizabilities and chelate forming properties in order to understand and influence chelation therapy and oxidative stress at the molecular level.

Full text of DOI:10.1016/j.cplett.2014.07.026

Chemical Physics Letters 610–611 (2014) 62–69
Contents lists available at ScienceDirect
Chemical Physics Letters
journal homepage: www.elsevier.com/locate/cplett
The species- and site-specific acid–base properties of penicillamine
and its homodisulfide
Arash Mirzahosseinia,b, András Szilvaya,b, Béla Noszála,b,∗
a
Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary
Research Group of Drugs of Abuse and Doping Agents, Hungarian Academy of Sciences, Hungary
b
a r t i c l e i n f o
a b s t r a c t
Penicillamine, penicillamine disulfide and 4 related compounds were studied by 1H NMR-pH titrations
and case-tailored evaluation methods. The resulting acid–base properties are quantified in terms of 14
macroscopic and 28 microscopic protonation constants and the concomitant 7 interactivity parame-
ters. The species- and site-specific basicities are interpreted by means of inductive and shielding effects
through various intra- and intermolecular comparisons. The thiolate basicities determined this way are
key parameters and exclusive means for the prediction of thiolate oxidizabilities and chelate forming
properties in order to understand and influence chelation therapy and oxidative stress at the molecular
level.
Article history:
Received 18 June 2014
In final form 10 July 2014
Available online 17 July 2014
©
2014 Elsevier B.V. All rights reserved.
1
. Introduction
2S)-2-Amino-3-methyl-3-sulfanylbutanoic acid, or penicil-
the thiolate oxidizability in the particular protonation stage of the
neighboring groups [9]. Penicillamine primarily metabolizes via its
disulfide form, and the pharmacological and therapeutic actions
are largely explained by its ability to take part in thiol–disulfide
exchange reactions. For example the formation of the mixed disul-
fide from penicillamine and cysteine is decisive for the treatment
of cystinuria as penicillamine disulfide and penicillamine-cysteine
disulfide are much more soluble than cystine and are thus elimi-
nated [2,10].
Although the acid–base properties of penicillamine have been
reported in a few papers [11–15], no data appeared on the
site-specific protonation constants of d-penicillamine and d-
penicillamine disulfide. Here we report the determination of all
the site-specific basicities for d-penicillamine, and its homod-
isulfide using 1H NMR-pH titrations on the parent molecules
and 4 synthesized auxiliary compounds. This is the first com-
plete microspeciation of these molecules, providing sine qua
non constituents for a comprehensive species- and site-specific
characterization of the complex, codependent acid–base and
thiol–disulfide equilibrium system, that is of crucial importance in
maintaining the intracellular redox homeostasis.
(
lamine (also known as 3-mercapto-d-valine, 3,3-dimethyl-d-
cysteine) is a nonproteinogenic amino acid containing a thiol group.
The three polar functional groups in penicillamine undergo charac-
teristic chemical reactions and differ in their ability to participate
in various chemical and biochemical reactions [1]. Penicillamine
was at first solely of interest as a key substance in the struc-
tural elucidation of penicillins and as a central building block in
their total synthesis [2]. Notwithstanding that its l-isomer is toxic;
d-penicillamine is widely used in medicine to treat Wilson’s dis-
ease [3], heavy metal poisoning [4], cystinuria [5] and rheumatoid
arthritis [6]. The thiol group as a typical weak acid occurs mainly
in neutral form in physiologic conditions, while the ammonium
and carboxylate groups deliver a zwitterionic structure. However,
it is the thiolate form that is directly engaged in thiol–disulfide
redox equilibria [7]. It has also been reported that thiolate basici-
ties are in correlation with the half-cell redox potentials [8]. Since
most biomolecules bear multiple protonation sites which proto-
nate in an overlapping fashion, site-specific characterization of
acid–base equilibria helps untangle the interactions between the
basic moieties. Such thiolate basicities are indirect indicators of
2. Materials and methods
2.1. Materials
∗ Corresponding author at: Department of Pharmaceutical Chemistry, Semmel-
weis University, H-1092 Budapest, H o˝ gyes E. u. 9, Hungary.
All chemicals (including d-penicillamine) were purchased from
Sigma, and were used without further purification.
E-mail address: noszal.bela@pharma.semmelweis-univ.hu (B. Noszál).
http://dx.doi.org/10.1016/j.cplett.2014.07.026
0
009-2614/© 2014 Elsevier B.V. All rights reserved.

A. Mirzahosseini et al. / Chemical Physics Letters 610–611 (2014) 62–69
63
(0.11 g, 99%). Mp: 253–260 C. 1H NMR (600 MHz, H O:D O, 95:5,
◦
2 2
2.2. NMR spectroscopy measurements
v/v) ı (ppm) 1.31 (3H, s, ␤CH ), 1.52 (3H, s, ␤CH ), 2.07 (3H, s, SCH ),
3
3
3
+
NMR spectra were recorded on a Varian 600 MHz spectrome-
3.67 (1H, s, ␣H); HRMS m/z [M+H] Calc 164.0745 Found 164.0687.
S-Methyl d-penicillamine methyl ester hydrochloride (4) was syn-
thesized in a similar fashion to compound 2 from 0.05 g (0.31 mmol)
◦
ter at 25 C. The solvent in every case was an aqueous solution
with H O:D O, 95:5, v/v (0.15 mol/l ionic strength), using DSS as
2
2
S-methyl d-penicillamine (3) to yield 0.06 g (98%) yellow oil. 1
H
the reference compound. The sample volume was 600 ␮l. In proton
NMR experiments pH values were determined by internal indica-
tor molecules optimized for NMR [16,17], and the water resonance
was diminished by double pulsed field gradient spin echo (nt = 16,
np = 64 000, acquisition time = 3.33 s, relaxation delay = 1.5 s).
NMR (600 MHz, H O:D O, 95:5, v/v) ı (ppm) 1.48 (3H, s, ␤CH ),
2
2
3
1.56 (3H, s, ␤CH ), 1.92 (3H, s, SCH ), 3.71 (3H, s, OCH ), 3.88 (1H,
3
3
3
+
s, ␣H); HRMS m/z [M+H] Calc 178.0902 Found 178.0930.
d-Penicillamine disulfide (5) was synthesized in situ by dissolv-
ing 0.10 g (0.67 mmol) d-penicillamine (1) into a 5% H O2 aqueous
2
2
.3. Data analysis
solution and adjusting the pH to 8. After stirring overnight the aque-
ous solution was used directly for NMR measurements. 1H NMR
(600 MHz, H2O:D2O, 95:5, v/v) ı (ppm) 1.44 (3H, s, ␤CH3), 1.55 (3H,
s, ␤CH3), 4.01 (1H, s, ␣H); HRMS m/z [M+H]+ Calc 297.0943 Found
297.1017.
For the analysis of NMR titration curves of proton chemical shifts
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 [18]:
ꢀ
d-Penicillamine disulfide dimethyl ester (6) was synthesized in
a similar fashion to compound 5 from 0.05 g (0.25 mmol) d-
penicillamine methyl ester hydrochloride (2). 1H NMR (600 MHz,
H O:D O, 95:5, v/v) ı (ppm) 1.50 (3H, s, ␤CH ), 1.57 (3H, s, ␤CH ),
n
log ˇ −i×pH
i
ıL +
ıH L × 10
i=1
i
ıobs(pH) =
ꢀ
(1)
n
log ˇ −i×pH
i
1
+
10
2
2
3
3
i=1
+
3
.66 (3H, s, OCH ), 4.19 (1H, s, ␣H); HRMS m/z [M+H] Calc 325.1256
3
where ıL is the chemical shift of the unprotonated ligand (L), ıH L
i
Found 325.1296.
values stand for the chemical shifts of successively protonated lig-
ands, where n is the maximum number of protons that can bind
to L, and ˇ is the cumulative protonation macroconstant, as exem-
plified in Eq. (1). The standard deviations of log ␤ values from the
regression analyses were used to calculate the Gaussian propaga-
tion of uncertainty to the other equilibrium constants derived in
Section 3.
3
. Results
Figure 1 shows the formulae of the molecules studied. Figure 2
represents the microscopic protonation schemes of penicillamine
A) and penicillamine disulfide (B). Macroequilibria (top lines) indi-
(
cate the stoichiometry of the successively protonated ligand and
the stepwise macroscopic protonation constants. In the microspe-
ciation schemes the 8 and 16 microspecies with their one-letter
symbols (a, b, . . ., h for penicillamine, and a, b, . . ., p [in italics] for
penicillamine disulfide), and the 12 and 32 microscopic protonation
2.4. TOF MS measurements
The exact mass of the synthesized and isolated compounds
was determined with an Agilent 6230 time-of-flight mass spec-
trometer equipped with a JetStream electrospray ion source in
constants are depicted (k , kO , kONS, . . .), for sake of consistency
penicillamine disulfide protonation constants are depicted in ital-
ics. The superscript of k indicates the protonating group while the
subscript (if any) shows the site(s) already protonated. N, S and O
symbolize the amino, thiolate and carboxylate sites, respectively.
Some examples of macro- and microconstants of penicillamine are:
O
N
positive ion mode. JetStream parameters: drying gas (N ) flow and
2
◦
temperature: 10.0 l/min and 325 C; nebulizer gas (N ) pressure:
2
1
0 psi; capillary voltage: 4000 V; sheath gas flow and tempera-
◦
ture: 325 C and 7.5 l/min. TOF MS parameters: fragmentor voltage:
1
70 V; skimmer potential: 170 V; OCT 1 RF Vpp: 750 V. Samples
were introduced (0.1–0.3 ␮l) by the Agilent 1260 Infinity HPLC
system (flow rate = 0.5 ml/min, 70% methanol–water mixture 0.1%
formic acid). Reference masses of m/z 121.050873 and 922.009798
were used to calibrate the mass axis during analysis. Mass spec-
tra were acquired over the m/z range 100–1000 at an acquisition
rate of 250 ms/spectrum and processed using Agilent MassHunter
B.02.00 software.
−
[
HL ]
[H L]
[H L]
2
2
K1 =
K2 =
K1K2 = ˇ2 =
(2)
(3)
−
+
−
+
2− +
2
[
[
L2 ][H ]
[HL ][H ]
[L ][H ]
[
d]
[f]
[h]
kO
=
k
N
=
k
S
ON
=
[f][H ]
+
O
+
+
a][H ]
[d][H ]
Concentrations of the various macrospecies comprise the sum
of the concentration of those microspecies that contain the same
number of protons. For example:
2.5. Synthetic protocols
−
d-Penicillamine methyl ester hydrochloride (2) was synthe-
[HL ] = [b] + [c] + [d]
(4)
(5)
sized by dissolving 0.10 g (0.67 mmol) d-penicillamine (1) in 5 ml
methanol and bubbling dry HCl gas into the solution for 15 min at
room temperature [19]. After stirring overnight at room tempera-
ture the reaction mixture was evaporated in vacuo to yield a white
[
H L] = [e] + [f] + [g]
2
The following equations show the relationships between the
micro- and macroconstants of penicillamine [21]:
◦
1
solid (0.13 g, 98%). Mp: 187–188 C; H NMR (600 MHz, H O:D O,
2
2
9
5:5, v/v) ı (ppm) 1.42 (3H, s, ␤CH ), 1.45 (3H, s, ␤CH ), 3.68 (1H,
N
S
O
3
3
K1 = k + k + k
(6)
(7)
(8)
+
s, ␣H), 3.79 (3H, s, OCH ); HRMS m/z [M+H] Calc 164.0745 Found
3
N
S
N
O
N
S
O
S
N
O
N
O S
O
1
64.0724.
K K = k k + k k + k kS = k kS + k kO + k k = . . .
1
2
N
N
S-Methyl d-penicillamine (3) was synthesized from 0.10 g
S
N
O
SN
S N O
S
(
0.67 mmol) d-penicillamine (1) using 54 ␮l (1.3 equiv.) methyl
K K K = k k k = k k k = . . .
1
2
3
SN
iodide and 0.02 g (1.3 equiv.) sodium hydride in 5 ml methanol
under N atmosphere [20]. After stirring overnight at room temper-
Eqs. (7) and (8) can be written in 2 and 6 different, equiva-
2
lent ways depending on the path of protonation. To characterize
all of the microscopic bacisities, the introduction and utilization of
auxiliary compounds are necessary.
ature the reaction mixture was evaporated in vacuo. The residual
oil was purified by column chromatography on silica gel (ethyl
acetate–hexane, 1:5, v/v) to afford compound 3 as a white solid

6
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A. Mirzahosseini et al. / Chemical Physics Letters 610–611 (2014) 62–69
Figure 1. The formulae of the compounds studied.
Table 1
Table of protonation constants.
Penicillamine
Penicillamine disulfide
Penicillamine
Penicillamine methyl ester
Penicillamine disulfide
Penicillaminedisulfidedimethylester
Macroscopic protonation constants
log K1
log K2
log K3
10.80 ± 0.01
8.07 ± 0.01
2.04 ± 0.01
log K1
log K2
8.99 ± 0.01
6.90 ± 0.01
log K1
log K2
log K3
log K4
9.48 ± 0.03
log K1
log K2
7.23 ± 0.02
6.32 ± 0.01
8.57 ± 0.03
2.34 ± 0.04
1.61 ± 0.04
S-methyl penicillamine
log K1
log K2
S-Methylpenicillaminemethylester
log K
9.53 ± 0.01
7.65 ± 0.01
2.04 ± 0.01
Microscopic Protonation Constants
N
log kSO
N
O
log k
log k
log k
10.78 ± 0.01
9.34 ± 0.01
5.02 ± 0.01
8.09 ± 0.01
3.14 ± 0.01
9.53 ± 0.01
3.92 ± 0.01
8.90 ± 0.01
8.24 ± 0.01
2.04 ± 0.01
6.99 ± 0.01
7.65 ± 0.01
log k
9.18 ± 0.03
4.29 ± 0.05
8.87 ± 0.03
3.92 ± 0.04
2.41 ± 0.05
8.81 ± 0.02
7.30 ± 0.05
4.16 ± 0.07
log k
2.04 ± 0.04
6.99 ± 0.03
2.28 ± 0.07
8.50 ± 0.01
3.79 ± 0.04
6.93 ± 0.02
1.91 ± 0.04
6.62 ± 0.01
NNꢀ
S
N
O
Nꢀ
log kO
log k
log k
log k
log k
log k
log k
log k
log k
Oꢀ N
O
log kOS
log kSN
Nꢀ
O
log k
log k
N
Oꢀ N
log kNS
O
S
Oꢀ
Nꢀ
N
ON
O
O
Oꢀ
log kN
log kON
log kN
ON
N
log kSN
log kOSN
log k
Nꢀ
O
OOꢀ
N
Oꢀ
log kO
ONNꢀ
Nꢀ
Oꢀ
log k
O
OOꢀ N
Interactivity parameters
log ꢀEN/S
log ꢀEN/O
1.25 ± 0.01
log ꢀES/O
1.10 ± 0.01
log ꢀEN/O
log ꢀEN/Oꢀ
1.88 ± 0.01
0.37 ± 0.04
log ꢀEN/Nꢀ
log ꢀEO/Oꢀ
0.31 ± 0.04
0.13 ± 0.06
1.88 ± 0.01

A. Mirzahosseini et al. / Chemical Physics Letters 610–611 (2014) 62–69
65
Figure 2. The protonation macro- and microequilibrium schemes of penicillamine (A) and penicillamine disulfide (B). N, S, O labels denote the amino, thiolate, and carboxylate
groups, respectively.
3
.1. The microscopic protonation constants of d-penicillamine
of the thiolate is “comparably lower” while basicity of the carbox-
ylate is “orders of magnitude lower” than that of the amino group.
Therefore, those microspecies, in which the carboxylate is proton-
ated but the amino is not protonated are “orders of magnitude
minor” ones. Similarly, those microspecies in which the thiolate is
protonated but the amino is not are the “relative minor” ones. The
“orders of magnitude minor” substances cannot be detected with
analytical methods like NMR or UV, but they may have an impor-
tant role in highly specific biochemical processes [22]. To elucidate
the complete microspeciation, auxiliary compounds that mimic the
d-Penicillamine was titrated (titration curve in Fig. 3) using 1
H
NMR under near physiological conditions to afford its macroscopic
protonation constants (Table 1). For the calculation of the 12 pro-
tonation microconstants through a multivariable equation system,
pieces of additional information are necessary. The quality and
quantity of the additional information depend on the acid–base
properties of the substance. It is chemically evident that basicities of
the sites decrease in the amino, thiolate, carboxylate order. Basicity

6
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A. Mirzahosseini et al. / Chemical Physics Letters 610–611 (2014) 62–69
Figure 3. The plot of 1H chemical shifts versus pH; (ꢀ) ␣ proton, (•) CH3 protons, (ꢁ) O-methyl protons, (ꢁ) S-methyl protons. (A) Penicillamine; (B) penicillamine methyl
ester; (C) S-methyl penicillamine; (D) S-methyl penicillamine methyl ester.
order of magnitude minor or relative minor substances, have to be
used. In this case, model compounds of the c, d and g microspecies
were synthesized: S-methyl penicillamine, penicillamine methyl
ester and S-methyl penicillamine methyl ester. The site in which
the methyl group is connected remains in neutral state, with prac-
tically identical electronic effects on the rest of the molecule as
COOH and SH groups [23].
N
S
O
S-methyl penicillamine
k
+ kS = K1
(13)
(14)
(15)
(16)
N
O
S
O
penicillamine methyl ester
k
+ k = K1
log k SN + log kO = log ˇ 2S -methyl penicillamine
SN
log k ON + log k = log ˇ2
S
penicillamine methyl ester
ON
The remaining microconstants (log k , log kNS, and log k O
N
)
The macroconstants of the auxiliary compounds (Table 1) were
N
1
also attained by H NMR titrations (titration curves in Fig. 3). The
were calculated using the amino/thiolate interactivity parameter
that could be obtained from the relationships in Eq. (8) and micro-
constants involved:
microscopic protonation constants of penicillamine were calcu-
lated using equations similar to (6) and (8) bearing in mind that
the macroscopic protonation constants of the model compounds
are components of the penicillamine microspeciation scheme.
For example the protonation constant of S-methyl penicillamine
methyl ester amino group is equivalent to kOSN. The equations uti-
lized to calculate the microscopic constants are as follows:
N
S-methyl penicillamine methyl ester
log ꢀEN/S = log kO − log K
log kN − log kSN
=
(17)
log ˇ 3p enicillamine = log k + log kN + log k
N
S
O
SN
log kS = log ˇ 3p enicillamine − log ˇ 2S -methyl penicillamine
(9)
=
log kN + log k NO + log kS
(18)
log kO = log ˇ 3p enicillamine − log ˇ 2p enicillamine methyl ester
ON
(10)
log k SO = log ˇ 2S -methyl penicillamine − log KS-methyl penicillamine methyl ester
The interactivity parameter shows to what extent the proto-
nation of site A reduces the basicity of site B, and vice versa. The
interactivity parameter is generally considered to be the most
invariant quantity in analogous moieties of different compounds
and also in various protonation states of the neighboring moiety
in the same molecule [24]. The protonation constants of penicil-
lamine with the interactivity parameters are compiled in Table 1.
(11)
log kS = log ˇ2
penicillamine methyl ester
O
−
log KS-methyl penicillamine methyl ester
(12)

A. Mirzahosseini et al. / Chemical Physics Letters 610–611 (2014) 62–69
67
Figure 4. The plot of 1H chemical shifts versus pH; (ꢀ) ␣ proton, (•) CH3 protons, (ꢁ)
O-methyl protons. (A) Penicillamine disulfide; (B) penicillamine disulfide dimethyl
Figure 5. The relative concentrations in logarithmic scale of the various
microspecies of penicillamine (A) and penicillamine disulfide (B) as a function of pH.
Letters a–h and a–p represent the microspecies of penicillamine and penicillamine
disulfide as defined in Figure 2.
ester.
The pH-dependent mole fraction diagram of the compounds is in
Figure 5.
Nꢀ
penicillamine disulfide
log k = log K2
+ log 2
(20)
(21)
(22)
N
3.2. The microscopic protonation constants of penicillamine
disulfide
O
NNꢀ
penicillamine disulfide
log k
log k
= log K3
− log 2
The microspeciation of penicillamine disulfide is simplified by
Oꢀ
= log K 4p enicillamine disulfide + log 2
two facts: (1) the symmetry of the molecular structure (the oppo-
site amino and carboxylate groups are chemically identical), and
ONNꢀ
In order to elucidate the minor pathway, an auxiliary com-
pound was synthesized and characterized. Penicillamine disulfide
dimethyl ester was used to model the microspecies where both car-
boxylates are protonated. Using the principles as above, additional
microscopic protonation constants of penicillamine disulfide were
attained:
(
2) the well-separated protonation pH range of the amino and
carboxylate groups. Fact (1) results in the equality of certain micro-
ꢀ
ꢀ
N
N
O
O
Nꢀ
N
Oꢀ
O
scopic protonation constants: k = k , k = k , k = k , k = k
,
,
,
N
= k
Nꢀ
Nꢀ
N
Nꢀ
O
Oꢀ
Nꢀ
N
N
Nꢀ
Oꢀ
O
O
Oꢀ
Nꢀ
N
ONꢀ
Oꢀ NNꢀ
k
k
k
= k , k = k , k = k , k = k , k
, k
= k
N
Nꢀ
O
Oꢀ
O
Oꢀ
Oꢀ
O
Oꢀ
NNꢀ
NNꢀ
Oꢀ N
O
Oꢀ
Nꢀ
N
O
N
Oꢀ
O
= k
, k = k
, k = k
, k
= k
, k
= k
Oꢀ N
ONꢀ
N
ON
Oꢀ Nꢀ
ON
Oꢀ Nꢀ
OOꢀ
OOꢀ
ONNꢀ
Nꢀ
= k
. Since the first and last two protonation steps in
OOꢀ N
OOꢀ Nꢀ
N
penicillamine disulfide dimethyl ester
penicillamine disulfide are practically separated (titration curve in
Fig. 4), they appear as independent systems. The first two proto-
nation steps are overwhelmingly those of the amino groups, since
the mono- and biprotonated ligands in which any of the two car-
boxylate groups is protonated are “orders of magnitude minor”.
Therefore the microscopic protonation constants of the major path-
way can be calculated using the following equations:
log k
log k
= log K1
− log 2
(23)
(24)
OOꢀ
Nꢀ
penicillamine disulfide dimethyl ester
= log K2
− log 2
OOꢀ N
The missing microconstants were calculated using the
amino/carboxylate interactivity parameter of penicillamine
(regarded as “same-side” amino/carboxylate interactivity param-
eter in penicillamine disulfide), and the other interactivity
parameters of penicillamine disulfide listed in Table 1:
log kN = log K 1p enicillamine disulfide − log 2
(19)

6
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A. Mirzahosseini et al. / Chemical Physics Letters 610–611 (2014) 62–69
S-methyl penicillamine methyl ester
N
log ꢀEN/O = log kS − log K
log kN − log kON
thiolate protonation shifts of the methyl groups, so much so that
one of the methyl groups is almost insensitive to the adjacent
thiolate protonation. This is another indication of the unique steric
properties of the two chemically distinct methyl groups.
=
(25)
The interactivity parameter is a good indicator of the basicity-
reducing effect on one of the sites when the other site protonates.
For example, the esterification of the carboxyl moiety decreases the
basicity of the amino and thiolate groups, while the S-methylation
has no effect on the carboxylate site, because the thiolate is
practically completely protonated when the carboxylate pro-
All of the macroscopic and microscopic protonation constants of
penicillamine disulfide with the interactivity parameters are listed
in Table 1. The pH-dependent mole fraction diagram of the various
microspecies is in Figure 5.
◦
The macroscopic and microscopic protonation constants (25 C,
0.15 mol/l ionic strength), and interactivity parameters of penicil-
S-methyl penicillamine
O penicillamine
tonates (K2
= kSN
). In penicillamine,
lamine, penicillamine disulfide and their model compounds in log
the interactivity parameters are in decreasing order as fol-
lows: amino/carboxylate, amino/thiolate and thiolate/carboxylate.
Because of the steric effects of the methyl groups, the thiolate-
containing interactivity parameters are smaller in penicillamine
than in cysteine, while no difference can be observed between their
amino/carboxylate interactivity parameters. Relative to the long
covalent distance, the interactivity parameter between the oppos-
ing amino and carboxylate groups is higher than expected. This
phenomenon might be caused by the flexible molecular structure
enabling Coulombic attraction between the protonated ammonium
and the opposing carboxylate. On the other hand, these oppos-
ing side interactivities are smaller than in cystine. Because of the
two shielding methyl groups, penicillamine disulfide is presumably
less flexible than cystine, which can explain the lower interactivity
parameters in penicillamine disulfide.
units ± s.d.
4
. Discussion
A profound submolecular acid–base evaluation of penicillamine
and penicillamine disulfide allows deeper insight compared to
the conventional macroscopic pKa values, especially when minor
microspecies are considered. Also, several remarkable peculiarities
can be observed when penicillamine microconstants are compared
to those of cysteine, the related molecule of analogous skele-
ton.
The first and second protonation constants of penicillamine are
predominated overlappingly by the amino and thiolate sites, the
amino being typically more favored. The carboxylate group pro-
tonates at much lower pH region. In fact, the monoprotonated
microspecies are mainly b and c in decreasing order of abun-
dance, while some 4 orders of magnitude smaller amount of d is
present. The biprotonated microspecies occurs mainly in the form
of e. Among the minor biprotonated species, the one protonated
at the amino and carboxylate groups is favored over the other
minor microspecies. When the carboxylate is protonated, and the
molecule takes on a second proton, the amino group is favored over
the negatively charged thiolate (as opposed to cysteine [9]). Due
to the electron-donating properties of the methyl groups of peni-
cillamine, the amino bacisity is higher in penicillamine compared
to that of cysteine. On the other hand, the bacisity of the thiolate
is lower in penicillamine, presumably due to steric hinderance of
the two methyl moieties. The basicity of the carboxylate is only
marginally higher in penicillamine.
5. Conclusions
The 28 site-specific basicities determined for penicillamine,
penicillamine disulfide and 4 related compounds is a significant
dataset to interpret the behavior of these therapeutically important
compounds in their biochemical, synthetic and analytical reactions.
Since thiolate basicities are related to their redox and chelating
properties, the 10 different thiolate protonation constants provide
sound means at the molecular level to predict thiolate oxidiz-
abilities, a key parameter to understand and influence oxidative
stress and heavy metal chelation and to produce custom-tailored
penicillamine-derived drug candidates.
Conflict of interest
ꢀ
In penicillamine disulfide the N and N amino groups proto-
nate parallel, followed by the also parallel protonation of the two
carboxylates. It is noteworthy that in the instance of biprotonated
disulfide (major f microspecies: both amino groups protonated) the
most favored minor microspecies is the one with opposite amino
and carboxylate groups protonated (h = i), due to the Coulombic
repulsion between hydronium ions and the ammonium ion, hence
the opposite carboxylate will be slightly more basic. By far the least
favored biprotonated species (k) is the one with both carboxylates
protonated. The bacisities of the amino and carboxylate groups are
also higher in penicillamine disulfide than in cystine due to the
electron-donating methyl groups.
The authors report no conflict of interest.
Acknowledgement
This research was supported by TÁMOP 4.2.1.B-09/1/KMR and
OTKA T 73804.
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