Rapid high-yield N-acylation of aminothiols: N-acetylglutathione and N-acetylhomocysteine and their thiol pKa values

Article abstract of DOI:10.1002/psc.2492

Methodology for the rapid N-acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical studies is described here. Glutathione disulfide (GSSG) and homocystine were diN-acetylated in ~100% yield in 0.1M aqueous NaHCO₃ (pH 8.5) at room temperature by 2.5equiv of the activated ester, N-hydroxysulfosuccinimidyl acetate, an efficient water-soluble acetylating reagent. Following acetone precipitation, diN-acetylGSSG was further purified and desalted on a strong anion-exchange (SAX) cartridge. DiN-acetylhomocystine was simultaneously purified and desalted on a C₁₈ cartridge. The N-acetylated aminothiols were generated using gel-immobilized tris(2-carboxyethyl)phosphine as a reductant, which obviated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N-acetylglutathione, which was purified on the SAX cartridge. pH titrations of N-acetylglutathione (8.99) and N-acetylhomocysteine (9.66) as well as those of commercially available N-acetylcysteine (9.53) and N-acetylpenicillamine (10.21) yielded pK_a(SH) values of importance for biological studies.

Full text of DOI:10.1002/psc.2492

Research Article
Received: 14 September 2012
Revised: 14 January 2013
Accepted: 16 January 2013
Published online in Wiley Online Library: 27 March 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2492
Rapid high-yield N-acylation of
aminothiols: N-acetylglutathione
and N-acetylhomocysteine and their
thiol pKa values
Biao Shen,a,b Cynthia Bazinc and Ann M. Englisha,b*
Methodology for the rapid N-acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical
studies is described here. Glutathione disulfide (GSSG) and homocystine were diN-acetylated in ~100% yield in 0.1 M aqueous
NaHCO3 (pH 8.5) at room temperature by 2.5 equiv of the activated ester, N-hydroxysulfosuccinimidyl acetate, an efficient
water-soluble acetylating reagent. Following acetone precipitation, diN-acetylGSSG was further purified and desalted on a
strong anion-exchange (SAX) cartridge. DiN-acetylhomocystine was simultaneously purified and desalted on a C18 cartridge.
The N-acetylated aminothiols were generated using gel-immobilized tris(2-carboxyethyl)phosphine as a reductant, which obvi-
ated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N-acetylglutathione,
which was purified on the SAX cartridge. pH titrations of N-acetylglutathione (8.99) and N-acetylhomocysteine (9.66) as well as
those of commercially available N-acetylcysteine (9.53) and N-acetylpenicillamine (10.21) yielded pKa(SH) values of importance
for biological studies. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: activated ester; glutathione; homocysteine; thiol pKa
Here, we describe the rapid N-acetylation of aminodisulfides in
~100% yield at room temperature in weakly basic aqueous
Introduction
media. The acetylating agent is the activated ester, NHSSA, which
Intracellular low-mass aminothiols, such as GSH, cysteine (Cys),
was developed as a water-soluble blocking agent for primary
and HCys, act as antioxidants and redox buffers with a multitude
amino groups in proteins [17–19]. Following N-acetylation, the
of physiological functions [1,2]. We have been investigating their
roles in trans-S-nitrosation [3–7], protein thionylation [6], and
their direct reactions with nitrogen oxides (NOx) [6,8]. For these
studies, we desired N-AcGSH and N-AcHCys, which are not
disulfides were purified and isolated as the free acids by solid-phase
extraction. Reduction of the disulfides on agarose-immobilized
TCEP yielded the pure N-acetylated aminothiols, and their elemen-
tal compositions were confirmed by exact mass measurements.
commercially available, so it was necessary to N-acylate the
We additionally report the acid dissociation constants of the
amino group in-house.
thiol groups [pKa(SH)] of the newly synthesized N-acetylated
Few methods for N-acylating aminothiols have been reported
aminothiols as well as those of N-AcCys and N-AcPen. Because
[9–13]. NMR evidence for the isolation of N-AcGSH and N-
formylGSH from overnight reactions of GSH with acetic
anhydride (Ac2O) in acetic and formic acid has been reported
[9], but product yields and elemental compositions were not
provided. Treatment of GSH with Ac2O in formic acid was also
reported to yield N-AcGSH, but the product was not character-
ized [10,11]. Because thiols are acetylated by Ac2O under basic
conditions, the disulfide GSSG was N-acetylated by this reagent
in water at pH 9.5 [12]. Multiple additions of Ac2O were necessary
during the 4-h reaction, and following DTT reduction, a product
identified by NMR as N-AcGSH was obtained in 55% yield [12].
GSH derivatives such as S-(4-bromobenzyl)GSH have been
N-acetylated by the Schotten–Baumann reaction over 1 day
with a yield of only 40% N-acetyl-S-(4-bromobenzyl)GSH after a
number of purification steps [13]. The disulfide cystine has also
been N-acetylated by Ac2O [14–16], but to the best of our
knowledge, the synthesis of N-AcHCys has not been reported
although N-AcHCys thiolactone is commercially available.
*
Correspondence to: Ann English, Department of Chemistry and Biochemistry,
Concordia University, 7141 Sherbrooke West, Montreal, Quebec, Canada
H4B 1R6. E-mail: ann.english@concordia.ca
a Department of Chemistry and Biochemistry, Concordia University, 7141
Sherbrooke Street West, Montreal, Quebec, Canada, H4B 1R6
b Centre for Biological Applications of Mass Spectrometry, 7141 Sherbrooke
Street West, Montreal, Quebec, Canada, H4B 1R6
c
Merck & Co., 351 N Sumneytown Pike, North Wales, PA 19454, USA
Abbreviations: DTT, dithiothreitol; GSH, glutathione; GSSG, glutathione disulfide;
HCys, homocysteine; Hcystine, homocystine; N-AcCys, N-acetylcysteine; N-AcGSH,
N-acetylglutathione; N-AcHCys, N-acetylhomocysteine; N-AcHcystine, N-
acetylhomocystine; N-AcPen, N-acetylpenicillamine; NEM, N-ethylmaleimide;
NHSS, N-hydroxysulfosuccinimide; NHSSA, N-hydroxysulfosuccinimidyl acetate;
SAX, strong anion-exchange; TCEP, tris(2-carboxyethyl)phosphine.
J. Pept. Sci. 2013; 19: 263–267
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

SHEN, BAZIN AND ENGLISH
their biological functions [1,2] depend in large part on their pKa
(SH) values, these are key properties of aminothiols. All pKa(SH)
values were determined by pH titration in 0.15 M KCl to mimic
physiological ionic strength. The results extend the number of
available pKa(SH) values, which are not abundant despite their
biological importance.
Potentiometric Determination of pKa Values
pH-metric titrations were carried out using the GLpKa potentiometric
pKa analyzer running RefinementPro V1.104 software (Sirius
Analytical Instruments, Beverly, MA, USA). Titrations were
performed in 0.15 M aqueous KCl at 25 ꢀC under argon flow to
remove CO2 in solution. Three independent titrations were
performed for each compound, and the experimental pKa values
were calculated by the RefinementPro software. Further details
are provided in the Supporting Information.
Materials and Methods
Materials
Results
DL-HCys, DL-Hcystine, NEM, L-GSH, L-GSSG, N-acetyl-L-cysteine
(N-AcCys), N-acetyl-D-penicillamine (N-AcPen), and DL-DTT were
purchased from Sigma. NHSSA and TCEP hydrochloride immobilized
on agarose gel (50% water slurry) and Handee Mini-Spin columns
(0.4 ml) were obtained from Thermo Scientific Pierce. SAX cartridges
(Bond Elut-SAX, 100 MG, 3 ML) were purchased from Agilent Varian
and Sep-Pak C18 cartridges (1 ml) from Waters. All solutions were
prepared using Nanopure water (MilliQ) from a Millipore system.
Reaction of NHSSA with GSSG
The free thiol and amino groups of GSH exhibit comparable
reactivity with NHSSA (see Reactions of NHSSA with GSH,
Supporting Information), so GSSG disulfide was used here to
avoid S-acetylation. Excess NHSSA was required for complete N-
acetylation of GSSG because the activated ester undergoes
competing hydrolysis at room temperature. For example, after
80-min reaction in 0.1 mM NaHCO3 (pH 8.5) with 1 equiv of
NHSSA (10 mM), HPLC analysis revealed that 5 mM GSSG was
75% diacetylated and 25% monoacetylated, whereas on reaction
with 2.5 equiv of NHSSA only diacetylated GSSG was detected
(Figure 1A) after just 30 min (Figure 1E). Peaks at m/z 655 and
m/z 697 in the ESI mass spectra (Figure 1(B)–(D)) correspond to
mono- and diacetylated GSSG, respectively. Thus, in a representative
acetylation, 2.5 equiv of NHSSA (6.5 mg) was added to 3.0 mg of
GSSG (~5 mM of the free acid form) in 1.0 ml of 0.1 M aqueous
NaHCO3 (pH 8.5). After 30 min at room temperature, the pH was
adjusted to 4 with 1 N HCl; DiN-AcGSSG was precipitated on the
addition of 2 Â 9.0 ml of ice-cold acetone, batch purified on a 3 ml
SAX cartridge and isolated as the free acid (see DiN-AcGSSG
purification, Supporting Information). DiN-AcGSSG (3.4 mg) was
recovered in ~98% yield, and the purity of the free acid was
confirmed by HPLC-UV (Figure S3D). ESI-MS analysis (Figure 2) con-
firmed the presence of the MNa+ (m/z 719) and MH+ (m/z 697) ions
of diN-AcGSSG in spectra of the acetone precipitate (spectrum B)
HPLC-UV, HPLC-MS, and ESI-MS Analyses
HPLC-UV analyses were performed on an Agilent Hypersil ODS
C18 column (4.6 Â 100 mm, 5 mm particles) attached to an Agilent
1090 HPLC with a diode-array detector (Agilent Technologies,
Inc., Santa Clara, CA). The solvents used for column equilibration
and the mobile phases are indicated in the figure legends. Samples
for HPLC-MS analysis (Figure 1) were diluted with water and infused
into the ESI source of an SSQ 7000 mass spectrometer (Thermo
Finnigan) by flow injection from the C18 column. Data from the
diode array and the SSQ 7000 were analyzed using ChemStation
(Agilent) and XCalibur (Thermo Finnigan) software, respectively.
Samples for direct ESI-MS analysis were diluted into 50%
aqueous acetonitrile with 0.2% formic acid and directly infused
into the Z-spray source of a Waters Micromass Q-ToF 2 mass
spectrometer. The instrument was operated in positive-ion mode
with the parameters given in the legend of Figure 2.
Figure 1. HPLC-UV (215 nm) and HPLC-MS analysis of GSSG acetylation by NHSSA in 0.1 M aqueous NaHCO3 (pH 8.5) at room temperature. HPLC
chromatograms of (A) GSSG before and after 80-min reaction with 1.0 and 2.5 equiv of NHSSA, and (E) GSSG after reaction with 2.5 equiv of NHSSA
for 5, 10, and 30 min. ESI mass spectra of GSSG (B) before (control) and after 80-min reaction with (C) 1.0 equiv of NHSSA and (D) 2.5 equiv of NHSSA.
Experimental conditions: 5 mM GSSG was reacted with 10 (1.0 equiv) or 25 mM (2.5 equiv) NHSSA for the times indicated and diluted tenfold with water
before injection onto the C18 HPLC column equilibrated with 10% aqueous acetonitrile/0.1% formic acid, which was also used as the mobile phase.
Samples were eluted at 1.5 ml/min (A–D) or 1.0 ml/min (E) into diode-array detector or the ESI source of the SSQ 7000 mass spectrometer. The ESI spray
voltage was 4.0 kV, and the source and capillary temperatures were 70 ꢀC and 250 ꢀC, respectively. Note that peak at m/z 350 in spectra C and D is the
doubly charged ion of diN-AcGSSG. Mass calibration of the SSQ 7000 was carried out with L-methionyl-arginyl-phenylalanyl-alanine acetate (MRFA) and
horse myoglobin (Sigma) at low and high mass, respectively.
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N-ACYLATION OF AMINOTHIOLS
100
%
A
B
+2Na
397
(N-AcHcystine)Na
F
100
%
282
+3Na
419
375
0
(diN-AcGSSG)Na
240
262
330 350
719
431
300
100
%
0
(diN-AcHcystine)H
741
353
100
0
G
H
(diN-AcGSSG)H
697
%
100
C
D
RA
%
0
0
(N-AcHCys)H
RA
178
100
(N-AcGSH)H
(N-AcHCys) H
160
350
100
%
355
%
(N-AcGSH) H
699
0
+NEM
303
0
+NEM
475
100
I
100
%
E
+Na
%
0
353
350
350
497
0
300
140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
400
450
500
550
600
650
700
750
800
850
m/z
m/z
Figure 2. ESI-MS analysis of diN-AcGSSG and diN-AcHcystine purification and reduction by gel-bound TCEP. Mass spectrum of (A) the 5 mM GSSG and
25 mM NHSSA reaction (crude products), (B) acetone-precipitated diN-AcGSSG following dissolution in water, (C) diN-AcGSSG following elution from the
SAX cartridge, (D) N-AcGSH following reduction on the TCEP gel, (E) the 5 mM N-AcGSH and 10 mM NEM reaction at pH 2.23, (F) the 2 mM Hcystine and
10 mM NHSSA reaction (crude products), (G) diN-AcHcystine following elution from the C18 cartridge, (H) N-AcHCys following reduction on the TCEP gel,
and (I) the 2 mM N-AcHCys and 4 mM NEM reaction at pH 2.77. Experimental conditions: the NHSSA and NEM reactions were carried out in 0.1 M NaHCO3
(pH 8.5) and water, respectively, at room temperature for 30 min. The diN-AcGSSG and diN-AcHcystine products were purified as described in Figures S3
and S4 and reduced on a TCEP gel (see Results). Before ESI-MS analysis, samples were diluted 10-fold to 20-fold into 50% aqueous acetonitrile/2% formic
acid and directly infused at a flow rate of 1 ml/min into the Z-spray source of the Q-ToF 2 mass spectrometer. Instrument settings were source block
temperature, 80 ꢀC, capillary voltage, 3.2 kV, cone voltage, 20 V, collision voltage, 5 V (no collision gas), MCP, 2.2 kV, and ToF, À9.1 kV. RA is the relative
abundance of the ions. The instrument was calibrated with human [Glu1]-fibrinopeptide B, and data analysis was performed using MassLynx 4.0
software (Waters Micromass).
and the SAX eluate (spectrum C), respectively. Note that no
diN-AcGSSG ions are visible in the spectrum of the crude products
(spectrum A) due to ion suppression.
a weak (M + 125)Na+ peak at m/z 497 and no remaining disulfide
peaks were detected (Figure 2E), indicative of complete reduction
of diN-AcGSSG to N-AcGSH. The TCEP gel was further washed with
0.2 ml of water; the washings (collected by centrifugation) were
analyzed by ESI-MS, and any residual disulfide was reduced on
fresh gel. DiN-AcGSSG was also reduced by DTT in solution, which
required an additional N-AcGSH purification step (See DiN-AcGSSG
homogenous reduction with DTT, Supporting Information).
Heterogeneous reduction of 1 mg diN-AcHcystine on the TCEP
column yielded MH+ (m/z 178), M2H+ (m/z 355), and (M + 125)H+
(m/z 303) of N-AcHCys (Figure 2H and I), the latter following
alkylation with NEM. The peak at m/z 160 in Figure 2H is assigned
to the (M À 18)H+ ion of N-AcHCys following loss of H2O in the
ESI source.
Exact mass measurements confirmed the elemental composition
of the N-acetylated aminothiols. The observed monoisotopic
masses of N-AcGSH and N-AcHCys differ by <1 ppm from their
calculated values (Table 1). HPLC-MS analysis indicated that
disulfide reduction on the TCEP column goes to completion and
gives the pure products (Figure 2). Thus, 2mg diN-AcGSSG and
1 mg diN-AcHcystine yielded the same quantities within
experimental error of N-AcGSH and N-AcHcys after the aminothiols
were dried on the SpeedVac at room temperature.
Reaction of NHSSA with Hcystine
Hcystine at ~2 mM (because of its low solubility [20]) was also
fully diacetylated by 2.5 equiv of NHSSA (data not shown). In a
representative reaction, 26 mg of NHSSA was added to 5.4 mg
of Hcystine in 10 ml of 0.1 M NaHCO3. After 30 min at room
temperature, 9.9 ml of the Hcystine/NHSSA reaction mixture
was acidified to pH 2.7 with a 0.1 ml of 10% aqueous formic acid,
batch purified on a 1-ml Sep-Pak C18 cartridge and isolated as the
free salt (see DiN-AcHcystine purification, Supporting Information).
DiN-AcHcystine (7.0mg) was obtained in ~98% yield, and its purity
was confirmed by HPLC-UV (Figure S4). The mass spectrum of
purified DiN-AcHcystine exhibited a dominant MH+ ion at
m/z 353 (Figure 2G), whereas sodiated ions dominate the
spectrum of the crude products (Figure 2F).
Disulfide Reduction
Purified diN-AcGSSG (~2 mg) was dissolved in 0.4 ml of water and
mixed with a 0.2 ml bed volume of agarose-bound TCEP that was
previously washed with 2 Â 0.5 ml of water. The gel suspension
was transferred to a Handee Mini-Spin column and vortexed at
room temperature for 1 h, and the N-AcGSH solution was
collected in a 2-ml microtube by centrifugation at 1000 xg for
1 min. Notably, TCEP and its oxidized form were not detected in
the mass spectrum (Figure 2D), which exhibits peaks
corresponding to the MH+ ion (m/z 350) and a noncovalent
protonated dimer (M2H+, m/z 699) of N-AcGSH. Thiol alkylation
with NEM yielded a dominant (M + 125)H+ peak at m/z 475 and
pKa(SH) Values
The thiol pKa values determined here for the four N-acetylated
aminothiols in Figure 3 fall in the range of 8.99À10.21 (Table 2).
N-AcPen (10.21) exhibits the highest pKa(SH) presumably because
of the electron-donating effect of the two methyl groups on its
b-carbon (Figure 3). However, we expected the pKa(SH) of N-AcGSH
to be higher than that of N-Cys (9.53) or N-AcHCys (9.66) because
the negative charges of its two carboxylate groups (Figure 3) are
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SHEN, BAZIN AND ENGLISH
Table 1. Exact mass measurements for GSH, HCys, and their N-acetylated derivativesa
Compound
Formula
Mr (u)
m/z of MH+
Error
(ppm)
Calculated
Measureda
GSH
C10H17N3O6S
307.08381
349.09437
135.03540
177.04596
308.09108
350.10165
136.04268
178.05324
308.09062
350.10167
136.04256
178.05329
À1.49
0.07
N-AcGSH
HCys
C12H19N3O7S
C4H9NO2S
C6H11NO3S
À0.83
0.29
N-AcHCys
aExact masses (Mr) were measured on an Agilent LC/MSD TOF MS. The compounds were diluted to ~100 mM in water (pH 5.5) and injected onto an
Agilent Zorbax SB column (4.6 Â 30 mm; 1.8 mm particles) attached to an Agilent 1100 HPLC. The column was equilibrated with solvent A at
70 ꢀC, and the compounds were eluted with a 4.5 min gradient of 5À95% B (A: 0.05% aqueous TFA; B: 100% acetonitrile) into the ESI source
at 3.5 ml/min. The mass spectrometer settings were fragmentor, 125 V, ESI capillary, 3.0 kV, gas temperature, 350 ꢀC, and drying gas flow,
12 l/min. Internal reference masses at m/z 121.050873 and 922.009798 were used for mass calibration of the instrument.
Figure 3. Structures of the N-acetylated aminothiols, the pKa(SH) values of which are reported here.
(long reaction times, multiple reagent addition, mixed solvent
Table 2. pKa(SH) values of N-acetylated aminothiolsa
systems or concentrated acids, and low yields). The side products,
NHSS and acetic acid formed on the competing hydrolysis of
NHSSA (Eqn 1) [21],
Compoundb
pKa (SH)
I (M)c
Reference
N-AcGSH
N-AcCys
8.99
9.52
0.15
0.30
0.15
0.15
0.30
0.30
0.15
This workd
[26]
This workd
This workd
[27]
9.53
N-AcHCys
N-AcPen
9.66
10.28
9.90
[26]
This workd
10.21
aThe pKa(SH) values listed here were determined by pH titration
(see Supporting Information).
bCompound abbreviations and structures are given in Figure 3.
cIonic strength.
dTitrations were performed in 0.15 M aqueous KCl under argon
flow (60 ml/min) to remove CO2 in solution (see Supporting
Information).
are readily removed from the N-acetylated disulfides by acetone
precipitation (Figure S3) or solid-phase extraction (Figure S4).
Notably, thiol reduction using gel-bound TCEP eliminated the
second purification step because both TCEP and its oxidized form
remained immobilized on the agarose gel. Immobilized TCEP is
odorless, effective at pH 4À9, and stable in aqueous solution.
Moreover, disulfide reduction can be quickly carried out just
before use to avoid thiol oxidation during storage.
Our facile N-acylation of aminothiols uses water-based reagents
commonly used in protein chemistry. Importantly, the methodology
can be adapted to construct new probes or sensors on 1-ethyl-3-
[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) activation
of a carboxylic acid bearing a spectroscopic, mass, affinity or other
tag. Addition of NHSS generates the activated NHSS ester that will
donate the tagged acyl group to a primary amine on the target of
interest. Furthermore, the NHSS ester can be isolated or reacted
with the target in situ [22].
unmatched at high pH (see Discussion). Additionally, we note that
our pKa values for commercially available N-AcCys and N-AcPen
are in good agreement with the literature values obtained by pH
titration (Table 2).
Discussion
N-Acetylation
The procedure described here for the preparation of N-AcGSH
and N-AcHCys involves the diN-acetylation, purification, and
reduction of their disulfides at room temperature in aqueous
media. The acetylating reagent used, NHSSA, rapidly (≤30 min)
N-acetylated the aminodisulfides at pH 8.5 as seen for GSSG in
Figure 1E. Use of the activated ester as an acyl donor circumvents
the drawbacks of the literature methods for aminothiol N-acylation
pKa(SH) Values of N-Acetylated Aminothiols
The pKa values of acidic protons are modulated by electrostatic and
inductive effects, the former operating through space and the latter
through bonds in the molecule [23]. Electron-withdrawing groups
have an acid-strengthening effect and depress pKa, whereas
electron-donating groups are acid-weakening and raise pKa.
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N-ACYLATION OF AMINOTHIOLS
6 Tao L, English AM. Protein S-glutathiolation triggered by decomposed
S-nitrosoglutathione. Biochemistry 2004; 43: 4028–38.
7 Tao L, Murphy ME, English AM. S-nitrosation of Ca(2+)-loaded and Ca
(2+)-free recombinant calbindin D(28K) from human brain. Biochemistry
2002; 41: 6185–92.
8 Shen B, English AM. Mass spectrometric analysis of nitroxyl-mediated
protein modification: comparison of products formed with free and
protein-based cysteines. Biochemistry (Mosc.) 2005; 44: 14030–44.
9 Levy EJ, Anderson ME, Meister A. On the synthesis and characterization
of N-formyl glutathione and N-acetyl glutathione. Anal. Biochem. 1993;
214: 135–7.
10 Utzinger GE, Strait LA, Tuck LD. The oxidation of glutathione with
formic acid and hydrogen peroxide. Experientia 1963; 19: 324–7.
11 Vander Jagt DL, Han LP. Deuterium isotope effects and chemically
modified coenzymes as mechanism probes of yeast glyoxalase-I.
Biochemistry 1973; 12: 5161–7.
12 Chen WJ, Graminski GF, Armstrong RN. Dissection of the catalytic
mechanism of isozyme 4-4 of glutathione S-transferase with alternative
substrates. Biochemistry 1988; 27: 647–54.
13 D’Silva C, Al-Timari A, Douglas KT. Synthesis of N-acylglutathione
derivatives by dimethylaminopyridine catalysis. Biochem. J. 1982;
207: 329–32.
14 Martin TA, Corrigan JR, Waller CW. N-Acylation of Cysteine. J. Org.
Chem. 1965; 30: 2839–40.
15 Martin TA, Causey DH, Corrigan JR. N-Acylcysteines. J. Med. Chem.
1968; 11: 625–7.
16 Xu H, Zhang Q, Kong X. New synthetic method for the N-acetyl-cysteine.
Jingxi Huagong 2000; 17: 205–7.
17 Salvucci ME. Covalent modification of a highly reactive and essential
lysine residue of ribulose-1,5-bisphosphate carboxylase/oxygenase
activase. Plant Physiol. 1993; 103: 501–8.
18 Lewis PN, Guillemette JG, Chan S. Histone accessibility determined by
lysine-specific acetylation in chicken erythrocyte nuclei. Eur. J.
Biochem. 1988; 172: 135–45.
19 Sondej M, Seok YJ, Badawi P, Koo BM, Nam TW, Peterkofsky A.
Topography of the surface of the Escherichia coli phosphotransferase
system protein enzyme IIAglc that interacts with lactose permease.
Biochemistry 2000; 39: 2931–9.
20 Ragone R. Homocystine solubility and vascular disease. FASEB J. 2002;
16: 401–4.
Thus, the sulfhydryl groups of the four N-acetylated aminothiols in
Figure 3 must experience varied electrostatic and inductive effects
given the spread in their pKa(SH) values (Table 2). For example, the
electron-donating inductive effect of dimethyl substitution at
the b-carbon of N-AcPen (Figure 3) has an acid-strengthening
effect on the thiol group and increases its pKa(SH) relative to
those of N-AcCys and N-AcHCys (Table 2).
Negative charges destabilize anions such as thiolates and
increase pKa(SH), whereas positive charges have the opposite
effect [24]. Thus, as mentioned previously, we anticipated that the
pKa(SH) of N-AcGSH (8.99) would be higher than that of N-AcCys
(9.53) because the former is a dianion when its SH group is titrated.
The proposed dominant electrostatic interactions involving the
thiol in GSH are SH/Gly-COOÀ (acid-weakening) and SÀ/NH3+
(acid-strengthening) [25]. The relatively low pKa(SH) suggests loss
or decreased SH/Gly-COOÀ (acid-weakening) interaction on GSH
N-acetylation. However, the SÀ moiety may act as an H-bond
acceptor from the acetylated nitrogen, which would be an acid-
strengthening interaction, albeit weaker than the SÀ/NH3+
interaction. Regardless of the origins of its relatively low pKa(SH),
the thiolate form of N-AcGSH will be ~3.5 times more abundant
in solution at physiological pH than that of the commonly used
antioxidant, N-AcCys.
Acknowledgements
The authors thank Dr. Angelo Filosa (Boehringer Ingelheim
Canada) for measuring the accurate masses reported here and
Alain Tessier (Centre for Biological Applications of Mass
Spectrometry, Concordia University) for his assistance in recording
the mass spectra. This work was funded in part by a research grant
from the Natural Sciences and Engineering Research Council
(NSERC) of Canada awarded to AME, who also holds a Concordia
University Research Chair.
21 http://www.piercenet.com/instructions/2161503.pdf (Accessed January
2013).
22 Badia A, Carlini R, Fernandez A, Battaglini F, Mikkelsen SR, English AM.
Intramolecular electron-transfer rates in ferrocene-derivatized glucose
oxidase. J. Am. Chem. Soc. 1993; 115: 7053–60.
References
1 Sies H. Glutathione and its role in cellular functions. Free Radic. Biol.
Med. 1999; 27: 916–21.
23 Perrin DD, Dempsey B, Serjeant EP. pKa predictions for organic acids
and bases. Chapman and Hall: New York, 1981.
2 Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism
and its implications for health. J. Nutr. 2004; 134: 489–92.
3 Tao L, English AM. Mechanism of S-nitrosation of recombinant human
brain calbindin D28K. Biochemistry 2003; 42: 3326–34.
4 Romeo AA, Capobianco JA, English AM. Superoxide dismutase targets
NO from GSNO to Cysbeta93 of oxyhemoglobin in concentrated
but not dilute solutions of the protein. J. Am. Chem. Soc. 2003;
125: 14370–8.
24 Bulaj G, Kortemme T, Goldenberg DP. Ionization-reactivity relationships
for cysteine thiols in polypeptides. Biochemistry 1998; 37: 8965–72.
25 Krezel A, Bal W. Structure-function relationships in glutathione and its
analogues. Org. Biomol. Chem. 2003; 1: 3885–90.
26 Friedman M, Cavins JF, Wall JS. Relative nucleophilic reactivities
of amino groups and mercaptide ions in addition reactions with
a,b-unsaturated compounds. J. Am. Chem. Soc. 1965; 87: 3672–82.
27 Backs SJ, Rabenstein DL. Nuclear magnetic resonance studies of
the solution chemistry of metal complexes. 16. Complexation of
trimethyllead by sulfhydryl-containing amino acids and related
molecules. Inorg. Chem. 1981; 20: 410–5.
5 Romeo AA, Capobianco JA, English AM. Heme nitrosylation of
deoxyhemoglobin by s-nitrosoglutathione requires copper. J. Biol.
Chem. 2002; 277: 24135–41.
J. Pept. Sci. 2013; 19: 263–267 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci