Structural Identification and Kinetic Analysis of the in Vitro Products Formed by Reaction of Bisphenol A-3,4-quinone with N-Acetylcysteine and Glutathione

Article abstract of DOI:10.1021/acs.chemrestox.7b00239

Bisphenol A (BPA) has received considerable attention as an endocrine disrupting chemical and a possible substrate for genotoxic metabolites. BPA metabolism leads to formation of electrophilic o-quinones cable of binding to DNA and other endogenous nucleophiles. We have structurally identified the products resulting from the reaction of bisphenol A-3,4-quinone (BPAQ) with N-acetylcysteine (NAC) and glutathione (GSH). The major and minor isomers are both the result of 1,6-conjugate addition and are produced almost instantly in high yield. Reactions using 1.3 equiv of GSH showed the presence of a bis-glutathionyl adduct which was not observed using higher GSH concentration relative to BPAQ. NAC reactions with BPAQ showed no bis-N-acetylcysteinyl adducts. Stopped-flow kinetic analysis reveals the 1,6-conjugate additions to be reversible with a forward free energy of activation of 9.2 and 7.8 kcal/mol for the NAC and GSH reactions, respectively. The bimolecular forward rate constant at 19.4 °C was approximately three time faster for GSH compared to NAC, 1547 vs 496 M^-1 s^-1. The free energy of activation for the reverse reactions were similar, 11.7 and 11.2 kcal/mol for NAC and GSH, respectively. We plan to use this model system to further explore the mechanism of adduct formation between sulfur nucleophiles and o-quinones and the resulting chemical properties of both NAC and GSH adducts.

Full text of DOI:10.1021/acs.chemrestox.7b00239

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Article
Structural Identification and Kinetic Analysis of the In Vitro Products Formed
by Reaction of Bisphenol A-3,4-quinone with N-Acetylcysteine and Glutathione
Douglas Edward Stack, John Albert Conrad, and Bejan Mahmud
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00239 • Publication Date (Web): 27 Dec 2017
Downloaded from http://pubs.acs.org on December 29, 2017
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Structural Identification and Kinetic Analysis of the In Vitro
Products Formed by Reaction of Bisphenol Aꢀ3,4ꢀquinone with
NꢀAcetylcysteine and Glutathione
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*
Douglas E. Stack , John A. Conrad, and Bejan Mahmud
Department of Chemistry, University of Nebraska at Omaha, Omaha, Nebraska, 68182,
United States
KEYWORDS Bisphenol A, o-quinones, conjugate addition, glutathione adducts
*Contact:
Phone: 402-554-3647; email: dstack@unomaha.edu; address: Department of Chemistry, University
of Nebraska at Omaha, 6001 Dodge Street, Durham Science Center, Omaha, NE 68182-0109
ORICD
Douglas E. Stack 0000-002-8253-860X
John A. Conrad 0000-0002-0588-8761
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Abstract:
Bisphenol A (BPA) has received considerable attention as an endocrine disrupting chemical and
a possible substrate for genotoxic metabolites. BPA metabolism leads to formation of
electrophilic oꢀquinones cable of binding to DNA and other endogenous nucleophiles. We have
structurally identified the products resulting from the reaction of bisphenol Aꢀ3,4ꢀquinone
(BPAQ) with Nꢀacetylcysteine (NAC) and glutathione (GSH). The major and minor isomers are
both the result of 1,6ꢀconjugate addition and are produced almost instantly in high yield.
Reactions using 1.3 equiv of GSH showed the presence of a bisꢀglutathionyl adduct which was
not observed using higher GSH concentration relative to BPAQ. NAC reactions with BPAQ
showed no bisꢀNꢀacetylcysteinyl adducts. Stoppedꢀflow kinetic analysis reveals the 1,6ꢀ
conjugate additions to be reversible with a forward free energy of activation of 9.2 and 7.8
kcal/mol for the NAC and GSH reactions, respectively. The bimolecular forward rate constant at
o
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9.4 C was approximately three time faster for GSH compared to NAC, 1547 vs 496 M s . The
free energy of activation for the reverse reactions were similar, 11.7 and 11.2 kcal/mol for NAC
and GSH, respectively. We plan to use this model system to further explore the mechanism of
adduct formation between sulfur nucleophiles and oꢀquinones and the resulting chemical
properties of both NAC and GSH adducts.
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Introduction
Bisphenol A (BPA) is a widely used industrial chemical prominent in the manufacture of
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plastics and epoxy resins. BPA and its metabolites can act as estrogen mimics and are classified
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as endocrine disrupting chemicals. Evidence for genotoxic effects in vitro have been revealed in
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studies involving mouse lymphoma , MCFꢀ7 and CHOꢀK1 cells. In vivo studies showed
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structural changes in bone marrow cells of mice.
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The major metabolites of BPA include glucuronic acid conjugates and catechols. Catechols
can undergo further oxidation to oꢀquinones (Scheme 1), known genotoxic compounds cable of
binding to the nucleosides of DNA. The monoꢀoꢀquinone of BPA, BPAꢀ3,4ꢀquinone (BPAQ)
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, 9
reacts with deoxyguanosine via 1,4ꢀconugate addition.
BPAQꢀDNA adducts have been
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detected in vitro and in vivo with either BPAQ exposure or metabolically activated BPA.
BPAQ chemistry has shown to be analogous to estrogen oꢀquinones, known genotoxic
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metabolites. Glutathione (GSH) is a prominent oꢀquinone scavenger. Several studies have
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shown the production BPAQꢀGSH adducts in vitro as analyzed by LC/MS
which confirmed
the presence of catecholꢀGSH products but do not indicate the regiochemistry of the conjugate
addition, namely 1,4ꢀ versus 1,6ꢀaddition. Soft sulfur nucleophiles show a preference for 1,6ꢀ
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addition with various oꢀquinones but 1,4ꢀaddition products are also observed.
Recently we have developed a large scale synthetic approach to the BPA metabolites, BPAQ,
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the diꢀoꢀquinone of BPA, the monocatechol of BPA, and the dicatechol of BPA. With this new
synthetic technique at hand, we sought to determine the major product(s) when BPAQ reacts
with NAC and GSH. Scheme 2 shows the three possible conjugate addition products when
BPAQ reacts with these sulfur nucleophiles. Products 2-SR and 5-SR are the result of 1,6ꢀ
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addition with respect to BPAQ C4 and C3 carbonyls, respectively. Adduct 6-SR would result
from 1,4ꢀaddition with respect to BPAQ C4 carbonyl.
BPAQ is a relatively stable oꢀquinone with a deep red color in organic solvents. We
recognized that the stability of BPAQ combined with its chromophore properties would make
kinetic analysis of the conjugate addition possible. Conjugate addition of sulfur nucleophiles to
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certain quinones has shown reversible characteristics indicating that sulfur detoxification may
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lead to other metabolic process.
Second order rate constants for both NAC and GSH
conjugate addition to various Michael acceptors has been measured and shown to be a function
of both steric and electronic factors of the electrophile in addition to the pKa of the thiol
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nucleophile.
Herein we described the synthesis and full structural characterization of the NAC and GSH
adducts of BPAQ. In addition, the measurement of secondꢀorder rate constants using stoppedꢀ
flow techniques and the resulting thermodynamic parameters are presented to establish a better
understanding of the reversible mechanism involved with thiol Michael additions to oꢀquinones.
Experimental Procedures
Chemicals and Reagents. 2ꢀIodoxybenzoic acid (IBX) was synthesized by the method of
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Frigerio , CAUTION! IBX is explosive under impact if heated to more than 200 C. BPAQ was
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made by IBX oxidation of BPA with modifications to product isolation (see below). Deuterated
NMR solvents were purchased from Cambridge Isotope (Andover, MA). All other chemicals and
solvents were purchased from Fisher Scientific Co. (Fair Lawn, NJ) or Aldrich Chemical Co.
(Milwaukee, WI) and used as received.
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Instrumentation. NMR analysis was obtained on a Bruker 400 MHz Avance III
spectrometer (Bruker, Billerica, MA, USA). HPLC was conducted on a Waters 2690 Separations
Module (Waters Corp. Milford, MA, USA) equipped with a Waters 2487 Dual λ Absorbance
Detector using solvent gradients consisting of acetonitrile and 0.5% aqueous formic acid on a
YMC, ODSꢀAQ C18 analytical column (5 ꢁm, 120Å, 250 mm × 4.5 mm, YMC America,
Allentown, PA, USA). Flash column purification of compounds was on a Biotage Isoler One
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(Biotage, Charlotte, NC) automated system using Biotage KPꢀShere 30 g and SNAP Ultra HP 12
g C18 columns. Stoppedꢀflow analysis was done on TgK Scientific, KinetAsyst system (TgK
Scientific, BradfordꢀonꢀAvon, UK). Reaction traces were fit to a sum of three exponentials using
KaleidaGraph (Synergy Software, Reading , PA, USA) . HRMS analysis was conducted at the
Nebraska Center for Mass Spectrometry (University of NebraskaꢀLincoln, Lincoln, NE).
Elemental analysis was obtained at MHW Labs (Phoenix, AZ).
Synthesis of 3-Hydroxy-5-NAC-BPA and 3- Hydroxy -2-NAC-BPA. BPA (0.250 g,
1
.10 mmol) was dissolved in 50 mL of a solvent mixture consisting of 3:2 CHCl :CH OH in a
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00 mL round bottom, equipped with a magnetic stir bar and brought to ꢀ15 C. To this solution
was added IBX 0.337 g (1.20 mmol) causing the solution to turn deep red after approximately 15
o
min. The mixture was stirred at ꢀ15 C for 12 h, transferred to a 250 mL separatory funnel and
the chloroform layer by washed 6X50 mL with 0.1M phosphate buffer, pH 6.0 to remove the oꢀ
iodobenzoic acid byproduct. The chloroform solvent was removed via rotovap and the crude was
placed in 50 mL of methanol. HPLC analysis of the crude methanol solution showed >95%
BPAQ which was used without further purification. Nꢀacetylcysteine, (235 mg, 1.43 mmol) was
dissolve in 5 mL of 0.1 M phosphate buffer, pH 6.2 and added in one portion to the BPAQ
solution. The mixture decolorized instantly from bright red to a light yellow color. The mixture
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was stirred an additional 10 min at rt , an HPLC sample was removed, and all solvents removed
via rotovap. The crude mixture was dissolved in1ꢀ2 mL of methanol and placed on a
preconditioned Biotage 30 g C18 flash column (see Figure S1 for flash chromatograph with
solvent gradient). Each collection tube was analyzed by HPLC on an analytical column and three
fractions were collected: one containing analytical grade 5-NAC, a middle fraction containing a
mixture of 5-NAC and 2-NAC, and fraction containing analytical grade 2-NAC. The middle
fraction was subject to a second separation using a 12g C18 SNAP Ultra column, solvents were
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removed and solids dried overnight under high vacuum at 50 C to afford a total of 319 mg
(81%) of 3ꢀhydroxyꢀ5ꢀNACꢀBPA and 27 mg (7%) of 3ꢀhydroxyꢀ2ꢀNACꢀBPA.
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3ꢀHydroxyꢀ5ꢀNACꢀBPA : needle white solid; H NMR (DMSOꢀd6, 400MHz): δ 1.52 (s, 6H),
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.84 (s, 3H), 2.95 (dd, J=13.4 and 9.2 Hz, 1H), 3.23 (dd, J=13.4 and 4.7 Hz, 1H), 4.28 (m, 1H),
.43 (d, J=2.2 Hz, 1H), 6.65 (d, J=8.7 Hz, 2H), 6.68 (d, J=2.2 Hz, 1H), 7.00 (d, J=6.7 Hz,2H)
8.24 (d, J=8.0 Hz), 9.18 (bs, phenolic OH partially exchanged), 12.67 (very bs, carboxylate OH).
1
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For H spectrum with structure assignment see Sꢀ4. C NMR (DMSOꢀd6, 100MHz): δ 22.39,
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0.71, 30.73, 33.76, 41.14, 51.70, 113.65, 114.59, 118.72, 119.69, 127.30, 140.81, 141.90,
1
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42.17, 144.60, 154.95, 169.27, 172.27. For C spectrum with structure assignment see S5.
Anal. Calcd for C H NO S: C, 59.25; H, 5.72; N, 3.45; S, 7.91. Found: C, 59.45; H, 5.84; N,
2
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.30; S, 8.18.
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3ꢀHydroxyꢀ2ꢀNACꢀBPA : white solid; H NMR (DMSOꢀd6, 400MHz): δ 1.58 (s, 3H), 1.62 (s,
3
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H), 1.83 (s, 3H), 2.51(m, 1H, peak obscured by residual DMSO), 2.69 (dd, J=12.5 and 4.7 Hz.
H), 4.04 (m, 1H), 6.57 (d, J=8.5 Hz, 1H), 6.76 (d, J=8.9 Hz, 1H), 6.79 (d, J=8.5 Hz, 1H), 6.86
1
(d, J=8.9 Hz, 1H), 7.98 (d, J=7.8 Hz), 9.17 (bs, 3H phenolic OH partially exchanged). For H
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spectrum with structure assignment see Sꢀ10. C NMR (DMSOꢀd6, 100MHZ): δ 22.34, 30.96,
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2.80, 52.05, 114.03, 114.44, 116.73, 142.12, 143.05, 143.74, 147.69, 154.26,169.16, 172.13.
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For C spectrum with structure assignment see Sꢀ11. HRMS (ESI+); calcd for
+
C H NO S[M+H] : 406.1246; found: 406.1253.
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Synthesis of 3-Hydroxy-2,5-diGSH-BPA, 3- Hydroxy -5-GSH-BPA, and 3- Hydroxy -2-
GSH-BPA. A 1.10 mmol solution of BPAQ was made as above except after workꢀup, BPAQ
was dissolved in 50 mL of 50:50 methanol: water solvent system. GSH, (572 mg, 1.43 mmol)
was dissolved in 5 mL of 0.1M phosphate buffer, pH 6.2 and added in one portion to the BPAQ
solution. The mixture was stirred at rt for 15 min and solvents removed via rotovap. The crude
solid was dissolved in 50 mL of water and extracted 3 X 25 mL with ethyl acetate to remove the
3ꢀhydroxyBPA reduction byproduct. The ethyl acetate extract was dried over magnesium sulfate
and solvent removed via rotvap. The aqueous phase was then reduced to 2 mL via rotovap and
place on a preconditioned Biotage 30 g C18 SANP Ultra flash column (see Figure S2 for flash
chromatograph with solvent gradient). Each collection tube was analyzed by HPLC on an
analytical column and four fractions containing pure 2,5- diGSH, a mixture of 2,5- diGSH and
5
-GSH, pure5-GSH, and pure 2-GSH were collected. The fraction containing the 2,5-GSH/5-
GSH mixture was subjected to a second separation using a Biotage 30 g C18 SANP Ultra flash
o
column, solvents were removed and solids dried overnight under high vacuum at 50 C to afford a
total of 151 mg of 3ꢀhydroxyꢀ2,5ꢀdiGSHꢀBPA (17%), 212 mg of 3ꢀhydroxyꢀ5ꢀGSHꢀBPA (36%),
4
3 mg of 3ꢀhydoxyꢀ2ꢀGSH (7%), and 81 mg of 3ꢀhydroxyBPA (32%)
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3ꢀHydroxyꢀ5ꢀGSHꢀBPA : offꢀwhite solid; H NMR (DMSOꢀd6, 400MHz): δ 1.51 (s, 6H), 1.93
(m, 2H), 2.33 (t, 2H, J=6.5Hz), 2.91 (dd, 1H, J=13.2 and 10.1 Hz), 3.23 (dd, 1H, J = 13.2 and 4.1
Hz), 3.37 (t, 1H, J=6.7Hz), 3.67 (m, 2H), 4.33 (m, 1H), 6.42 (d, 1H, J=2.1Hz), 6.61ꢀ6.68 (3H,
arene protons 6 and 3’ overlap), 6.99 (d, 2H, J=8.7Hz), 8.42 (d, 1H, J=7.9 Hz), 8.52 (t, 1H,
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J=5.7Hz). For H spectrum with structure assignment see Sꢀ15. C NMR (DMSOꢀd6,
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00MHZ): δ 26.73, 30.75, 30.78, 31.45, 34.52, 41.11, 41.29, 52.36, 53.10, 113.91, 114.62,
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118.87, 120.29, 140.72, 141.97, 142.21, 144.76, 155.01, 170.65, 170.88, 171.03, 171.78. For C
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spectrum with structure assignment see Sꢀ16. HRMS (ESI+); calcd for C H N O S [M+H] :
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49.1859; found: 549.1871.
3ꢀHydroxyꢀ2ꢀGSHꢀBPA : offꢀwhite solid; H NMR (DMSOꢀd6, 400MHz): δ 1.57 (s, 3H), 1.63
s,3H), 1.91 (m, 2H), 2.30 (t, 2H, J=7.0Hz), 2.41 (dd, 1H, J=12.0 and 10.3 Hz), 2.62 (dd, 1H, J =
2.1 and 4.7 Hz), 3.40 (t, 1H, J=6.4Hz), 3.65 (m, 2H), 4.16 (m, 1H), 6.59 (d, 2H, J=8.7Hz), 6.76
(d, 1H, J=8.1Hz), 6.79 (d, 2H, J=8.7Hz), 6.86 (d, 1H, J=8.1Hz) 8.05 (d, 1H, J=7.5 Hz), 8.52 (t,
1
(
1
1
13
1
H, J=5.6Hz). For H spectrum with structure assignment see Sꢀ18. C NMR (DMSOꢀd6,
1
00MHZ): δ 26.73, 30.93, 31.44, 32.33, 35.99, 41.19, 42.81, 52.51, 53.17, 114.49, 114.67,
1
3
116.82, 120.32, 126.75, 142.07, 143.14, 143.93, 147.75, 154.46, 170.56, 170.98, 171.66. For C
+
spectrum with structure assignment see Sꢀ19. HRMS (ESI+); calcd for C H N O S [M+H] :
2
5
31
3
9
5
49.1859; found: 549.1870.
1
3ꢀHydroxyꢀ2,5ꢀdiGSHꢀBPA : offꢀwhite solid; H NMR (D O, 400MHz): δ 1.49 (s, 3H), 1.52 (s,
2
3
H), 2.03ꢀ2.23 (m, 6H), 3.16 (dd, 1H, J=14.5 and 8.2 Hz), 3.34 (dd, 1H, J=14.4 and 5.0 Hz), 3.72
(
m, 2H), 3.86 (m, 2H), 3.95 (m, 2H), 4.03 (dd, 1H, J=8.2 and 5.9 Hz), 4.35 (dd, 1H, J=8.0 and
1
5.0Hz), 6.66 (d, 2H, J=8.8Hz), 6.95 (d, 2H, J=8.7Hz). For H spectrum with structure assignment
13
see Sꢀ20. C (D O, 100MHz, chemical shifts calibrated with post methanol addition): 26.12,
2
2
6.16, 31.20, 31.61, 31.64, 31.94, 35.22, 35.59, 41.63, 41.72, 43.83, 5.97, 52.99, 53.41, 115.46,
118.39, 119.78, 120.38, 123.81, 128.43, 143.92, 144.07, 145.90, 147.93, 153.58, 163.37, 163.72,
1
3
1
72.28, 172.94, 173.38, 174.53, 174.62. For C spectrum with structure assignment see Sꢀ21.
+
HRMS (ESI+); calcd for C H N O S2 [M+H] : 855.2541; found: 855.2554.
35
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Stopped-flow Kinetic Analysis. A 125 µM BPAQ solution (final concentration) in 50%
methanol was mixed with various concentration of Nꢀacetylcysteine (0.050 M, 0.10 M, and
0.125 M; final concentration) in 0.050 M phosphate buffer pH 6.2 and 50 % methanol using a
1
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stoppedꢀflow spectrophotometer (TgK Scientific). Concentration dependence was carried out at
o
5
different temperatures: 4.6 ºC, 9.6 ºC, 14.5 ºC, and 19.4 C. Reactions were monitored at 400
nm. Reaction traces were fit to a sum of three exponentials using KaleidaGraph (Synergy
Software). GSH reactions under the same conditions with the following changes: BPAQ
concentration 65 ꢀM; GSH concentrations 40, 20, and 10 mM.
Results
Reaction of BPAQ with NAC and NMR Analysis of Mercapturate Products. When BPAQ
is dissolved in methanol and an aqueous solution of NAC (1.3 equiv, phosphate buffer, pH 6.2)
is added, rapid decolorization of the red BPAQ solution is observed. Figure 1 shows the HPLC
chromatograph of the crude mixture indicating the presence of two product peaks along with
trace amounts of reduced 3ꢀhydroxyBPA (3ꢀOHBPA). The major product (89%) was 5-NAC the
minor product was 2-NAC (11%); both products are the result of 1,6ꢀconjugate addition (see
Scheme 2). Of the three possible isomers in Scheme 2, the most easily distinguished by 1ꢀD
NMR is 2-SR since the vicinal arene protons H5 and H6 on the catechol ring would show a
distinct coupling pattern when compared to 5-SR or 6-SR. Discrimination between 5-NAC and a
possible 6-NAC isomer required a combination of both 1ꢀD and 2ꢀD NMR analysis.
Heteronuclear (CꢀH) multiple bond correlation (HMBC) proved to be the most direct 2ꢀD NMR
technique in establishing the structure of 5-NAC from 6-NAC. HMBC optimized for three bond
correlation would show three cross peaks between the quaternary, saturated carbon connecting
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the two arene rings to arene protons in structure 5-NAC, while 6-NAC would show only two
correlations. Figure 2A shows the portion of the HMBC spectrum where these correlation occur
clearly showing three cross peaks consistent with isomer 5-NAC. Several noteworthy differences
in chemical shifts between the 5-NAC and the 2-NAC isomers were also observed. The
methylene protons alpha to the sulfur atom on the NAC moiety were shifted downfield relative
to NAC on the 5-NAC adduct while the same signals of the 2-NAC isomer were shifted upfield
relative to NAC (Figure 2B). The proton beta to the sulfur atom in the 2-NAC isomer displayed a
significant upfield shift relative to NAC while the 5ꢀNAC shifted upfield slightly. The 5-NAC
isomer showed significant throughꢀspace NOE correlations between arene proton H6 to both the
aforementioned alpha and beta protons. These observation proved useful in establishing the
structure of the larger GSH adducts (vida infra).
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Reaction of BPAQ with GSH and NMR Analysis of Glutathione Adducts. When BPAQ
was reacted with an aqueous solution of GSH under similar conditions used with NAC, several
significant differences were observed. The chromatograph in Figure 3 shows the presence of four
significant peaks when 1.3 equiv of GSH was mixed with BPAQ. The peak corresponding to 3ꢀ
OHBP, the result of BPAQ reduction, is observed in much larger quantities when compared to
the NAC reaction. Moreover, an early eluting peak was identified as the bisꢀglutathionyl adduct
of BPAQ, 2,5-diGSH. Figure 3 also shows reactions done a smaller, 0.5 mM scale of BPAQ
with increasing equivalents of GSH. At fiveꢀfold excess of GSH, little of the di-2,5-GSH adduct
or the reduced 3ꢀOHBPA catechol were observed. Only monoꢀadducted adducts were observed
for NAC when using 1.3, 2.0.or 5.0 equiv of NAC. For the large scale reaction using 1.3 equiv of
GSH, isolated yields produced 17% 2,5-diGSH, 36% 5-GSH, 7% 2-GSH, and 32% 3-OHBPA.
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NMR analysis of the 5-GSH and the 2-GSH adducts was similar to the NAC adducts. The
NMR properties of the NAC adducts was useful in establishing the location of the two GSH
moieties in 2,5-diGSH; specifically, distinguishing between a 2,5ꢀdiadduct versus a 2,6ꢀ or 5,6ꢀ
diadduct. First, two resolved sets of methylene protons alpha to the sulfur atom were observed,
one shifted downfield and one shifted upfield relative to GSH. This is consistent with a 2,5ꢀ
disubstitution profile (vida supra, Figure 2B). Also, throughꢀspace NOE experiments shows only
one of these methylene signals correlates to the lone remaining arene proton on the catechol ring,
specifically the methylene protons shifted downfield. Thus, the 2,5-diGSH adduct is the result of
two separate 1,6ꢀconjugated additions. No products, monoꢀ or diꢀadducted, were the result of
1,4ꢀaddition conjugate addition.
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Kinetic Analysis of the BPAQ-NAC Reaction and BPAQ-GSH Reaction. Using the distinct
chromophore of the BPAQ, we sought to explore the kinetics of NAC and GSH conjugate
addition to BPAQ by following the disappearance of BPQA at 400 nm. Figure 4A shows the
decrease in the absorbance when a 125 ꢀM solution of BPAQ in 50% methanol was mixed with
various concentrations of NAC (0.050 M, 0.10 M, and 0.125 M; final concentration) in 0.050 M
phosphate buffer pH 6.2 and 50% methanol using a stoppedꢀflow spectrophotometer. To simplify
the exponential decay, the reaction was made pseudoꢀfirst order using a large excess of NAC. A
similar process was performed using GSH using more dilute solutions due to the reduced
solubility of GSH in 50% methanol when compared to NAC. Fitting software indicates the decay
is the sum of three different exponentials indicating three separate phases. The first phase, which
represents 86% of absorbance decrease, is both NAC concentration and temperature dependent.
The second phase is NAC concentration independent and temperature dependent. The third
phase is both temperature and NAC concentration independent which suggest it is not part of the
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reaction process but an artifact of how components were mixed. Reaction with GSH displayed
the same three exponential decays.
Analysis of the first phase was carried out by plotting the observed rate constants as a function
of NAC or GSH concentration generating liner plots the slope of which corresponds to the
0
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forward rate constants, k , of the bimolecular conjugate addition (Figure 4B). Nonꢀzero
1
intercepts indicates a reversible process and the value of these nonꢀzero intercept represents the
reverse rate constant, k . Both NAC reactions and GSH reaction displayed a reversible profile
ꢀ1
with GSH forward rate constants approximately three time faster than NAC. Each NAC
concentration was carried out at four temperatures to obtain an Arrhenius plots of both the
forward and reverse reactions. Only three different temperatures were conducted with GSH as
insolubility limited lower temperatures (Supporting Information, Figures S3 and S4). The free
energy of activation for the forward reaction was calculated at 9.2 and 7.8 kcal/mol for the NAC
‡
and GSH reactions, respectively. These ꢁG values are similar with computational calculations,
1
0.0 kcal/mol, that model the reaction of GSH with BPAQ even though the modeled reaction
24
assumed a 1,4ꢀconugate addition . Error analysis for the line slopes was between 4ꢀ6% while the
‡
yꢀintercepts were between 7ꢀ10%; the final ꢁG values reported are ca. ± 0.5 kcal/mol. The free
energy of activation for the reverse process was similar for both NAC and GSH, 11.7 and 11.2
kcal/mol, respectively (see Figure 4C). The kinetic data of the first phase indicates a reversible
1,6ꢀaddition, which is NAC concentration dependent. The second phase observed, Figure 4A, is
likely from the proton transfers involved in the tautomerization to the final catechol product.
These proton transfers would be temperature dependent while independent of the concentration
of NAC, which is consistent with our data.
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Discussion. If BPAQ is a significant metabolite formed by exposure to BPA, the most
2
5ꢀ27
abundant biomarker would likely be mercapturates in the form of the NAC adducts.
This
work provides a simple and efficient methodology in the synthesis of both the NAC and GSH
adducts of BPAQ. Both nucleophiles showed exclusive preference for 1,6ꢀaddition in lieu of 1,4ꢀ
addition. The formation of a bisꢀglutathionyl adduct with GSH as opposed to NAC, which
showed no bisꢀNꢀacetylꢀcysetinyl adducts, is of interest in that bisꢀadduct formation implies
differences in the oxidation potential of GSH and NAC adducts. The mechanism for bisꢀadduct
formation likely involves oxidation of the newly formed catechol adduct by unreacted oꢀquinone,
BPAQ (Scheme 3). This would explain the concurrent increase of 3ꢀOHBPA observed in the
GSH reaction were 2,5-diGSH is produced as compared to the NAC reaction were very little of
the 3ꢀOHBPA catechol is observed. This also implies that the 5-GSH adduct has a lower
oxidation potential when compared to the 5-NAC adduct. Macedo et al., using cyclic
voltammetry, showed GSH adducts formed from the oꢀquinones of αꢀmethyldopamine and Nꢀ
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methylꢀαꢀdopamine had lower oxidation potentials (30 to 65 mV), when compared to the
2
8
corresponding NAC adducts. This suggest that GSH imparts a stronger electron donating effect
when compare to NAC increasing k in Scheme 3 which facilitates formation of 2,5-diGSH. If
2
k is greater than k , increasing GSH concentration relative to BPAQ would decrease the rate of
1
2
5
-GHS-BPAQ formation and subsequent formation of 2,5-diGSH.
Kinetic data shows the reversible nature of conjugate addition of both NAC and GSH to
BPAQ. The forward rate constant for GSH reaction was significantly faster than NAC reaction.
Freeman et al., also using stoppedꢀflow analysis, observed much faster conjugate addition of
GSH relative to NAC with α,βꢀunsaturated nitro compounds derived from fatty acids and
2
1
postulated this was function of thiol acidity. GSH, pKa 8.8, is more acidic than NAC, pKa
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9
.5. Thus, at a pH of 6.2, the effective concentration of thiolate anion would much larger with
GSH solutions compared to NAC. Properties of other GSH and NAC adducts have suggested
that the sulfur adduction process is reversible. GSH adducts formed between the oꢀquinone of 4ꢀ
hydroxyequilenin, which exists in the oxidized oꢀquinone form, liberated 4ꢀhydroxyequilenin
0
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18
and GSH when exposed to NADPH reduction. Isomeric GSH adducts of the oꢀquinone formed
30
from quercetin oxidation were shown to interconvert when allowed to equilibrate in solution.
GSH adducts of allyl and benzyl isothiocyanate conjugation were shown to release the
31
isothiocyanate electrophile in vitro. GSH conjugation is generally considered a detoxification
process used to sequester reactive electrophiles such a s oꢀquinones. However GSH conjugates
formed from various quinones display toxicity in certain tissues and many of these GSH adducts
32ꢀ35
stem from pꢀ or oꢀquinones.
Is there a correlation between the reversible nature of quinone
conjugate addition and the subsequent toxicity of GSH adducts? The BPAQ adducts synthesized
in this work, efficiently made at large scale, and generated from a relatively stable quinone with
a distinctive chromophore, could serve as a platform for further studies to this effect.
Funding Sources
Partial support of the work was provided by Funds for Undergraduate Scholarly Experiences
(
FUSE), University of Nebraska at Omaha.
Supporting Information
Chromatography conditions for the separation of NAC and GSH adducts; 1ꢀD and 2ꢀD NMR
spectra of NAC and GSH adducts; Arrhenius plots used to calculate free energies of activation
(
PDF). This material is available free of charge via the Internet at http://pubs.acs.org.
Abbreviations
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BPA, bisphenol A; BPAQ, bisphenol Aꢀ3,4ꢀquinone; NAC, Nꢀacetyl cysteine; GSH,
glutathione; IBX, 2ꢀiodoxybenzoic acid; HMBC, heteronuclear multiple bond correlation; 3ꢀ
OHBPA, 3ꢀhydroxybisphenol A;
1
1
1
1
1
1
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2
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2
2
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References
(
1) Staples, C. A., Dome, P. B., Klecka, G. M., Oblock, S. T., and Harris, L. R. (1998) A
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(
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(
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pot synthesis of the monocatechol, monoꢀoꢀquinone, dicatechol, and diꢀoꢀquinone of bisphenol
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(17) Chandrasena, R. E. P., Edirisinghe, P. D., Bolton, J. L., and Thatcher, G. R. J. (2008)
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and glutathioneꢀsꢀtransferase. Chem. Res. Toxicol. 21, 1324ꢀ1329.
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22) Chen, J., Jiang, X., Carroll, S. L., Huang, J., and Wang, J. (2015) Theoretical and
experimental investigation of thermodynamics and kinetics of thiolꢀmichael addition reactions:
A case study of reversible fluorescent probes for glutathione imaging in single cells. Org. Lett.
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24) Kolšek, K., Sollner Dolenc, M., and Mavri, J. (2013) Computational study of the
(
(
reactivity of bisphenol Aꢀ3,4ꢀquinone with deoxyadenosine and glutathione. Chem. Res.
Toxicol. 26, 106ꢀ111.
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1992) Mercapturic acids, protein adducts, and DNA adducts as biomarkers of electrophilic
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acids as biomarkers of exposure to electrophilic chemicals: Applications to environmental and
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determination by liquid chromatography/mass spectrometry and their use in toxicant
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(28) Macedo, C., Branco, P. S., Ferreira, L. M., Lobo, A. M., Capela, J. P., Fernandes, E.,
Bastos, M. d. L., and Carvalho, F. (2007) Synthesis and cyclic voltammetry studies of 3,4ꢀ
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29) Trujillo, M., and Radi, R. (2002) Peroxynitrite reaction with the reduced and the oxidized
forms of lipoic acid: New insights into the reaction of peroxynitrite with thiols. Arch. Biochem.
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(30) Boersma, M. G., Vervoort, J., Szymusiak, H., Lemanska, K., Tyrakowska, B., Cenas, N.,
SeguraꢀAguilar, J., and Rietjens, I. M. C. M. (2000) Regioselectivity and reversibility of the
glutathione conjugation of quercetin quinone methide. Chem. Res. Toxicol. 13, 185ꢀ191.
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31) Bruggeman, I. M., Temmink, J. H. M., and Van Bladeren, P. J. (1986) Glutathioneꢀ and
cysteineꢀmediated cytotoxicity of allyl and benzyl isothiocyanate. Toxicol. Appl. Pharmacol.
3, 349ꢀ359.
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conjugates. Chem. Res. Toxicol. 10, 1296ꢀ1313.
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33) Monks, T. J., Anders, M. W., Dekant, W., Stevens, J. L., Lau, S. S., and Van Bladeren, P.
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34) Lau, S. S., Jones, T. W., Highet, R. J., Hill, B. A., and Monks, T. J. (1990) Differences in
(
the localization and extent of the renal proximal tubular necrosis caused by mercapturic acid
and glutathione conjugates of 1,4ꢀnaphthoquinone and menadione. Toxicol. Appl. Pharmacol.
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04, 334ꢀ350.
35) Lau, S. S., Hill, B. A., Highet, R. J., and Monks, T. J. (1988) Sequential oxidation and
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glutathione addition to 1,4ꢀbenzoquinone: Correlation of toxicity with increased glutathione
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Figure Legends
Figure 1. HPLC chromatograph of the reaction of BPAQ (1.0 equiv) and NAC (1.3 equiv), in
CH OH:H O 90:10, rt at 10 min.
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Figure 2. A. Portion of the HMBC spectrum of 5-NAC showing three cross peaks between the
saturated quaternary carbon and arene protons H2, H6 , and H2’. B. Chemical shifts of the αꢀ
methylene and βꢀmethide protons (relative to sulfur) in NAC, 5-NAC, and 2-NAC.
Figure 3. HPLC chromatographs of the reaction of BPAQ (1.0 equiv) and GSH (1.3, 2.0, and 5.0
equiv), in 50:50 CH OH:H O, 10 min at rt. HPLC conditions similar to Figure 1 except solvent
3
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gradient was 20% acetonitrile: 80% 0.2% formic acid for 2 min. then linear gradient to 100%
acetonitrile at 15 min.
Figure 4. A. The decrease in absorption of a 125ꢀM solution of BPAQ in 50% methanol when
mixed with various, excess concentrations of NAC. B. The observed rate constants as a function
of NAC concentration run at four different temperatures. C. Forward and reverse rate constants
for the reaction BPAQ with NAC and GSH at various temperatures. D. Free energies of
activation, derived from Arrhenius plots, for both forward and reverse conjugate additions.
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Scheme Legends
Scheme 1. Metabolism of BPA and conjugation with sulfur nucleophiles.
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Scheme 2. Possible isomers from the reaction of BPAQ with sulfur nucleophiles. 2-SR and 5-SR
are products of 1,6ꢀaddition while 6-SR would result from 1,4ꢀaddition.
Scheme 3. Possible mechanism for the formation of 2,5-diGSH from reaction of BPAQ with
GSH.
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