Oxidation of dopamine in the presence of cysteine: Characterization of new toxic products

Article abstract of DOI:10.1021/tx960145c

Previous studies demonstrated that oxidation of dopamine (DA) in the presence of L-cysteine (CySH) at pH 7.4 gives a complex mixture of cysteinyl conjugates of the neurotransmitter that can be easily further oxidized to a number of dihydrobenzothiazines (DHBTs) along with unidentified yellow products. In this investigation, three of these products have been identified. 7-(2-Aminoethyl)-5-hydroxy-1,4-benzothiazine-3-carboxylic acid (BT-1) is formed as a result of oxidation of 5-S-cysteinyldopamine (5-S-CyS- DA) and 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3- carboxylic acid (DHBT-1). Regioisomers 6-(2-aminoethyl)1,8,9,10- tetrahydrobenzo[1,2-b:4,3-b']bis[1,4]thiazine-9-carboxylic acid (12) and 6- 2-aminoethyl)1,2,3,10-tetrahydrobenzo[1,2-b:4,3-b']bis[1,4]thiazine-2- carboxylic acid (13) are formed by oxidation of 2,5-bi-S-cysteinyldopamine (2,5-bi-S-CyS-DA), 6-S-cysteinyl-7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H- 1,4-benzothiazine-3-carboxylic acid (DHBT-2), and 6-S-cysteinyl-8-(2- aminoethyl)-3,4-dihydro-5-hydroxy-2H- 1,4-benzothiazine-3-carboxylic acid (DHBT-6). 2,5-Bi-S-CyS-DA, DHBT-2, and DHBT-6 are major early products of DA oxidation in the presence of CySH. However, because these three compounds are the most easily oxidized products formed in this reaction, they are subsequently transformed into 12 and 13, the latter regioisomer always being the major product. Both 12 (LD₅₀ = 18.5 μg) and 13 (LD₅₀ = 1.5 μg) are lethal when administered into the brains of mice and evoke hyperactivity and tremor. The potential relevance of the in vitro chemistry described in this and earlier reports to reactions that might occur in neuromelanin-pigmented dopaminergic neurons in Parkinson's disease is discussed.

Full text of DOI:10.1021/tx960145c

Chem. Res. Toxicol. 1997, 10, 147-155
147
Articles
Oxid a tion of Dop a m in e in th e P r esen ce of Cystein e:
Ch a r a cter iza tion of New Toxic P r od u cts
Xue-Ming Shen, Fa Zhang, and Glenn Dryhurst*
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019
Received August 15, 1996X
Previous studies demonstrated that oxidation of dopamine (DA) in the presence of L-cysteine
(CySH) at pH 7.4 gives a complex mixture of cysteinyl conjugates of the neurotransmitter
that can be easily further oxidized to a number of dihydrobenzothiazines (DHBTs) along with
unidentified yellow products. In this investigation, three of these products have been identified.
7-(2-Aminoethyl)-5-hydroxy-1,4-benzothiazine-3-carboxylic acid (BT-1) is formed as a result
of oxidation of 5-S-cysteinyldopamine (5-S-CyS-DA) and 7-(2-aminoethyl)-3,4-dihydro-5-
hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1). Regioisomers 6-(2-aminoethyl)-
1,8,9,10-tetrahydrobenzo[1,2-b:4,3-b′]bis[1,4]thiazine-9-carboxylic acid (12) and 6-(2-aminoethyl)-
1,2,3,10-tetrahydrobenzo[1,2-b:4,3-b′]bis[1,4]thiazine-2-carboxylic acid (13) are formed by
oxidation of 2,5-bi-S-cysteinyldopamine (2,5-bi-S-CyS-DA), 6-S-cysteinyl-7-(2-aminoethyl)-3,4-
dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-2), and 6-S-cysteinyl-8-(2-
aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-6). 2,5-Bi-
S-CyS-DA, DHBT-2, and DHBT-6 are major early products of DA oxidation in the presence of
CySH. However, because these three compounds are the most easily oxidized products formed
in this reaction, they are subsequently transformed into 12 and 13, the latter regioisomer
always being the major product. Both 12 (LD50 ) 18.5 µg) and 13 (LD50 ) 1.5 µg) are lethal
when administered into the brains of mice and evoke hyperactivity and tremor. The potential
relevance of the in vitro chemistry described in this and earlier reports to reactions that might
occur in neuromelanin-pigmented dopaminergic neurons in Parkinson’s disease is discussed.
cysteinyldopamine (5-S-CyS-DA; major product) and 2-S-
cysteinyldopamine (2-S-CyS-DA; minor product) (Scheme
1). Both 5-S- and 2-S-CyS-DA are more easily oxidized
than DA (1, 2). Thus, under conditions where DA is
oxidized, these cysteinyl conjugates are also further
oxidized. In the presence of free CySH, the in vitro
oxidations of 5-S-CyS-DA and 2-S-CyS-DA follow several
pathways, one of which leads to 2,5-bi-S-cysteinyldopam-
ine (2,5-bi-S-CyS-DA) and thence 2,5,6-tri-S-cysteinyl-
dopamine (2,5,6-tri-S-CyS-DA) by the pathways shown
in Scheme 1. However, o-quinones such as 2 and 3,
proximate oxidation products of 5-S-CyS-DA and 2-S-
CyS-DA, respectively, can also undergoes intramolecular
cyclization reactions (1-3). To illustrate, intramolecular
cyclization of 2 yields o-quinone imine 5 that can chemi-
cally oxidize 5-S-CyS-DA to 2 with concomitant formation
of radical 6. Disproportionation of 6 then gives dihy-
drobenzothiazine (DHBT)-12 and 5. Similar reaction
pathways lead to DHBT-5 from 2-S-CyS-DA and DHBT-2
and DHBT-6 from 2,5-bi-S-CyS-DA (Scheme 1) (2).
Details of the reaction pathways leading to these and
other DHBTs have been presented elsewhere (2). How-
ever, not only are most cysteinyl conjugates of DA more
easily oxidized than the neurotransmitter but so also are
the resulting DHBTs. In our earlier investigations it was
noted that oxidations of DA in the presence of free CySH
gave, in addition to cysteinyldopamines and DHBTs,
additional bright yellow products. Formation of these
In tr od u ction
Recent reports from this laboratory have described the
influence of L-cysteine (CySH)1 on the in vitro oxidation
chemistry of the catecholaminergic neurotransmitter
dopamine (DA) at neutral pH (1-3). These studies have
demonstrated that CySH diverts the normal oxidation
of DA to dark brown/black melanin polymer (4, 5) by
scavenging the proximate oxidation product of the neu-
rotransmitter, DA-o-quinone (1), to form initially 5-S-
* Address correspondence to this author: Tel (405) 325-4811; Fax
(405) 325-6111; E-mail gdryhurst@chemdept.chem.uoknor.edu.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
1
Abbreviations: Parkinson’s disease, PD; substantia nigra, SN;
dopamine, DA; dopamine-o-quinone, 1; L-cysteine, CySH; glutathione,
GSH; glutathione disulfide, GSSG; 5-S-cysteinyldopamine, 5-S-CyS-
DA; homovanillic acid, HVA; cerebrospinal fluid, CSF; γ-glutamyl
transpeptidase, γ-GT; 2-S-cysteinyldopamine, 2-S-CyS-DA; 2,5-bi-S-
cysteinyldopamine, 2,5-bi-S-CyS-DA; 2,5,6-tri-S-cysteinyldopamine,
2,5,6-tri-S-CyS-DA; dihydrobenzothiazine, DHBT; 7-(2-aminoethyl)-
3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid, DHBT-
1; 6-S-cysteinyl-7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-ben-
zothiazine-3-carboxylic acid, DHBT-2; 6-S-cysteinyl-8-(2-aminoethyl)-
3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid, DHBT-
6; pyrolytic graphite electrode, PGE; saturated calomel reference
electrode, SCE; nuclear magnetic resonance, NMR; fast atom bombard-
ment mass spectrometry, FAB-MS; high-performance liquid chroma-
tography, HPLC; 7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine-3-
carboxylic acid, BT-1; retention time, tR; trifluoroacetic acid, TFA; 6-(2-
aminoethyl)-1,8,9,10-tetrahydrobenzo[1,2-b:4,3-b]bis[1,4]thiazine-9-
carboxylic acid, 12; 6-(2-aminoethyl)-1,2,3,10-tetrahydrobenzo[1,2-b:
4,3-b′]bis[1,4]thiazine-2-carboxylic acid, 13; 6-(2-aminoethyl)-1,2,3,8,9,10-
hexahydrobenzo[1,2-b:4,3-b′]bis[1,4]thiazine-9-carboxylic acid, 14; 6-(2-
aminoethyl)-1,2,3,8,9,10-hexahydrobenzo[1,2-b:4,3-b′]bis[1,4]thiazine-
2-carboxylic acid, 15; heteronuclear multiple-quantum coherence,
HMQC; heteronuclear multiple bond connectivity, HMBC; peak po-
tential, Ep; dimethyl sulfoxide, Me2SO.
2 Abbreviations used for various dihydrobenzothiazines (DHBTs) are
identical to those employed in an earlier paper (2).
S0893-228x(96)00145-2 CCC: $14.00 © 1997 American Chemical Society

148 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Shen et al.
Sch em e 1
yellow products was favored by long oxidation times or
when DA was oxidized in the presence of relatively low
concentrations of CySH. This suggested that these
products are formed by oxidation of one or more DHBTs
or by alternative oxidative pathways from cysteinyl-
dopamine precursors. However, we were unable to
isolate and identify any of these yellow secondary prod-
ucts in our earlier investigation.
Our interest in investigating the influence of CySH on
the oxidation chemistry of DA stems from a number of
lines of evidence that have led us to propose that similar
reactions might occur in neuromelanin-pigmented dopam-
inergic neurons in the substantia nigra (SN) pars com-
pacta in Parkinson’s disease (PD). Dopaminergic SN
cells are pigmented with black neuromelanin as a result
of the autoxidation of cytoplasmic DA to 1 that oxida-
tively polymerizes (6, 7). Similar to other neuronal cell
bodies, dopaminergic SN neurons normally contain little
or no CySH or glutathione (GSH) (8, 9). However, it has
been reported that γ-glutamyl transpeptidase (γ-GT) is
significantly upregulated in the Parkinsonian SN (10).
γ-GT is the only known mammalian enzyme that can
cleave the γ-glutamyl bond of GSH to give CySH (11) and
plays a key role in the translocation of CySH and hence
GSH into cells, including neurons (12). Based, in part,
on such factors we have proposed (1, 2) that a very early
event in the pathogenesis of PD might be the γ-GT-
mediated translocation of CySH, biosynthesized and
exported along with GSH by nigral glial cells (13, 14),
into neuromelanin-pigmented SN neurons (1, 2). Such
a γ-GT-mediated translocation of CySH into these cells
might account not only for the decreased neuromelanin
content of surviving pigmented SN neurons (15-17) by
scavenging o-quinone 1 but also for the irreversible loss
of nigral GSH that is not accompanied by a corresponding
increase of glutathione disulfide (GSSG) (18-20), the
increased 5-S-CyS-DA/DA concentration ratio in the SN
(21), and the increased 5-S-CyS-DA/homovanillic acid
(HVA) ratio in cerebrospinal fluid (CSF) (22), all of which
occur in PD. Furthermore, we have also speculated that
one or more of the metabolites that should be formed in
the cytoplasm of SN cells as a consequence of the CySH-

Oxidation of Dopamine in the Presence of L-Cysteine
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 149
(FAB-MS) employed a VG Instruments (Manchester, U.K.)
ZAB-E spectrometer. Thermospray mass spectra were obtained
with a Kratos (Manchester, U.K.) MS25/RFA spectrometer
equipped with a thermospray source. UV-visible spectra were
recorded on a Hewlett-Packard (Palo Alto, CA) Model 8452A
diode array spectrophotometer.
High -P er for m a n ce Liqu id Ch r om a togr a p h y. HPLC em-
ployed two Gilson (Middleton, WI) gradient systems equipped
with either dual Model 302 pumps (10 mL pump heads) or dual
Model 306 pumps (25 mL pump heads). Both systems were
computer-controlled and were equipped with Rheodyne (Cotati,
CA) Model 7125 injectors and UV detectors set at 254 nm.
Preparative-scale, reversed phase columns (Bakerbond C18, 10
µm, 250 × 21.2 mm) were used. Four mobile phase solvents
were employed for HPLC. Solvent A was deionized water.
Solvent B was a 1:1 solution of MeCN and deionized water.
Solvent C was prepared by adding concentrated TFA to deion-
ized water until the pH was 2.15. Solvent D was prepared by
adding TFA to a 1:1 solution of MeCN and deionized water until
the pH was 2.15.
mediated diversion of the neuromelanin pathways might
include endotoxins that contribute to the selective de-
generation of nigrostriatal dopaminergic neurons in PD.
Whether any cysteinyldopamines or DHBTs are indeed
dopaminergic neurotoxins remains to be established
although 2,5-bi-S-CyS-DA and several DHBTs are toxic
(lethal) when administered into the brains of mice (1, 2).
However, the various cysteinyl conjugates of DA and
resultant DHBTs discovered in our previous investiga-
tions represent the products formed in the early stages
of oxidation of the neurotransmitter in the presence of
CySH. These putative nigral metabolites are more easily
oxidized than DA at physiological pH and, hence, if
formed in the cytoplasm of SN neurons would be expected
to undergo further oxidation reactions. Accordingly, the
principal goal of the present investigations was to identify
the major products responsible for the bright yellow color
that develops as a result of more prolonged oxidation of
DA in the presence of CySH. As in previous investiga-
tions (1-3), electrochemical methods have been employed
to generate o-quinone 1 from DA. Because 1 is formed
as a result of chemical (6, 7), enzymatic (9), and electro-
chemical (1-5) oxidations of DA, it was judged reasonable
to expect that the results of this and earlier studies are
probably relevant to the chemistry that is speculated to
occur in the cytoplasm of SN neurons in PD.
HPLC method I employed solvents A and B and the following
gradient: 0-10 min, 100% solvent A; 10-98 min, linear
gradient to 24% solvent B; 98-101 min, linear gradient to 100%
solvent B; 101-113 min, 100% solvent B. The flow rate was
constant at 7 mL min-1
.
HPLC method II employed solvents C and D and the following
gradient: 0-97 min, linear gradient from 100% solvent C to
38% solvent D; 97-100 min, linear gradient to 100% solvent D;
100-113 min, 100% solvent D. The flow rate was constant at
7 mL min-1
.
Ma ter ia ls a n d Meth od s
Oxid a tion Rea ction P r oced u r e. DA-HCl (5.7 mg; 1.0 mM)
and CySH (1.82-7.28 mg; 0.5-2.0 mM) were dissolved in 30
mL of phosphate buffer (pH 7.4; µ ) 0.2) and the resulting
solution was electrolyzed at 50-100 mV for 60 min. Upon
termination of the reaction, the entire volume of the yellow
product solution was pumped onto the reversed phase column
and reactants and products were separated using HPLC method
I. The solutions eluted under the chromatographic peaks
corresponding to major products were collected separately and
immediately frozen at -80 °C (dry ice). Following several
repetitive experiments, the combined solutions containing each
product was purified several times using HPLC method I. The
resulting solutions containing purified products were then
freeze-dried.
Spectroscopic and chemical evidence in support of the pro-
posed structures of products is presented below. Complete
chemical names are provided for each new compound. However,
for simplicity, the slightly modified atom numbering systems
for 12-15 shown in Scheme 2 and Table 1 are employed to
assign resonances in 1H and 13C NMR spectra. The purity of
each new compound (12-15) was estimated to be g99% on the
basis of analytical HPLC using a reversed phase systems and
both UV (254 nm) and electrochemical (0.80 V vs Ag/AgCl;
glassy carbon detector electrode) detectors.
Ca u tion . Compound 12 and particularly compound 13, in
addition to 2,5-bi-S-CyS-DA, DHBT-1, DHBT-2, and DHBT-6,
are lethal when administered into the brains of mice and, hence,
should be regarded as potentially hazardous to humans and
therefore handled with care. Such chemicals should always be
handled with protective gloves and in a hood.
Ch em ica ls. Dopamine hydrochloride (DA-HCl) and L-cys-
teine (CySH) were obtained from Sigma (St. Louis, MO). HPLC
grade acetonitrile (MeCN) was obtained from EM Science
(Gibbstown, NJ ). Concentrated trifluoroacetic acid (TFA) was
obtained from Aldrich (Milwaukee, WI). 2,5-Bi-S-cysteinyl-
dopamine (2,5-bi-S-CyS-DA), 7-(2-aminoethyl)-3,4-dihydro-5-
hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1), 6-S-
cysteinyl-7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-ben-
zothiazine-3-carboxylic acid (DHBT-2), and 6-S-cysteinyl-8-(2-
aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-car-
boxylic acid (DHBT-6) were electrochemically synthesized,
isolated, and purified as described previously (2).
Electr och em istr y. Voltammograms were obtained at a
pyrolytic graphite microelectrode (PGE; Pfizer Minerals, Pig-
ments and Metals Division, Easton, PA) having an approximate
surface area of 6 mm2 as described previously (1-3). A conven-
tional three-electrode voltammetric cell was used with a plati-
num wire counter electrode and a saturated calomel reference
electrode (SCE). Cyclic voltammetry was carried out with a
BAS-100A (Bioanalytical Systems, West Lafeyette, IN) electro-
chemical analyzer. Controlled-potential electrolyses employed
a Princeton Applied Research Corp. (Princeton, NJ ) Model 173
potentiostat. A three-compartment cell was used in which the
working, counter, and reference electrode compartments were
separated with a Nafion membrane (type 117, DuPont Co.,
Wilmington, DE). The working electrode consisted of several
plates of pyrolytic graphite having a total surface area of ∼180
cm2. The counter electrode was platinum gauze, and a SCE
reference electrode was used. The solution in the working
electrode compartment was vigorously bubbled with N2 and
stirred with a Teflon-coated magnetic stirring bar. All poten-
tials are referenced to the SCE at ambient temperature (22 (
2 °C).
7-(2-Am in oeth yl)-5-h ydr oxy-1,4-ben zoth iazin e-3-car box-
ylic Acid (BT-1). In the HPLC mobile phase (method I; ∼6%
MeCN in water), BT-1 gave a bright yellow solution (λmax ) 342,
∼300 shoulder (sh), 246, 216 nm). The thermospray mass
spectrum of this solution gave m/z ) 253 (MH+, 100%).
A
comparison of these spectral data and the cyclic voltammetric
behavior of BT-1 with model compounds previously permitted
a tentative structure for this benzothiazine to be assigned (3).
However, efforts to isolate BT-1 and to obtain spectroscopic
evidence (NMR, FAB-MS) to directly confirm this structure were
unsuccessful owing to the instability of this compound. How-
ever, similar structures have been elucidated as a result of
borohydride reduction of benzothiazines to the corresponding
dihydrobenzothiazines that can be isolated (23). Accordingly,
a vigorously stirred solution of freshly chromatographed BT-1
dissolved in the HPLC mobile phase (method I) was treated with
sodium borohydride as described elsewhere (23). During addi-
tion of NaBH4 the bright yellow color of BT-1 disappeared to
give a colorless solution. After 10 min, the solution (pH 7-8)
Sp ectr oscop y. NMR spectra were recorded on either a
Varian (Palo Alto, CA) XL-300 or VXR-500 spectrometer. Low-
and high-resolution fast atom bombardment mass spectrometry

150 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Shen et al.
Sch em e 2
was adjusted to pH 2.15 with TFA and then pumped onto the
preparative reversed phase column. Using HPLC method II,
the reduced form of BT-1 eluted at a retention time (tR) of 66
min (λmax at pH 2.15 ) 304, 230 nm). The eluent containing
this compound was freeze-dried to give a white solid. In
phosphate buffer (pH 7.4) λmax (log ꢀmax M-1 cm-1) ) 306 (3.38)
and 234 (4.38) nm. FAB-MS (3-nitrobenzyl alcohol matrix) gave
m/z ) 255.0818 (MH+, 65%, C11H15N2O3S; calcd m/z ) 255.0803).
1H NMR (300 MHz; Me2SO-d6) gave δ 9.58 (s, 1H, C(5)OH), 7.70
(br s, 3H, NH3+), 6.36 (d, J ) 2.1 Hz, 1H, C(6)H), 6.31 (d, J )
2.1 Hz, 1H, C(8)H), 5.24 (br s, 1H, N(4)H), 4.34 (dd, J ) 5.7, 3.3
Hz, 1H, C(3)H), 3.15 (dd, J ) 12.6, 3.3 Hz, 1H, C(2)H), 3.07
(dd, J ) 12.6, 5.7 Hz, 1H, C(2)H), 2.96-2.88 (m, 2H, C(R)H2),
2.63-2.56 (m, 2H, C(â)H2). The chromatographic tR (method
II), FAB-MS, and 1H NMR spectra of the reduction product of
BT-1 were virtually identical to those of an authentic sample
of DHBT-1. Borohydride reduction of BT-1 (molar mass 252 g)
would be expected to yield a racemic mixture of DHBT-1 (molar
mass 254 g) and hence supports the structure proposed for the
former compound.
6-(2-Am in oeth yl)-1,8,9,10-tetr a h yd r oben zo[1,2-b:4,3-b′]-
bis[1,4]th ia zin e-9-ca r boxylic Acid (12). Compound 12 was
isolated as a yellow solid. In phosphate buffer (pH 7.4; µ ) 1.0)
the UV-visible spectrum of 12 exhibited bands at λmax (log ꢀmax
,
M-1 cm-1) ) 390 (3.28), 334 (3.18), 288 sh (3.57), 264 sh (3.94),
and 244 (4.13) nm. The thermospray mass spectrum of a
solution of 12 dissolved in the HPLC (method I) mobile phase
gave m/ z ) 310 (MH+, 58%). FAB-MS (thioglycerol/glycerol
matrix) gave m/z ) 310.0706 (MH+, 5%, C13H16N3O2S2; calcd
m/z ) 310.0684). 1H NMR (300 MHz; D2O) gave δ 7.88 (t, J )
4.2 Hz, 1H, C(2′)H), 6.86 (s, 1H, C(5)H), 4.25 (dd, J ) 5.4, 3.6
Hz, 1H, C(9′)H), 3.21-3.07 (m, 6H, C(10′)H2, C(1′)H2, C(R)H2),
2.93-2.85 (m, 2H, C(â)H2). The low solubility (D2O, CD3OD,
Me2SO-d6, pyridine-d5) of 12 precluded more extensive studies
of its NMR spectra. Additional evidence in support of the
proposed structure of 12 was obtained by reducing a vigorously
stirred solution of this compound dissolved in the HPLC mobile
phase (method I) with NaBH4 until the bright yellow color had
disappeared. After 10 min, the resulting solution (pH 7-8) was
adjusted to pH 2.15 with TFA and then pumped onto the

Oxidation of Dopamine in the Presence of L-Cysteine
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 151
Ta ble 1. 1H (500 MHz), 13C (125 MHz), HMQC, a n d HMBC Ch em ica l Sh ift Assign m en ts (p p m ) for 15 a n d Ca r bon s to
Wh ich Lon g-Ra n ge Con n ectivity Is Obser ved in HMBC Exp er im en ta
1′
2′
HOOC
S
1
β
2
HN
NH2
3′
8′
6
5
α
3
HN
4
S
9′
10′
15
assignment
1H (J in Hz)
13C
HMQC (13C)
HMBC (13C)
â
2.80 (t, 7.5, 2H)
3.03 (dd, 13.0, 3.5, 1H)
3.09 (t, 7.5, 2H)
3.14 (m, 2H)
3.25 (dd, 13.0, 4.5, 1H)
3.67 (m, 2H)
29.68
24.86
38.00
22.33
24.86
29.68
24.86
38.00
22.33
24.86
42.53
52.58
118.12
131.88 (C-6), 118.12 (C-5), 112.97 (C-1), 38.00 (C-R)
174.54 (COOH), 112.97 (C-1), 52.58 (C-2′)
131.88 (C-6), 29.68 (C-â)
1′
R
10′
1′
9′
2′
5
6
1
121.86 (C-4), 42.53 (C-9′)
112.97 (C-1), 52.58 (C-2′)
117.13 (C-3), 22.33 (C-10′)
174.54 (CdO), 133.533 (C-2)
117.13 (C-3), 112.97 (C-1), 29.68 (C-â)
42.53
52.58
4.57 (dd, 4.5, 3.5, 1H)
6.51 (s, 1H)
118.12
131.88
112.97
133.53
117.13
121.86
174.54
2
3
4
COOH
a
15 was dissolved in D2O for all experiments.
preparative reversed phase column. Using HPLC method II,
the reduced form of 12, i.e., 14, eluted at tR ) 87 min. In the
HPLC mobile phase (pH 2.15) 14 exhibited a spectrum with λmax
) 324, 284 sh, 252 nm. The eluent containing 14 was freeze-
dried to give a very pale yellow solid. In phosphate buffer (pH
nitrobenzyl alcohol matrix) gave m/z ) 312.0833 (MH+, 100%,
C13H18N3O2S2; calcd m/z ) 312.0840). 1H NMR (300 MHz; D2O)
gave δ 6.52 (s, 1H, C(5)H). 4.57 (dd, J ) 4.8, 3.0 Hz, 1H, C(2′)H),
3.63-3.59 (m, 2H, C(9′)H2), 3.28 (dd, J ) 13.2, 4.8 Hz, 1H,
C(1′)H), 3.18-3.06 (m, 5H, C(1′)H, C(10′)H2, C(R)H2), 2.89-2.82
(m, 2H, C(â)H2). These spectral data suggested that 15 was
6-(2-aminoethyl)-1,2,3,8,9,10-hexahydrobenzo[1,2-b:4,3-b′]bis-
[1,4]thiazine-2-carboxylic acid. Further evidence to support the
proposed structure of 15 and hence 13 will be presented
subsequently.
7.4), 14 exhibited a UV spectrum at λmax (log ꢀmax, M-1 cm-1
)
318 (3.37), 286 sh (3.82), and 256 (4.40) nm, calculated as the
2TFA salt (2). FAB-MS (3-nitrobenzyl alcohol matrix) gave m/z
) 312.0846 (MH+, 40%, C13H18N3O2S2; calcd m/z ) 312.0840).
1H NMR (300 MHz; D2O) gave δ 6.59 (s, 1H, C(5)H), 4.54 (dd,
J ) 4.8, 3.0 Hz, 1H, C(9′)H), 3.60-3.55 (m, 2H, C(2′)H2), 3.22
(dd, J ) 13.2, 4.8 Hz, 1H, C(10′)H), 3.18-3.10 (m, 3H, C(10′)H,
C(R)H2), 3.08-3.04 (m, 2H, C(1′)H2), 2.87-2.80 (m, 2H, C(â)-
H2). The assignment of 1H resonances were confirmed by
homonuclear decoupling and 2D COSY experiments. These
spectral data were interpreted to indicate that 14 is 6-(2-
aminoethyl)-1,2,3,8,9,10-hexahydrobenzo[1,2-b:4,3-b′]bis[1,4]-
thiazine-9-carboxylic acid formed by borohydride reduction of
the C(2′)dN(3′) double bond of 12 (see later discussion).
6-(2-Am in oeth yl)-1,2,3,10-tetr a h yd r oben zo[1,2-b:4,3-b]-
bis[1,4]th ia zin e-2-ca r boxylic Acid (13). Compound 13 was
isolated as a yellow solid. In phosphate buffer (pH 7.4), 13
An im a ls. Outbred Adult Male Mice of the HSD:ICR albino
strain (Harlan Sprague-Dawley, Madison, WI) weighing 32 (
5 g were employed. Animals were housed 10 per cage, allowed
free access to Purina rat chow and water, and maintained on a
12 h light/dark cycle with lights on a 7:00 a.m. Experimental
mice were treated with test compounds dissolved in either 5
µL of isotonic saline (0.9% NaCl in deionized water) or 5 µL of
30% Me2SO in aqueous isotonic saline. Prior to drug adminis-
tration, animals were anesthesized with ether for 45-55 s.
Injections were performed freehand with the point of puncture
being 3 mm anterior to the interaural line, 1 mm left lateral of
the midline, and 3 mm perpendicular to the scalp as described
previously (1, 2). Control animals were treated with 5 µL of
vehicle alone under otherwise identical conditions. All animal
procedures were approved by the Institutional Animal Care and
Use Committee at the University of Oklahoma.
exhibited a UV-visible spectrum with λmax (log ꢀmax, M-1 cm-1
)
at 386 (3.35), 332 (3.30), 290 sh (3.64), 264 sh (4.06), and 242
(4.22) nm. The thermospray mass spectrum of a solution of 13
dissolved in the HPLC mobile phase (method I) gave m/z ) 310
(MH+, 100%). FAB-MS (thioglycerol/glycerol matrix) gave m/z
) 310.0711 (MH+, 6%, C13H16N3O2S2; calcd m/z ) 310.0684).
1H NMR (300 MHz; D2O) gave δ 7.83 (t, J ) 4.2 Hz, 1H, C(9′)H),
6.54 (s, 1H, C(5)H), 4.27 (dd, J ) 5.4, 3.9 Hz, 1H, C(2′)H), 3.21-
3.11 (m, 6H, C(1′)H2, C(10′)H2, C(R)H2), 2.96-2.84 (m, 2H, C(â)-
H2). The low solubility of 13 in D2O (and all other common
deuterated solvents) resulted in a very weak spectrum which
precluded more extensive NMR studies. Additional evidence
for the proposed structure of 13 was obtained by reducing a
solution of this yellow compound dissolved in the HPLC method
I mobile phase with NaBH4 as described for 12 (23). The
resulting colorless solution (pH 7-8) was adjusted to pH 2.15
with TFA and then pumped onto the preparative reversed phase
column. Using HPLC method II, the reduced form of 13, i.e.,
15, eluted at tR ) 85 min. In the HPLC mobile phase (pH 2.15)
15 exhibited a UV spectrum with λmax ) 322, 282 sh, and 250
nm. This solution was freeze-dried to give a very pale yellow
solid. In phosphate buffer (pH 7.4) 15 exhibited a UV spectrum,
λmax (log ꢀmax, M-1 cm-1) at 318 (3.32), 284 sh (3.82), and 254
(4.40) nm, calculated as the 2TFA salt (2). FAB-MS (3-
Resu lts
Oxid a tion of DA in th e P r esen ce of CySH.
A
chromatogram (HPLC method I) of the product solution
formed following controlled-potential electrooxidation of
DA (1.0 mM) in the presence of CySH (1.0 mM) in
phosphate buffer (pH 7.4; µ ) 0.2) at an applied potential
of 50 mV for 60 min is shown in Figure 1A. Under these
chromatographic conditions, cysteinyldopamines and most
DHBTs formed in the reaction (described in detail
elsewhere) (1, 2) coeluted under the peaks at tR < 20 min.
However, four major products were well separated and,
hence, could be isolated and spectroscopically identified:
DHBT-1, BT-1, and regioisomers 12 and 13. HPLC
analysis (method I) of the product solution at earlier
stages of the reaction (<60 min) revealed that the
chromatographic peaks at tR < 20 min and for DHBT-1

152 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Shen et al.
drying step. Based upon a comparison of the UV-visible
spectrum, thermospray mass spectrum, and cyclic vol-
tammetric behavior with those of more stable model
compounds and the fact that it is formed as a result of
oxidation of 5-S-CyS-DA and DHBT-1, a tentative
structure for BT-1 was proposed in a previous study (3).
This structure was confirmed on the basis that BT-1
could be reduced by NaBH4 to the racemate of DHBT-1
(see Materials and Methods), a method employed by other
investigators to elucidate the structure of similar ben-
zothiazines (23).
P r ecu r sor s of a n d Ch em ica l Evid en ce for th e
Str u ctu r es of 12 a n d 13. The proposed structures of
12 and 13 suggested that the progenitor of these regioi-
somers was 2,5-bi-S-CyS-DA. The latter cysteinyl con-
jugate is a major early product of oxidations of DA in the
presence of CySH. However, because of its ease of
oxidation, 2,5-bi-S-CyS-DA is the precursor of 2,5,6-tri-
S-CyS-DA (particularly in the presence of large molar
excesses of CySH), DHBT-2 (minor product), DHBT-6
(major product) (2). The HPLC revealed that oxidations
(100 mV) of 2,5-bi-S-CyS-DA, DHBT-2, and DHBT-6 in
phosphate buffer (pH 7.4) all yield 12 (minor) and 13
(major). Furthermore, using HPLC method I, 2,5-bi-S-
CyS-DA, DHBT-2, and DHBT-6 all eluted under the
peaks at tR < 20 min. These are the peaks that decrease
and disappear upon oxidation of DA in the presence of
CySH at 100 mV (Figure 1B) or as a result of more
prolonged oxidations at lower applied potentials (data not
shown). Taken together, these results indicate that 2,5-
bi-S-CyS-DA, DHBT-2, and DHBT-6 are precursors of 12
and 13 and provide unequivocal evidence for sites of
attachment of the sulfur residues in the latter com-
pounds.
F igu r e 1. HPLC chromatograms (method I) of the product
solutions formed following controlled-potential electrooxidation
of DA (1.0 mM) and CySH (1.0 mM) in phosphate buffer (pH
7.4; µ ) 0.2) for 60 min at (A) 50 and (B) 100 mV.
(tR ) 39 min) were significantly larger than those shown
in Figure 1A. Correspondingly, peaks for BT-1, 12, and
13 were smaller. These observations suggested that BT-
1, 12, and 13 were secondary products formed by further
oxidation of cysteinyldopamines and/or DHBTs. Oxida-
tion of DA (1.0 mM) in the presence of CySH (1.0 mM)
in phosphate buffer (pH 7.4) for 60 min at a more positive
applied potential (e.g., 100 mV) resulted in higher yields
of BT-1, 12, and 13 (Figure 1B). However, under these
more strongly oxidizing conditions, DHBT-1 disappeared
and the chromatographic peaks at tR < 20 min decreased
and disappeared. The yields of 12 and 13 were maximal
when DA (1.0 mM) was oxidized in the presence of 2.0
mM CySH for 60 min at an applied potential of 100 mV.
Under these conditions additional minor product peaks
were observed in chromatograms of the reaction solution.
In the presence of higher concentrations of CySH (g3
mM), under otherwise identical conditions, the yields for
these additional unidentified products increased. How-
ever, 12 and 13 remained as major reaction products.
Controlled-potential electrooxidations of DA (1.0 mM) in
the presence of CySH (0.5-5.0 mM) at potentials ranging
from 0 to 100 mV always gave 12 and 13 as products.
However, at low applied potentials (0 mV), very long
electrolyses (>120 min) were necessary before significant
yields of BT-1, 12, and 13 were observed. In view of the
fact that BT-1, 12, and 13 were formed under all
conditions when DA (1.0 mM) was oxidized (0-100 mV)
in the presence of CySH (0.5-5.0 mM) at pH 7.4, this
investigation focused on the identification of these com-
pounds.
Sp ectr oscop ic Evid en ce for th e Str u ctu r es of 12
a n d 13. Based on high-accuracy FAB-MS results, both
12 and 13 had a molar mass of 309 g and molecular
formula C13H15N3O2S2. The preceding chemical evidence
indicated that 12 and 13 were both formed as a result of
oxidations of 2,5-bi-S-CyS-DA, DHBT-2, and DHBT-6.
Thus, the sulfur atoms in 12 and 13 must be attached to
C-1 and C-4 (corresponding to the C-2 and C-5 positions,
respectively, in the original residue of DA). Furthermore,
the molar masses of 12 and 13 (309 g) indicate that upon
oxidation (2e, 2H+) both cysteinyl residues of 2,5-bi-S-
CyS-DA and the single cysteinyl residues of both DHBT-2
and DHBT-6 must undergo intramolecular cyclization
(condensation) and that loss of one carboxyl group occurs.
The similarities between the UV-visible and 1H NMR
(300 MHz) spectra of 12 and 13 together with their
identical molar masses and the fact that they are both
formed by oxidations of 2,5-bi-S-CyS-DA, DHBT-2, and
DHBT-6 clearly support the conclusion that these com-
pounds are regiosomers derived from a common precursor
that can decarboxylate from two different sites. Because
of the low solubilities of both 12 and 13 in all common
deuterated solvents (D2O, CD3OD, Me2SO-d6, pyridine-
d5), it was not possible to employ further NMR experi-
ments to arrive at definitive structures directly using
either compound. Accordingly, 12 and 13 were reduced
by NaBH4 to 14 and 15, respectively (see Materials and
Methods). The latter compounds were appreciably more
soluble. Spectroscopic information on these compounds,
In an earlier investigation (3), it was demonstrated
that BT-1 is formed as a result of oxidation of both 5-S-
CyS-DA and DHBT-1 at pH 7.4. However, attempts to
isolate pure samples of BT-1 were unsuccessful owing to
its decomposition particularly during the final freeze-
1
including H NMR (300 MHz) spectra, are presented in
Materials and Methods. However, these spectra do not
allow unequivocal structure assignments. Because of the
significantly higher yields of 13, and hence 15, additional

Oxidation of Dopamine in the Presence of L-Cysteine
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 153
NMR experiments focused on the latter compound. A
summary of the 1H NMR (500 MHz), 13C (125 MHz),
heteronuclear multiple-quantum coherence (HMQC), and
heteronuclear multiple-bond connectivity (HMBC) NMR
spectra of 15 is presented in Table 1. Homonuclear
decoupling and 2D COSY experiments were consistent
with coupling between the resonances assigned to C(R)-
H2 and C(â)-H2, C(1′)-H2 and C(2′)-H, and C(9′)-H2
and C(10′)-H2 (data not shown). The following discus-
sion of the structure determination of 15 relies mainly
1
on long-range H-13C connectivity spectra (HMBC). To
assist in the discussion, the proposed structure of 15 and
atom numbering system is shown in Table 1. Starting
at the most upfield protons, C(â)H2 at 2.80 ppm (t, 2H),
connectivity is observed to C-R (38.00 ppm), nonproto-
nated carbons at 131.88 (C-6) and 112.97 (C-1) ppm, and
a protonated carbon (C-5) at 118.12 ppm. Connectivity
between C(R)H2 and a nonprotonated carbon at 131.88
ppm confirms the point of attachment of the ethylamino
side chain at C-6. Similarly, connectivity between C(5)H
(s, 6.51 ppm) and a methylene carbon at 29.68 ppm (C-
â) and a nonprotonated carbon at 112.97 ppm (C-1)
permits assignments of the C-1 and C-5 positions. Con-
nectivity between two nonequivalent methylene protons
at C-1′ (3.03 ppm, dd; 3.25 ppm, dd) and a nonprotonated
carbon at 112.97 ppm (C-1), a protonated carbon at 52.58
(C-2′), and the carboxylic acid carbonyl carbon at 174.54
ppm together establish that the carboxyl group-contain-
ing dihydrobenzothiazine residue is connected 1,2-b to
the aromatic ring. Connectivity between the methylene
protons at C-9′ (3.67 ppm, m, 2H) and the methylene
carbon at 22.33 ppm (C-10′), a nonprotonated carbon at
117.13 ppm (C-3) and between C(5)H (6.51 ppm, 1H) and
a nonprotonated carbon at 117.13 ppm (C-3) confirm that
the carboxyl group-free dihydrobenzothiazine residue in
15 is connected 4,3-b′ to the aromatic ring. The structure
assigned to 15, together with the spectroscopic informa-
tion provided for its oxidized form, 13, lead to the
conclusion that the latter compound possesses a carboxyl-
substituted dihydrobenzothiazine residue connected 1,2-b
to the aromatic ring. Accordingly, the unsaturated
carboxyl residue-free 1,4-benzothiazine residue in 13
must be attached 4,3-b′ to the aromatic ring. 12,
therefore, is a regioisomer of 13 with the carboxyl residue
attached at C-9′ in the dihydrobenzothiazine residue
attached 4,3-b′ to the aromatic ring.
Cyclic Volta m m etr y of 12 a n d 13. Cyclic voltam-
mograms of 12 and 13 (1.0 mM) in phosphate buffer (pH
7.4; µ ) 1.0) at a sweep rate of 50 mV s-1 are presented
in Figure 2. Thus, 12 exhibits three voltammetric
oxidation peaks at peak potentials (Ep) of 128, 421, and
586 mV (Figure 2A). 13 exhibits three oxidation peaks
at Ep ) 155, 473, and 596 mV (Figure 2B).
P r elim in a r y Biologica l Exp er im en ts. 12 was dis-
solved in 70:30 (v/v) isotonic saline/Me2SO; 13 was
dissolved in isotonic saline. Solutions of each compound
dissolved in 5 µL of vehicle were injected into the vicinity
of the left lateral ventricle while mice were maintained
under ether anesthetic (see Materials and Methods for
procedures employed). The LD50 values, used as a
measure of toxicity and defined as the dose of injected
compound (expressed as free base) at which 50% of
treated animals died, were determined using the statisti-
cal method of Dixon (24) in order to minimize the number
of animals needed (∼25). All animals that died did so at
times of e1 h after administration of 12 or 13. Because
BT-1 could not be isolated in a pure form, biological
F igu r e 2. Cyclic voltammograms at the PGE of 1.0 mM
solutions of (A) 12 and (B) 13 in phosphate buffer (pH 7.4; µ )
1.0) at a sweep rate of 50 mV s-1
.
studies on this compound were not carried out. However,
both 12 and 13 were lethal, having experimental LD50
values of 18.5 ( 1.3 µg (578 ( 41 µg/kg of body weight)
(mean ( standard deviation) and 1.5 ( 1.1 µg (47 ( 34
µg/kg of body weight), respectively. Doses of 12 ranging
from 15 to 25 µg, and of 13 ranging from 1 to 10 µg, were
employed to determine LD50 values. Solutions of 12 and
13 in vehicle were freshly prepared prior to administra-
tion to animals. HPLC analysis (method I) of these
solutions after animal experiments were completed re-
vealed insignificant decomposition.
The neurobehavioral responses evoked by 12 and 13
were similar to those evoked by other lethal DHBTs (1,
2). Thus, following an LD50 dose, animals exhibited
extreme hyperactivity characterized by episodes of rapid
running, jumping, and rolling along the head-tail axis
that was accompanied by repetitive squeaking. Episodes
of severe trembling occurred. Control animals treated
with 5 µL of vehicle alone exhibited none of these
neurobehavioral responses and all survived.
Discu ssion
Previous studies (1-3, 9) established that CySH can
divert the in vitro oxidation of DA to melanin polymer
(5) by scavenging o-quinone 1 to give principally 5-S-CyS-
DA along with much smaller amounts of 2-S-CyS-DA.
The facile oxidation of these cysteinyl conjugates then
leads to more highly substituted cysteinyldopamines and
DHBTs, some of which are shown in Scheme I. The
results of this and earlier studies (3) together reveal that
o-quinone imine 5, formed by oxidation of both 5-S-CyS-
DA and DHBT-1, rearranges (tautomerizes) to yellow
BT-1 (Scheme 2). The latter 1,4-benzothiazine is a major
product of oxidation of DA in the presence of CySH.
Based upon the structures and yields of products
formed when DA is oxidized in the presence of free CySH
at pH 7.4 it is clear that 2,5-bi-S-CyS-DA, formed

154 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Shen et al.
primarily by nucleophilic addition of CySH to o-quinone
2, is a major precursor of many identified products.
However, yields of 2,5-bi-S-CyS-DA are low at any stage
of the oxidation reaction (2) owing to the great ease of
oxidation of this conjugate. The oxidation of 2,5-bi-S-
CyS-DA generates o-quinone 4 that can undergo in-
tramolecular cyclizations to o-quinone imines 7 and 8
(Scheme 2) (2). In order to account for formation of
DHBT-2 and DHBT-6 when 2,5-bi-S-CyS-DA is oxidized
even in the absence of free CySH, it is clear that 7 and
8 can chemically oxidize this cysteinyl conjugate by the
reaction pathways shown in Scheme 2 and discussed in
detail elsewhere (2). However, the present study estab-
lishes that oxidations of 2,5-bi-S-CyS-DA, DHBT-2, and
DHBT-6 also result in formation of the regioisomers 12
and 13, the latter always being the major product.
Accordingly, it can be concluded that intramolecular
cyclizations of both cysteinyl residues of 4, formed by
oxidation (2e, 2H+) of 2,5-bi-S-CyS-DA, leads to the
tricyclic o-diimine 11. Decarboxylation of 11 from C-2′
or C-9′, the preferred route, then leads to regioisomers
12 and 13, respectively (Scheme 2). Borohydride reduc-
tion of 12 and 13 to 14 and 15, respectively, provides a
valuable method to convert these sparingly soluble ben-
zothiazines into much more soluble dihydrobenzothiaz-
ines. Spectroscopic evidence, particularly on 15, permits
unequivocal structure assignment and hence the struc-
ture of 13. The bright yellow color that progressively
develops during oxidations of DA in the presence of CySH
can be traced primarily to BT-1, 12, and 13, i.e., products
derived 5-S-CyS-DA and from 2,5-bi-S-CyS-DA, DHBT-
2, and DHBT-6, respectively.
The upregulation of nigral γ-GT (10), irreversible loss
of GSH (18-20), and increased 5-S-CyS-DA/DA concen-
tration ratio (21) are all compatible with the hypothesis
that an elevated translocation of CySH into neuromela-
nin-pigmented SN neurons might occur in PD. The
observation that surviving dopaminergic cells are less
heavily pigmented in the Parkinsonian SN compared to
age-matched controls (15-17) would also be expected, by
analogy with the results of in vitro studies, as a conse-
quence of the diversion of the neuromelanin pathway by
CySH. The in vitro products of oxidation of DA in the
presence of free CySH include not only 5-S-CyS-DA but,
as a consequence of the fact that this conjugate is more
easily oxidized than the neurotransmitter (1, 2), many
additional cysteinyldopamines, DHBTs, BT-1, 12, and 13.
Whether a similarly complex mixture of metabolites is
formed in the cytoplasm of neuromelanin-pigmented SN
neurons in PD remains to be established. However, that
the initial step in formation of such metabolites occurs
in PD is supported by the increased 5-S-CyS-DA/DA ratio
in the SN (21) and the increased 5-S-CyS-DA/HVA ratio
in CSF (22). Furthermore, a number of lines of evidence
support the conclusion that 5-S-CyS-DA is formed as a
result of oxidation of cytoplasmic DA (21, 25, 26).
Particularly in the cytoplasm of pigmented SN neurons,
in which DA is autoxidized to form neuromelanin (6, 7),
5-S-CyS-DA must almost certainly undergo further oxi-
dation. In the presence of free CySH such an in vivo
oxidation reaction would lead to the more complex
cysteinyl dopamines, DHBTs, BT-1, 12, and 13 that are
formed in the in vitro reaction. The results presented
in this and earlier reports (1, 2) indicate that 2,5-bi-S-
CyS-DA, DHBT-1, DHBT-2, DHBT-5, DHBT-6 (and other
DHBTs), 12, and 13 are toxic (lethal) when administered
into the ventricular system of mice and evoke a charac-
teristic neurobehavioral response. Interestingly, 13 (LD50
) 1.5 µg), the major product of oxidation of 2,5-bi-S-CyS-
DA (LD50 ) 37 µg), DHBT-2 (LD50 ) 70 µg), and DHBT-6
(LD50 ) 17 µg), is the most toxic compound discovered in
these investigations. Thus, it appears that as the oxida-
tion reactions of DA in the presence of CySH progress
through cysteinyldopamines and DHBTs to 13 increas-
ingly toxic putative metabolites are formed. These
observations raise the possibility that one or more of
these putative nigral metabolites might be endotoxins
that contribute to SN cell death in PD. However, there
is currently no evidence that any of these compounds are
dopaminergic neurotoxins or that they are formed in the
Parkinsonian brain. Nevertheless, all other factors being
equal, intraneuronal formation of 5-S-CyS-DA, other
cysteinyldopamines, DHBTs, BT-1, 12, and 13 would be
expected to be most extensive in those neurons that
sustain the highest basal levels of DA autoxidation, i.e.,
SN cells normally most heavily pigmented with neu-
romelanin. Thus, it may be significant that such neurons
appear to be preferentially vulnerable to degeneration
in PD (15-17).
In summary, three new compounds, BT-1, 12, and 13,
have been identified as products formed in the relatively
late stages of the in vitro oxidation of DA in the presence
of CySH. The immediate precursor of BT-1 is DHBT-1.
The immediate precursors of 12 and 13 are 2,5-bi-S-CyS-
DA, DHBT-2, and DHBT-6. The results presented and
hypothesis advanced in this paper are based on the
influence of CySH on the in vitro electrochemically driven
oxidation of DA, the toxicity of many resulting products,
and other known changes that are known to occur in the
Parkinsonian brain. While it is plausible to expect that
similar chemistry would accompany the autoxidation of
DA in pigmented dopaminergic SN cells as a result of
the hypothesized γ-GT-mediated translocation of CySH
into these neurons, this remains to be experimentally
verified. Furthermore, while a rather remarkable num-
ber of products formed from the electrochemical oxidation
of DA in the presence of CySH are relatively potent toxins
in mouse brains, particularly 13, it must be emphasized
that it is not yet known whether any of these putative
nigral metabolites evoke degeneration of SN neurons and
therefore might play a role in the pathogenesis of PD.
Ack n ow led gm en t. This work was supported by
Grant NS-29886 from the National Institutes of Health.
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