470 Alguindigue, Lemma, and Ashby
EXPERIMENTAL
fides were quantitative (based on CSSCN) as deter-
1
mined by H NMR.
Syntheses
1
S-(2-Hydroxyethylthio)-L-cysteine (2-MESSC). H
Materials. L-cysteine (97%), D-penicillamine
NMR (0.5 M DCl/0.5 M HCl, 399.96 MHz) δ: 4.53
3
3
(
97–102%),
2-mercaptoethanol,
3-mercapto-
(dd, 1H, J(2-3) = 4.3 Hz, J(2-3ꢁ) = 7.7 Hz, H-2), 3.91
(t, 2H, J(7-6) = 5.9 Hz, J(7-6ꢁ) = 5.9 Hz, H-7), 3.43 (dd,
1H, J(3-3ꢁ) = 15.2 Hz, J(3-2) = 4.3 Hz, H-3), 3.28 (dd,
1H, J(3ꢁ-3) = 15.2 Hz, J(3ꢁ-2) = 7.7 Hz, H-3 ), 2.97 (t,
3
3
propionic acid (99+%), L-gluthathione (99%),
and a 35% (w/w) solution of deuterium chloride
in deuterium oxide (99% D) were used as received
from Aldrich-Sigma (Milwaukee, Wisconsin). Deu-
2
3
2
3
ꢁ
3
3
1H, J(6-7) = 5.9 Hz, H-6); 2.96 (t, 1H, J(6ꢁ-7) = 5.9 Hz,
13
ꢁ
13
1
terium oxide (99.9% D) and thiocyanate (98% C)
were used as received from Cambridge Isotope
Laboratories, Inc., (Andover, Massachusetts). All
other chemicals were American Chemical Society
certified grade or better.
H-6 ); C{ H} NMR (0.5 M DCl/0.5 M HCl, 100.58
MHz) δ 170.7 (C1), 59.1 (C7), 51.9 (C2), 39.4 (C6),
36.9 (C3).
S-(β-Carboxyethylthio)-L-cysteine (3-MPASSC).
H NMR (0.5 M DCl/0.5 M HCl, 399.96 MHz) δ:
1
3
3
4
3
3
.51 (dd, 1H, J(2-3) = 4.5 Hz, J(2-3ꢁ) = 7.6 Hz, H-2),
2
3
.42 (dd, 1H, J(3-3ꢁ) = 15.1 Hz, J(3-2) = 4.5 Hz, H-3),
Preparation of Cysteinyl Thiocyanate. Solutions
2
3
.29 (dd, 1H,
J
(3ꢁ-3) = 15.1 Hz,
J
(3ꢁ-2) = 7.6 Hz,
of thiocyanogen in CCl were prepared by oxidation
4
ꢁ
3
H-3 ), 3.04 (t, 2H, J(6-7) = 6.5 Hz, H-6), 2.88 (t, 2H,
of lead(II) thiocyanate with bromine, as described
in the literature [1]. Lead(II) thiocyanate was pre-
pared as a white solid by reacting ice-cold solutions
3
13
1
J
(7-6) = 6.5 Hz, H-7); C{ H} NMR (0.5 M DCl/0.5
M HCl, 100.58 MHz) δ 176.1 (C8), 170.2 (C1), 51.4
(C2), 36.3 (C3), 33.1 (C7), 31.6 (C6).
of Pb(NO
3
)
2
and NaSCN (two-fold excess) for about
0 min. The product was dried under vacuum for
days. The concentration of (SCN) in CCl was de-
L-Cysteine-D-penicillamine Disulfide (PENSSC).
3
3
1
H NMR (0.5 M DCl/0.5 M HCl, 399.96 MHz) δ:
2
4
3
3
−
1
4.50 (dd, 1H, J(2-3) = 4.9 Hz, J(2-3ꢁ) = 6.2 Hz, H-2),
termined spectrophotometrically, ε296 nm = 140 M
cm [2]. The compound CSSCN was prepared by
extracting 1.1 molar equivalents of (SCN) into a
.0 M DCl solution that contained L-cysteine. The
3
−
1
4.25 (s, 1H, H-7), 3.47 (d, 1H, J(3-2) = 6.2 Hz, H-3),
3
ꢁ
3
6
.48 (d, 1H, J(3ꢁ-2) = 4.9 Hz, H-3 ), 1.63 (s, 3H, H-
2
13 1
a), 1.53 (s, 3H, H-6b); C{ H} NMR (0.5 M DCl/0.5
1
M HCl, 100.58 MHz) δ 170.4 (C1), 169.7 (C8), 59.7
(C7), 52.2 (C2), 51.2 (C6), 39.1 (C3), 25.9 (C6a), 22.6
(C6b).
yield of CSSCN was typically 90%, as determined
1
by H NMR (0.5 M DCl/0.5 M HCl, 399.96 MHz)
3
3
δ: 4.63 (dd, 1H, J(2-3) = 4.7 Hz, J(2-3ꢁ) = 7.3 Hz, H-2),
1
2
3
L-Cysteine-glutathione Disulfide (GSSC). H NMR
3
3
.85 (dd, 1H, J(3-3ꢁ) = 15.7 Hz, J(3-2) = 4.7 Hz, H-3),
2
3
ꢁ
(0.5 M DCl/0.5 M HCl, 399.96 MHz) δ: 8.50 (d,
.72 (dd, 1H, J(3ꢁ-3) = 15.7 Hz, J(3ꢁ-2) = 7.3 Hz, H-3 );
3
3
13
1
J
(12-13) = 8.0 Hz, NH12), 8.43 (br. t, J(15-16) = 6 Hz,
C{ H} NMR (0.5 M DCl/0.5 M HCl, 100.58 MHz)
3
3
NH15), 4.80 (ddd, 1H,
J
(13-12) = 8.0 Hz,
J
(13-6ꢁ)
=
=
=
δ 169.3 (C1), 112.4 (SCN), 51.3 (C2), 38.1 (C3).
3
3
9
4
6
(
(
(
(
(
.0 Hz, J(13-6) = 4.5 Hz, H-13), 4.52 (dd, 1H, J(2-3)
3
3
.2 Hz,
J
(2-3ꢁ) = 7.7 Hz, H-2), 4.15 (t, 1H,
J
(8-9)
General Procedure for the Preparation of
Symmetrical Disulfides. The disulfides of 2-
mercaptoethanol, 3-mercaptopropionic acid, and
3
.5 Hz, H-8), 4.05 (m, 2H, H-16), J(16-15) = 6 Hz, 3.46
2
3
dd, 1H, J(3-3ꢁ) = 15.2 Hz, J(3-2) = 4.2 Hz, H-3), 3.32
2
3
dd, 1H, J(6-6ꢁ) = 14.2 Hz, J(6-13) = 4.5 Hz, H-6), 3.27
D-penicillamine were prepared by extracting (SCN)
2
2
3
ꢁ
dd, 1H, J(3ꢁ-3) = 15.2 Hz, J(3ꢁ-2) = 7.7 Hz, H-3 ), 3.07
into 1.0 M DCl containing 2 equivalents of the indi-
vidual thiols. The resulting symmetrical disulfides
2
3
ꢁ
dd, 1H, J(6ꢁ-6) = 14.2 Hz, J(6ꢁ-13) = 9.0 Hz, H-6 ), 2.67
13
1
m, 2H, H-10), 2.28 (m, 2H, H-9); C{ H} NMR (0.5
1
were identified by comparing their H NMR spectra
M DCl/0.5 M HCl, 100.58 MHz) δ 174.6 (C11), 173.0
C17), 172.6 (C14), 171.5 (C7), 170.5 (C1), 52.5 (C13),
with authentic samples. The yields were quantitative
by NMR.
(
5
3
2.3 (C8), 51.8 (C2), 41.3 (C16), 38.1 (C6), 36.7 (C3),
1.1 (C10), 25.4 (C9).
General Procedure for the Preparation of Mixed
Disulfides. The mixed disulfides of L-cysteine
with 2-mercaptoethanol, 3-mercaptopropionic acid,
D-penicillamine, and L-glutathione were prepared by
reacting stoichiometric amounts of the thiols with
CSSCN. Solutions of the thiols were prepared in
NMR Studies
NMR experiments were carried out on a Varian
Inova 400 MHz Spectrometer with VNMRJ2.1B
software. 3-(Trimethylsilyl)-1-propane sulfonic acid,
sodium salt (DSS, δ = 0.015 ppm), and 1,4-dioxane
1.0 M HCl and mixed with equal volumes of CSSCN
1
solutions to make 15–20 mM mixed disulfide. The
yields of the corresponding unsymmetrical disul-
(δ = 66.6 ppm) were used as references for H and
13
C NMR measurements, respectively. Assignments
Heteroatom Chemistry DOI 10.1002/hc