438
H. Han et al.
Arch. Pharm. Chem. Life Sci. 2010, 8, 429–439
separated by thin-layer chromatography on a 10 ꢂ 20 cm plate,
eluted three times in ethyl acetate/hexane (1:1, v/v). The struc-
tures of the products were confirmed by a gCOSY measurement.
Product A: 3,4,5-trimethoxybenzaldehyde: 1H-NMR (CDCl3) d
trypsin/tubulin ratio) for 48 h at 378C. Digestion with pepsin was
carried out on samples pretreated with 6 M guanidine ꢃ HCl in
100 mM NH4HCO3, pH 7.4, for 15 min at 378C. After proteolytic
digestion, peptides were desalted and fractionated by use of
ZipTip microchromatography tips, followed by elution with
25, 50, and 75% acetonitrile in H2O, containing 0.1% trifluoro-
acetic acid. Mass spectrometry was carried out on a Voyager-DE
STR MALDI-TOF in reflector (positive) mode, with a-cyano-4-
hydroxycinnamic acid matrix. Samples were mass calibrated
by a mixture of four protein standards. Analysis of recorded
spectra was carried out with Applied Biosystems Data Explorer
software. We made use of the website tools at Protein Prospector
(University of California, San Francisco) and ExPASy (Swiss
Institute of Bioinformatics) websites for predicting peptide masses
and data analyses.
(ppm): 3.93 (s, 6H, 2 OCH3), 3.41 (s, 3H, OCH3), 7.13 (s, 2H, C(2)H,
þ
–
C(6)H), 9.87 (s, 1H, HC O); MS (MALDI) m/z: 197.079 [M þ H]
–
(theor.: 197.081).
Product B: N-acetyl-S-[2-oxo-2-(3,4,5-trimethoxyphenyl)ethyl]-
1
–
–
3
cysteamine: H-NMR (CDCl ) d (ppm): 2.01 (s, 3H, CH C O),
3
2.76 (t, J ¼ 6.0 Hz, 2H, CH2CH2S), 3.49 (dt, J ¼ 6.0, 6.5 Hz, 2H,
–
CH N), 3.85 (s, 2H, SCH C O), 3.93 (s, 6H, 2 OCH ), 3.94 (s, 3H,
2
–
2
3
OCH3), 6.1 (br s, 1H, NH), 7.24 (s, 2H, arom. C(2)H C(6)H); MS
(MALDI) m/z: 328.120 [M þ H]þ (theor.: 328.121); 350.103
[M þ Na]þ (theor.: 350.102).
Reaction of 4a with N-acetyl-L-cysteine
A solution of N-acetyl-L-cysteine (0.11 M) in 0.4 mL 50 mM
Na2HPO4 in D2O was adjusted to pD 7.7 and was allowed to react
with 1 equivalent of 4a in 0.4 mL CD3CN at 378C. The reaction
was followed by 1H-NMR and was found to be 30% complete after
1 h. After 24 h, the reaction was 100% complete. The structures
of the products were determined by gCOSY, gHMBC, and gHMQC
measurements. 1H-NMR (50 mM phosphate-buffered D2O/CD3CN)
We thank the Arizona Biomedical Research Commission and the Del E.
Webb Foundation for financial support. We also thank Professor G. Robert
Pettit of the Cancer Research Institute at ASU (Arizona State University) for
screening compound 4a against cancer cell lines in vitro (Table 1), John
Lopez of the Proteomics and Protein Chemistry Facility at ASU for many
informative discussions regarding the MALDI experiments, Ruben Munoz
for performing the tubulin polymerization assay and the National Cancer
Institute Developmental Therapeutics Program for carrying out the 60-cell-
line anticancer screening of 4a.
d
cysteine, 5, presumably mono-deuterated a to the ketone
(ppm)
N-acetyl-S-[2-oxo-2-(3,4,5-trimethoxyphenyl)ethyl]-L-
carbonyl due to quenching of the enolate by D2O (Scheme 2):
–
1.97 (s, 3H, CH C O), 2.87 (dd, J ¼ 8.5 Hz, 14.0 Hz, 1H SCH HCH),
–
3
a
The authors have declared no conflict of interest.
3.04 (dd, J ¼ 4.0 Hz, 14.0 Hz, 1H, SCHHbCH), 3.84 (s, 3H, OCH3),
3.898 (s, 7H, 2 OCH3, CH2CHaN), 4.35 (dd, J ¼ 8.5 Hz, 4.0 Hz,
–
1H, C OCHDS), 7.29 (s, 2H, arom. C(2)H C(6)H), and 3,4,5-
–
trimethoxybenzaldehyde: 3.85 (s, 3H, OCH3), 3.904 (s, 6H, 2
References
OCH ), 7.25 (s, 2H, C(2)H C(6)H), 9.82 (s, 1H, HC O); 13C-NMR
–
–
3
(phosphate-buffered D2O/CD3CN) d (ppm) N-acetyl-S-[2-oxo-2-
(3,4,5-trimethoxyphenyl)ethyl]-L-cysteine: 22.7, 37.9, 45.5, 54.7,
56.5, 62.7, 107.0, 131.6, 142.6, 153.3, 172.9, 176.4, 196.5;
and 3,4,5-trimethoxybenzaldehyde: 56.5, 60.9, 107.7, 132.1,
143.6, 153.9, 193.9. After removal of buffer salts by reverse-
phase C18-silica gel column chromatography (acetonitrile/
water, 1:1 v/v, containing 0.1% trifluoroacetic acid), mass spec-
trometry was carried out. MS (MALDI) m/z: 373.107 [M-d1 þ H]þ
(theor.: 373.118), 395.093 [M-d1 þ Na]þ (theor.: 395.100), 374.119
[M-d2 þ H]þ (theor.: 374.124, presumably formed by exchange
on standing in buffer), 396.101 [M-d2 þ Na]þ (theor.: 396.106).
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