T. Chatterji, K. S. Gates / Bioorg. Med. Chem. Lett. 13 (2003) 1349–1352
1351
and a final elution with 0:100 water/acetonitrile for 20 min.
LC/MS and LC/MS/MS experiments were carried out using a
Thermoseparations liquid chromatograph interfaced to a Fin-
nigan TSQ 7000 triple quadrupole instrument operated in
negative ion electrospray mode. Components of the mixture
were separated by C18 reverse-phase chromatography as
described above, except at a flow rate of 0.8 mL/min and with
NH4OAc (0.01%) added to the mobile phase. Instrument set-
tings included a capillary temperature of 350 ꢀC and capillary
voltage of 2.5 kV. LC/MS analysis of the mixture shown in
Figure 1 is as follows: 8.6 min, corresponds to 2-mercap-
toethanol (identified by retention time and coinjection of
authentic material); 12.4 min, m/z 212.9, corresponds to
[M+OAc]À for 7, x=0; 17.7 min, m/z 244.9, corresponds
to [M+OAc]À for 7, x=1; 21.8 min, m/z 276.9, corresponds to
[M+OAc]À for 7, x=2; 26.1 min, m/z 309.0, corresponds
to [M+OAc]À for 7, x=3; 30.2 min, m/z 341.0, corresponds to
[M+OAc]À for 7, x=4 (the retention times of all preceeding
peaks correspond to that of authentic samples prepared as
described in ref 23); 33.5 min, m/z 367.1, corresponds to
[M+OAc]À for 5 x=2, y=2; m/z 36.4 min, m/z 399.1, corre-
sponds to [M+OAc]À for 5 x=2, y=3; 38 and 39 min, m/z
431.2 corresponds to [M+OAc]À for either 5 x=3, y=3 or 5
x=2, y=4; 43.0 min, m/z 463.2, corresponds to [M+OAc]À
for 5 x=3, y=4; 47.1 min, m/z 521.4, corresponds to
[M+OAc]À for 6 x,y,z=2; 50.4 min, m/z 553.3, corresponds to
[M+OAc]À for 6 x,y=2, z=3, or 6 x,z=2, y=3; 59.2 min,
corresponds to starting material 4. For information regarding
the formation of anionic adducts (e.g., [M+OAc]À ) in nega-
tive ion electrospray mass spectroscopy, see: Cai, Y.; Cole,
R. B. Anal. Chem. 2002, 74, 985. Kamel, A. M.; Brown, P. R.;
Munson, B. Anal. Chem. 1999, 71, 5481.
16. In the reaction of 100 equiv of 2-mercaptoethanol with 4,
the aromatic dithiol product (8) was characterized as its
dimethylthioether derivative using the following protocol: to a
solution of 4 (21.5 mg, 0.086 mmol) in chloroform (1 mL) was
added 2-mercaptoethanol (603 mL, 8.6 mmol, 100 equiv) and
triethylamine (2.4 mL, 0.017 mmol, 0.2 equiv). The reaction
was agitated on a vortex mixer for 5 min and allowed to stir
for 30 min at 25 ꢀC. The mixture was evaporated to dryness
under reduced pressure to yield a pale yellow oil that was
mixed with hexane (3 mL) to yield a biphasic mixture. The
hexane layer (top layer) containing the 3,4-dimercaptotoluene
was separated from the bottom layer (containing 2-mercap-
toethanol) and the hexane extract was evaporated under
reduced pressure to yield a pale yellow oil. The oil was
redissolved in benzene (1 mL) and the resulting solution
mixed with an aqueous solution of NaOH (100 mL of a 2.7-
mM solution in water), followed by methyl iodide (14 mL,
0.224 mmol), and the phase-transfer catalyst Aliquat-336
(80 mL, 0.175 mmol). The reaction mixture was stirred for 1 h
at 50 ꢀC and then extracted with ether (3 Â 2 mL). The
extract was dried over sodium sulfate, filtered, and evaporated
under reduced pressure. The resulting colorless oil was sub-
jected to flash column chromatography on silica gel eluted
with 5:1 hexane/ethyl acetate to yield the derivatized product,
Scheme 2.
thiol groups could play a role in the biological activity
of the pentathiepin antibiotics. For example, the pre-
viously observed inhibition of protein kinase C by pen-
tathiepin-containing natural products2 may involve
modification of critical cysteine residues on the
enzyme.29,30 Finally, our findings raise the possibility
that hydrogen sulfide generated in the reaction of
endogenous thiols with pentathiepin antibiotics could
contribute to the production of oxygen radicals31 and to
the overall cytotoxicity32 of these compounds.
Acknowledgements
We thank the National Institutes of Health (CA83925)
for partial financial support of this work. In addition,
we thank Dmitri Zagorevski (University of Missouri)
for LC/MS analysis.
References and Notes
1. Davidson, B. S.; Molinski, T. F.; Barrows, L. R.; Ireland,
C. M. J. Am. Chem. Soc. 1991, 113, 4709.
2. Compagnone, R. S.; Faulkner, D. J.; Carte, B. K.; Chan,
G.; Freyer, A.; Hemling, M. E.; Hofmann, G. A.; Mattern,
M. R. Tetrahedron 1994, 50, 12785.
3. Searle, P. A.; Molinski, T. F. J. Org. Chem. 1994, 59, 6600.
4. Chatterji, T.; Gates, K. S. Bioorg. Med. Chem. Lett. 1998,
8, 535.
5. Lee, H. F. L.; Chan, A. S. C.; Li, T. Chem. Commun. 2002,
2112.
6. Evans, M. B.; Saville, B. Proc. Chem. Soc. 1962, 18.
7. Vineyard, B. D. J. Org. Chem. 1967, 32, 3833.
8. Myers, A. G.; Cohen, S. B.; Kwon, B. M. J. Am. Chem.
Soc. 1994, 116, 1255.
9. Meister, A.; Anderson, M. E. Ann. Rev. Biochem. 1983, 52,
711.
10. Computational studies that consider the attack of thiols
on the pentathiepin ring system have recently been reported:
Greer, A. J. Am. Chem. Soc. 2001, 123, 10379.
11. Sato, R.; Ohyama, T.; Kawagoe, T.; Baba, M.; Nakajo,
S.; Kimura, T.; Ogawa, S. Heterocycles 2001, 55, 145.
12. Chenard, B. L.; Harlow, R. L.; Johnson, A. L.; Vla-
duchick, S. A. J. Am. Chem. Soc. 1985, 107, 3871.
13. Bosser, G.; Anouti, M.; Paris, J. J. Chem. Soc. Perkin
Trans. 2 1996, 1993.
14. Kustos, M.; Steudel, R. J. Org. Chem. 1995, 60, 8056.
15. Separations were conducted using a C18 Rainin Dynamax
Microsorb column (100 A sphere size, 5 mm pore size, 25 mm
length, 10 mm id) eluted with a linear gradient of 100:0 to
61.5:38.5 water/acetonitrile over 40 min, followed by a linear
gradient of 61.5:38.5 to 0:100 water/acetonitrile over 20 min
3,4-bis(methylthio)toluene as
a
white crystalline solid
(Rf=0.67, 12.9 mg, 80% yield). Spectral data for this material
matched with that described in the literature: Root, M. J.;
Sullivan, B. P.; Meyer, T. J.; Deutsch, E. Inorg. Chem. 1985,
24, 2731. Release of hydrogen sulfide in this reaction was
measured as follows: to a solution of 4 (300 mg, 1.2 mmol) in
chloroform (1 mL) was added 2-mercaptoethanol (8.42 mL,
120 mmol, 100 equiv) and triethylamine (33 mL, 0.24 mmol,
0.2 equiv). A stream of nitrogen gas was bubbled into the reac-
tion mixture and the effluent passed through solution of lead
acetate (350 mg in 5 mL water, 0.184 mM) to yield a black pre-
cipitate (PbS). Control reactions utilizing 4 alone or thiol alone
did not produce a black precipitate. The black precipitate was