Selective reduction of peptide isothiazolidin-3-ones

Article abstract of DOI:10.1021/ol062077j

(Diagram presented) Isothiazolidinones are a rare but potentially important chemical moiety in biochemistry. We report the identification of several thiol, phosphinate, and carbon nucleophiles that form covalent adducts by addition to the sulfenamide sulf

Full text of DOI:10.1021/ol062077j

ORGANIC
LETTERS
2006
Vol. 8, No. 25
5697-5699
Selective Reduction of Peptide
Isothiazolidin-3-ones
Timothy P. Shiau,* Daniel A. Erlanson, and Eric M. Gordon†
Sunesis Pharmaceuticals, Inc., 341 Oyster Point BouleVard,
South San Francisco, California 94080
tpshiau@yahoo.com
Received August 22, 2006
ABSTRACT
Isothiazolidinones are a rare but potentially important chemical moiety in biochemistry. We report the identification of several thiol, phosphinate,
and carbon nucleophiles that form covalent adducts by addition to the sulfenamide sulfur. This reduction is selective for isothiazolidinones
over similar peptide disulfides. We synthesized a coumarin-based thioacid nucleophile which shows a marked fluorescence increase after
addition to an isothiazolidinone sulfenamide bond.
PTP-1B is an archetypical protein tyrosine phosphatase
(PTP)1 and has been implicated in a wide variety of diseases
including type II diabetes2 and obesity.3 Attempts to develop
cell-permeable active site4 inhibitors have met with limited
success due to the anionic nature of most inhibitors. Although
researchers have discovered inhibitors that target an allosteric
site distal to the active site5 as well as a covalent modifier
that targets Cys121,6 a second allosteric site, alternative
strategies of inhibition are needed to realize the therapeutic
potential of this target.
Recently, a new oxidized form of PTP-1B was observed
crystallographically, in which the active site cysteine forms
an isothiazolidin-3-one (also referred to as an isothiazolidi-
none or sulfenamide) (Scheme 1).7 PTP-1B undergoes
Scheme 1. Isothiazolidin-3-one Form of PTP-1B
* Present address: NovaCal Pharmaceuticals, 5980 Horton St., Suite 550,
Emeryville, California 94608.
† Palantir Consulting, 955 Channing Ave., Palo Alto, California 94301.
(1) Reviews: (a) Ostman, A.; Bohmer, F. D. Trends Cell Biol. 2001,
11, 258-266. (b) Ukkola, O.; Santaniemi, M. J. Int. Med. 2002, 251, 467-
475. (c) Burke, T. R.; Lee, K. Acc. Chem. Res. 2003, 36, 426-433.
(2) (a) Byon, J. C. H.; Kusari, A. B.; Kusari, J. Mol. Cell. Biochem.
1998, 182, 101-108. (b) Gum, R. J. et al. Diabetes 2003, 52, 21-28.
(3) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins,
S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan, C. C.;
Ramachandran, C.; Gresser, M. J.; Tremblay, M. L.; Kennedy, B. P. Science
1999, 283, 1544-1548.
reversible oxidation of the active site cysteine upon exposure
to reactive oxygen species both in biochemical8 and cellular9
models, and direct evidence for a PTP-1B sulfenic acid had
(7) (a) Salmeen, A.; Andersen, J. N.; Myers, M. P.; Meng, T. C.; Hinks,
J. A.; Tonks, N. K.; Barford, D. Nature 2003, 423, 769-772. (b) Montfort,
R. L. M.; Congreve, M.; Tisi, D.; Carr, R.; Jhoti, H. Nature 2003, 423,
773-777.
(8) (a) Barford, D. Curr. Opin. Struct. Biol. 2004, 14, 679-686. (b)
Tonks, N. K. Cell 2005, 121, 667-670.
(9) (a) Saurin, A. T.; Neubert, H.; Brennan, J. P.; Eaton, P. Proc. Natl.
Acad. Sci. 2004, 101, 17982-17987. (b) Meng, T. C.; Buckley, D. A.; Galic,
S.; Tiganis, T.; Tonks, N. K. J. Biol. Chem. 2004, 279, 37716-37725.
(4) For a recent review, see: Bialy, L.; Waldmann, H. Angew. Chem.,
Int. Ed. 2005, 44, 3814-3839.
(5) Wiesmann, C.; Barr, K. J.; Kung, J.; Zhu, J.; Erlanson, D. A.; Shen,
W.; Fahr, B. J.; Zhong, M.; Taylor, L.; Randal, M.; McDowell, R. S.;
Hansen, S. K. Nat. Struct. Biol. 2004, 11, 730-737.
(6) Hansen, S. K.; Cancilla, M. T.; Shiau, T. P.; Kung, J.; Chen, T.;
Erlanson, D. A. Biochemistry 2005, 44, 7704-7712.
10.1021/ol062077j CCC: $33.50
© 2006 American Chemical Society
Published on Web 11/16/2006

been previously reported.10 However, it remains unclear how
the protein protects itself against irreversible oxidation to
the sulfinic and sulfonic acid forms. The isothiazolidin-3-
one provides a plausible mechanism to prevent overoxidation.
Because this isothiazolidinone represents an unusual
chemical moiety in biochemistry, we sought to develop a
set of chemical probes that could be used to detect the
presence of the PTP-1B sulfenamide inside the cell or as
potential “warheads” for inhibitors. We first needed a
chemical model of the isothiazolidinone.
Although an isothiazolidinone-containing small molecule
has been previously reported as a chemical model of oxidized
PTP-1B,11 this model is not based on a peptide backbone.
We turned to a class of molecules first reported by Morin
and colleagues12 as a potentially more biologically relevant
model of the PTP-1B sulfenamide. Cystinyl peptide disul-
fides 1a,b can be oxidized with bromine to give the
corresponding dipeptide isothiazolidinones 2a,b (Scheme 2).
and oxygen-based nucleophiles with alkyl, aromatic, and acyl
variants of each. In general, we found that oxygen- and
nitrogen-based nucleophiles were too weak, whereas sulfur-
and phosphorus-based nucleophiles were sufficient to break
the sulfenamide bond. The regiochemistry of these com-
pounds was verified by 1H NMR, 13C NMR, and IR spectra
(see Supporting Information).
Next, we sought to eliminate those nucleophiles that were
so potent that they would react with disulfides as well as
with sulfenamide bonds. By cross-screening the reactive
nucleophiles with the disulfide 1b, we were able to determine
which nucleophiles were chemoselective for the sulfenamide
moiety. The results are shown in Table 1.
Table 1. Cross-Screening of 1b and 2b with Nucleophiles
Scheme 2. Synthesis of Dipeptide Sulfenamides
Although the reaction is very sensitive to stoichiometry, the
best results were generally obtained with a significant excess
of bromine, slow warming, and reductive workup with
aqueous Na2S2O3.
Because isothiazolidinones are rare in biochemistry, we
sought to identify nucleophiles that would specifically react
with the isothiazolidinone under physiological conditions.
Our strategy depended on the electrophilicity of the isothia-
zolidinone, focusing on nucleophiles that were too weak to
react with common physiological electrophiles such as
disulfides (Scheme 3).
Scheme 3. Screening Strategy for Nucleophiles
Interestingly, all thiophenols, even electron-deficient ones,
failed to achieve any selectivity.
Because fluorescence spectroscopy is one of the most
sensitive and straightforward detection systems, we sought
to generate a fluorescent probe in which nucleophile addition
to a sulfenamide would alter the chromophore.
Isothiazolidinone 2b in ethanolic solution was treated with
a variety of sulfur-based, phosphorus-based, nitrogen-based,
(10) Denu, J. M.; Tanner, K. G. Biochemistry 1998, 37, 5633-5642.
(11) Sivaramakrishnan, S.; Keerthi, K.; Gates, K. S. J. Am. Chem. Soc.
2005, 127, 10830-10831.
(12) Morin, R. B.; Gordon, E. M.; Lake, J. R. Tetrahedron Lett. 1973,
14, 5213-5216.
Coumarin-3-carboxylic acids possess an intramolecular
hydrogen bond between the acid functionality and the
5698
Org. Lett., Vol. 8, No. 25, 2006

2-carbonyl.13 Because the internal ester is intimately linked
to the fluorescent properties of the molecule, we reasoned
that coversion of a coumarin-3-thioacid to a disulfide would
alter the fluorescent properties of the coumarin.
increase in fluorescence only when the coumarin-cysteine
conjugate is formed, with an excitation maximum at 392 nm
and an emission maximum at 422 nm (Figure 1).
Coumarin-3-thioacid (5) was synthesized in two steps from
commercially available coumarin-3-carboxylic acid. Forma-
tion of the acid chloride in neat thionyl chloride followed
by reaction with concentrated NaSH yielded the thioacid 5
in 45% overall yield (Scheme 4).
Scheme 4. Synthesis of Coumarin Thioacid
The coumarin thioacid shows a reactivity profile similar
to that for the thiobenzoic acid on which the structure was
based (product 3a, Table 1); it reacts with the sulfenamide
quantitatively within 1 h but shows absolutely no reactivity
with the disulfide even after 48 h at room temperature
(Scheme 5).
Figure 1. Excitation (solid) and emission (dashed) spectra of 6
(red) and 5 (green).
In conclusion, we have applied a screening strategy to
identify several classes of nucleophiles which chemoselec-
tively react with isothiazolidinones but not disulfides. One
nucleophile, a thioacid, was modified to act as a fluorescent
detector of isothiazolidinones. This strategy could be used
to develop PTP-1B inhibitors as well as to elucidate the
biological role of isothiazolidinones with fluorescence spec-
troscopy.
Scheme 5. Synthesis of the Coumarin-Peptide Conjugate
Acknowledgment. We acknowledge Small Business
Innovative Research Grant R43-DK063764 for partial fi-
nancial support of this project.
Supporting Information Available: Experimental pro-
cedures and characterizations of compounds 3a-l, 5, and 6
(1H NMR, 13C NMR, MS). This material is available free of
charge via the Internet at http://pubs.acs.org.
Fluorescence spectroscopy of the coumarin-cysteine
conjugate (6) and coumarin thioacid (5) shows a marked
(13) Dobson, A. J.; Gerkin, R. E. Acta Crystallogr. C 1996, 52, 3081-
3083.
OL062077J
Org. Lett., Vol. 8, No. 25, 2006
5699