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65
in the corresponding bisulfite salts, with bisulfite 7d (R2 =
cyclohexylmethyl) being the most potent. In order to determine
the nature of the active species, the behavior of aldehyde 6a and
its corresponding bisulfite salt 7a was examined by mass spectros-
copy. In separate experiments, compounds 6a and 7a were dis-
solved in dimethyl sulfoxide and diluted 1–1000 in either
acetonitrile or water and examined by MS and tandem MS–MS. In
acetonitrile the expected peaks for aldehyde 6a were 404.4 M+H+
(dominant peak) and 426.3 M+Na. The mass spectra of bisulfite salt
7a using negative mode detection, showed a dominant peak at
484.5 for (Mꢀ1)ꢀ, a loss of H+ from the sulfonic acid moiety.
Aldehyde 6a in aqueous solution showed peaks corresponding to
the aldehyde (404.6), the aldehyde + sodium (426.4) and hydrated
aldehyde + sodium (444.2) in positive mode. In water, bisulfite ad-
duct 7a displayed a dominant peak at 484.5 in negative mode and
the relative intensities of this parent ion and other ions remained
unchanged over 24 h (a time course study was carried out). In the
case of 6a, the hydrated form was the dominant species after only
5 min exposure to water, while 7a remains unchanged as the bisul-
fite form after 24 h. The results indicate that the bisulfite adduct of
7a is stable in aqueous solution; however, in buffer solution, pH 7.4,
compound 7a gradually dissociates into the corresponding alde-
hyde 6a within an hour, rapidly becoming hydrated. These observa-
tions are in agreement with the results of X-ray crystallographic
studies showing that incubation of bisulfite adduct 7a with norovi-
rus 3CLpro in buffer solution results in the formation of an enzyme–
aldehyde complex, with the active site cysteine residue covalently
bonded to the carbonyl carbon of aldehyde 6a.34 Lastly, the variable
stability of the bisulfite adducts in buffer solution has precluded
accurate determination of the IC50 values of the inhibitors.
11. Dou, D.; He, G.; Mandadapu, S. R.; Aravapalli, S.; Kim, Y.; Chang, K.-O.; Groutas,
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25. Aldehyde bisulfite adducts have been previously used in the inhibition of
glycollate oxidase (Corbett, J. R.; Wright, B. J. Phytochemistry 1971, 10, 2015)
and thrombin (Ruterbories, K. J.; Shuman, R. T. U.S. Patent 5,436,229, 1995).
26. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157. The
residues on the N-terminus side of the peptide bond that is cleaved are
designated P1–Pn and those on the C-terminus side are designated 0P10–Pn0. The
corresponding active site subsites are designated S1–Sn and S10–Sn
.
27. Dragovich, P. S.; Prins, T. J.; Zhou, R.; Webber, S. E.; Marakovits, J. T.; Fuhrman,
S. A.; Patick, A. K.; Matthews, D. A.; Lee, C. A.; Ford, C. E.; Burke, B. J.; Rejto, P. A.;
Hendrickson, T. F.; Tuntland, T.; Brown, E. L.; Meador, J. W.; Ferre, R. A.; Harr, J.
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Kjell, D. P.; Slattery, B. J.; Semo, M. J. J. Org. Chem. 1999, 64, 5722. Briefly,
aldehyde or
a-ketoamide (1.24 mmol) was dissolved in ethyl acetate (2 mL)
and a solution of sodium bisulfite (1.12 mmol) in ethanol (1 mL) and water
(0.4 mL) was added. The mixture was heated to 40 °C with stirring. After the
mixture was stirred for 2 h, it was allowed to cool to room temperature. The
solution was filtered and the solid residue was washed with ethanol (5 mL).
The filtrate was dried over anhydrous sodium sulfate, filtered, and
concentrated, leaving an oily residue. Treatment with ether gave a solid (60–
80% yield).
In summary, the utilization of bisulfite adducts of transition
state inhibitors of cysteine and serine proteases in the in vitro
and cell-based inhibition of norovirus 3CL protease has been
described for the first time.
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