Solid-phase synthesis of peptide vinyl sulfones as potential inhibitors and activity-based probes of cysteine proteases

Article abstract of DOI:10.1021/ol0275567

(Matrix presented) Peptide vinyl sulfones were prepared from 2-chlorotrityl resin-bound phenolic amino vinyl sulfones in high yield and purity. This method enables the convenient synthesis of peptide vinyl sulfones having different amino acids at the P

Full text of DOI:10.1021/ol0275567

ORGANIC
LETTERS
2003
Vol. 5, No. 5
737-740
Solid-Phase Synthesis of Peptide Vinyl
Sulfones as Potential Inhibitors and
Activity-Based Probes of Cysteine
Proteases
,†,‡
Gang Wang,† Uttamchandani Mahesh,‡ Grace Y. J. Chen,†,‡ and Shao Q. Yao*
Departments of Chemistry and Biological Sciences, National UniVersity of Singapore,
3 Science DriVe 3, Singapore 117543, Republic of Singapore
chmyaosq@nus.edu.sg
Received December 26, 2002
ABSTRACT
Peptide vinyl sulfones were prepared from 2-chlorotrityl resin-bound phenolic amino vinyl sulfones in high yield and purity. This method
enables the convenient synthesis of peptide vinyl sulfones having different amino acids at the P1 position. It also allows efficient synthesis
of vinyl sulfone-containing, activity-based probes of cysteine proteases used in a proteomic experiment.
Cysteine proteases are an important class of enzymes
involved in the hydrolysis of peptide amide bonds. They play
vital roles in numerous physiological processes such as
arthritis, osteoporosis, Alzheimer’s disease, cancer cell
invasion, and apoptosis.1 Many low-molecular weight inhibi-
tors of cysteine proteases such as epoxysuccinyl derivatives,
peptidyl Michael acceptors, (acyloxy)methyl ketones, and
halomethyl compounds have been reported.2 Recently, pep-
tidyl vinyl sulfones and their derivatives were found to
inactivate cysteine proteases selectively.3 They function by
mimicking the peptide substrates of cysteine proteases. Upon
binding to the active site of the enzyme, the vinyl sulfone
moiety of the inhibitor acts as a Michael acceptor and reacts
with the nucleophilic cysteine residue located within the
active site, resulting in the formation of a covalently linked,
irreversible enzyme-vinyl sulfone complex. The advantage
of such mechanism-based, covalent reactions has led to their
application in mechanistic studies of proteases and protea-
somes,4 protein engineering and modifications,5 and more
recently, activity-based protein profilings.4,6
Typically, peptide vinyl sulfones have been prepared by
methods based on conventional solution-phase peptide
synthesis,1,3,4a whereby individual amino acid-containing
vinyl sulfones were synthesized, followed by sequential
couplings of additional amino acid residues to make up the
full-length peptide chain. The whole synthesis was done in
solution, making the process inefficient and time-consuming.
More recently, a number of solid-phase strategies have been
(4) (a) Bogyo, M.; McMaster, J. S.; Gaczynska, M.; Tortorella, D.;
Goldberg, A. L.; Ploegh, H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6629-
6634. (b) Bogyo, M.; Shin, S.; McMaster, J. S.; Ploegh, H. L. Chem. Biol.
1998, 5, 307-320. (c) Nazif, T.; Bogyo, M. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 2967-2972. (d) Groll, M.; Nazif, T.; Huber, R.; Bogyo, M. Chem.
Biol. 2002, 9, 655-662.
† Department of Chemistry.
‡ Department of Biological Sciences.
(1) (a) Otto, H. H.; Schirmeister, T. Chem. ReV. 1997, 97, 133-171. (b)
Roush, W. R.; Gwaltney, S. L., II; Cheng, J.; Scheidt, K. A.; McKerrow,
J. H.; Hansell, E. J. Am. Chem. Soc. 1998, 120, 10994-10995.
(2) Hernandez, A. A.; Roush, W. R. Curr. Opin. Chem. Biol. 2002, 6,
459-465.
(3) Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bromme, D. J. Med. Chem.
1995, 38, 3193-3196
(5) Walsh, C. T. Enzymatic Reaction Mechanisms; Freeman: New York,
1979.
(6) Liau, M. L.; Panicker, R. C.; Yao, S. Q. Tetrahedron Lett. 2003, 44,
1043-1046.
10.1021/ol0275567 CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/12/2003

Scheme 1. Solid-Phase Synthesis of Peptide Vinyl Sulfonesa
a Reagents and conditions: (a) N,O-Dimethylhydroxylamine, DCC/HOBt/DIEA, DMF, 5 h at rt, 80-99%. (b) LiAlH4, THF, 15-20 min
at 0 °C, 52-90%. (c) 4 (1 equiv), NaH, THF, 1.5 h at rt, 59-78%. (d) Pyridine, Cs2CO3, THF, 12-18 h at rt, 0.43-0.52 mmol/g loading.
(e) Fmoc solid-phase peptide synthesis.
developed.7,4b By taking advantage of Kenner’s safety catch
strategy,8 Overkleeft et al. first synthesized the N-terminal
peptide fragment of the vinyl sulfone on a solid support,
followed by nucleophilic cleavage/ligation using a desired
vinyl sulfone-containing, C-terminal amino acid. Deprotec-
tion of the resulting product followed by HPLC purification
gave the final peptide vinyl sulfone in 20-40% yield.7 This
method is intrinsically inefficient and low yielding, due to
the generation of a fully protected peptide product following
the cleavage/ligation step.9 Consequently, this makes it
difficult to synthesize vinyl sulfones having longer peptide
chains. Alternatively, Nazif and Bogyo reported a solid-phase
method for generating positional-scanning combinatorial
libraries of peptide vinyl sulfones.4c By attaching a vinyl
sulfone-containing aspartic acid onto a Rink amide resin via
its side-chain carboxylic acid, these workers were able to
generate P2-P4 positional-scanning tetrapeptidic vinyl sul-
fone libraries while holding the P1 position constant.
However, this strategy is limited only to synthesis of peptide
vinyl sulfones having carboxyl side chains at the P1 positions
(e.g. Asp and Glu).
Herein, we report a facile yet efficient solid-phase method
that may be used for the preparation of peptide vinyl sulfones
having any amino acid at the P1 position. By anchoring the
vinyl sulfone-derivatized P1 amino acid residue onto 2-
chlorotrityl resin via the phenolic alcohol moiety of the vinyl
sulfone (Scheme 1), this strategy allows the generation of
peptide vinyl sulfones from any peptide sequence, either
individually or combinatorially, with high yield and ef-
ficiency. Furthermore, this strategy may be used for facile
synthesis of activity-based probes to specifically target
cysteine proteases in a crude proteome mixture.4,6 With the
increasing emphasis in the field of proteomics, this method
thus provides a valuable tool for (1) the generation potential
inhibitors of cysteine proteases critical for diseases and (2)
the identification/profiling of new cysteine proteases in an
organism.
Our strategy took advantage of peptide vinyl sulfones
containing a phenol group adjacent to the vinyl sulfone
moiety. It had been previously shown that having a phenyl
or phenolic group (instead of a methyl group) next to the
vinyl sulfone not only does not compromise the inhibitory
activity of the peptide vinyl sulfone toward its targeting
enzyme but, in many cases, actually enhances the potency
of the inhibitor.4b We reasoned that, by modification of the
P1 amino acid with a phenolic vinyl sulfone, followed by
loading the resulting product (e.g., 5 in Scheme 1) onto a
suitable solid support via the phenolic alcohol, any peptide
vinyl sulfone may be potentially synthesized with high
efficiency using conventional solid-phase peptide synthesis.
We chose to develop such a strategy on the basis of Fmoc
chemistry, as it is the preferred method for solid-phase
peptide synthesis. Our first task was to synthesize N-
fluorenylmethoxycarbonyl (Fmoc)-R-amino aldehydes. They
were conveniently synthesized by a well-established, two-
step procedure, involving the transformation of N-Fmoc-R-
amino acid to the corresponding Weinreb amide, followed
by reduction with LiAlH4.10 The N-Fmoc-R-amino acids 1
were first coupled with N,O-dimethylhydroxylamine to give
the resulting amides 2 in 80-99% yields, followed by LiAlH4
reduction at 0 °C to give the aldehydes 3. Aldehydes
corresponding to all 20 natural amino acids were synthesized,
most of which afforded the desired products in good yields
(52-90%; Table 1). Examination of the products showed
that most aldehydes were free of racemization and had high
purities, except for proline and arginine, the former giving
a racemic mixture and the latter forming an intramolecular
ring.11 Subsequent steps were carried out with five randomly
(7) (a) Overkleeft, H. S.; Bos, P. R.; Hekking, B. G.; Gordon, E. J.;
Ploegh, H. L.; Kessler, B. M. Tetrahedron Lett. 2000, 41, 6005-6009. (b)
Kessler, B. M.; Tortorella, D.; Altun, M.; Kisselev, A. F.; Fiebiger, E.;
Hekking, B. G.; Ploegh, H. L.; Overkleeft, H. S. Chem. Biol. 2001, 8, 913-
929.
(8) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem. Soc.,
Chem. Commun. 1971, 636-637.
(9) Backes, B. J.; Ellman, J. A. Curr. Opin. Chem. Biol. 1997, 1, 86-
93.
(10) (a) Lubell, W. D.; Rapoport, H. J. Am. Chem. Soc. 1987, 109, 236-
239. (b) Zemlicka, H.; Murata, M. J. Org. Chem. 1976, 41, 3317-3321.
(11) Ho, P. T.; Ngu, K. Y. J. Org. Chem. 1993, 58, 2313-2316.
738
Org. Lett., Vol. 5, No. 5, 2003

Scheme 2. Synthesis of the Activity-Based Probea
Table 1. Yield of N-Fmoc-R-amino Aldehydes 3
3
yield (%)
3
yield (%)
Fmoc-Val
Fmoc-Asp(tBu)
Fmoc-Leu
Fmoc-Tyr(tBu)
Fmoc-Glu(tBu)
Fmoc-Ser(tBu)
Fmoc-His(Trt)
Fmoc-Ile
52
86
81
85
52
80
67
79
80
Fmoc-Gly
Fmoc-Ala
89
81
87
64
90
89
78
76
87
Fmoc-Lys(Boc)
Fmoc-Trp(Boc)
Fmoc-Phe
Fmoc-Cys(Trt)
Fmoc-Asn(Trt)
Fmoc-Gln(Trt)
Fmoc-Thr(tBu)
Fmoc-Met
chosen amino aldehydes. N-Fmoc-R-amino aldehydes, 3,
were reacted with 4-hydroxy-thiophenyl-methyl-diethylphos-
ponate, 4, prepared as previously reported,4b to give phenolic-
N-Fmoc-R-amino-vinyl sulfones 5 in a Horner-Emmons
condensation reaction. The reaction was carried out smoothly
in the presence of NaH at room temperature, generating both
the trans isomer (e.g., 5a in Scheme 1) as the predominantly
1
major product (>90% as judged by H NMR and HPLC),
and the cis isomer, 5b, with acceptable yield (59-78% for
5a + 5b). No racemization of 5 at its chiral center was
observed, as judged by both H NMR and HPLC, in good
a Reagents and conditions: (a) 20% piperidine in DMF, 30 min.
(b) HBTU (4 equiv), HOBt (4 equiv), DIEA (8 equiv), Fmoc-AA-
OH (4 equiv), preactivation for 3 min, then coupling for 2 h. (c)
Cy3 (3 equiv), DIC (3 equiv), HOBt (3 equiv), DIEA (6 equiv),
12-18 h. (d) Reagent R.
1
agreement with previous reports for similar reactions.10a
Compounds 5a and 5b were proven to be inseparable by
TLC and flash chromatography and therefore used as a
mixture for the subsequent attachment to the solid support.
We chose 2-Cl-trityl resin because the phenolic alcohol on
5 can be loaded onto this solid support under mild conditions
with high efficiency. In addition, the resin is compatible with
Fmoc chemistry. A number of loading strategies were tested.
It was found that using cesium carbonate together with
pyridine as a mild base allowed different phenolic-N-Fmoc-
R-amino-vinyl sulfones 5 to be loaded onto the resin with
high efficiency (loading level ) 0.43-0.52 mmol/g; Table
2).
from the resin and directly analyzed by reverse-phase HPLC
and mass spectrometry to assess its purity: more than 90%
of products generated were the desired products (Figure 1),
Table 2. Yield of 5 and the Loading Efficiency on 2-Cl-Trityl
Resin
5
yield (%)
6 loading efficiency (mmol/g)
Fmoc-Leu
74
78
72
59
67
0.44
0.43
0.52
0.48
0.45
Fmoc-Lys(Boc)
Fmoc-Tyr(tBu)
Fmoc-Asp(tBu)
Fmoc-Phe
Figure 1. HPLC analysis of the crude peptide vinyl sulfone.
We next examined the utility of these vinyl sulfone-
containing resins in the solid-phase synthesis of peptide vinyl
sulfones. As an example, N-Fmoc-R-Leu-vinyl sulfone resin
was used as the solid support, and a potential tetrapeptide
inhibitor (e.g., H2N-Gly-Tyr-Leu-Leu-vinyl sulfone) was
synthesized using standard Fmoc-based peptide chemistry
(see Scheme 2 for a representative synthetic strategy). Upon
completion of the synthesis, the resulting product was cleaved
in which both the cis and trans isomers of the peptide vinyl
sulfone were observed, in good agreement with previous
reports.4 In addition, no epimerized product was detected,
further indicating that the Horner-Emmons condensation
(e.g., 3 f 5 in Scheme 1) was indeed racemization-free.
We then extended the strategy to the solid-phase synthesis
of a vinyl sulfone-containing, activity-based probe that targets
Org. Lett., Vol. 5, No. 5, 2003
739

cysteine proteases. The design of the probe was as previously
described.6 Briefly, in addition to the vinyl-sulfone reactive
unit, the probe also contains a tetrapeptide recognition unit,
as well as a Cy3-containing fluorescence unit, which
facilitates the detection of proteins upon labeling by the probe
(Scheme 2). The probe 7 was synthesized using the N-Fmoc-
R-Tyr(tBu)-vinyl sulfone resin. The Cy3 dye was conven-
iently coupled onto the solid support at the end of peptide
synthesis, using the standard DIC/HOBt/DIEA strategy.
Upon cleavage of the resin, the resulting probe was precipi-
tated with cold ether and purified to homogeneity by HPLC.
The probe was subsequently tested for selective labeling
of cysteine proteases on the basis of their enzymatic activity
(Figure 2). A panel of 12 enzymes was used, of which two
were cysteine proteases. Only cysteine proteases (i.e., lanes
4 and 5) were selectively labeled, indicating the specific
nature of the probe. To further confirm that the fluorescence
labeling of the cysteine proteases by the probe is due to their
enzymatic activity, the enzymes were first inactivated by heat
and then treated with the probe followed by SDS-PAGE
analysis. No labeling of the cysteine proteases was observed,
indicating that the enzymatic activity is a prerequisite for
the labeling reaction to occur.
Figure 2. Activity-based protein profiling using probe 7. Lane 1:
Type I-S Alkaline Phosphatase, from bovine intestine. Lane 2: Type
VIII Alkaline Phosphatase, from rabbit intestine. Lane 3: Type IV
Alkaline Phosphatase, from porcine intestinal mucosa. Lane 4:
Chymopapain, from papaya latex. Lane 5: Papain, from papaya
latex. Lane 6: R-Chymotrypsin. Lane 7: â-Chymotrypsin. Lane
8: γ-Chymotrypsin. Lane 9: Proteinase K, from tritirachium album.
Lane 10: Subtilisin, from bacillus licheniformis. Lane 11: Lysozyme,
from chicken egg white. Lane 12: Lipase, from Candida rugosa.
For details of the labeling reaction, see Supporting Information.
identification of new cysteine proteases and the generation
of their potential inhibitors.
In conclusion, we have successfully developed a facile and
efficient approach that allows the solid-phase synthesis of
peptide vinyl sulfones using standard Fmoc chemistry. This
method circumvents numerous limits imposed by previously
reported solid-phase methods.4b,7 Peptide vinyl sulfones
having different amino acids at the P1 position may be readily
synthesized in high yield and purity. The method may be
used to synthesize vinyl sulfone-containing, activity-based
probes to profile cysteine proteases in a proteome. Conse-
quently, this strategy may provide a useful tool for the
Acknowledgment. Funding support was provided by the
National University of Singapore (NUS) and the Agency for
Science, Technology and Research (A*STAR) of Singapore.
S.Q.Y. is the recipient of the 2002 Young Investigator Award
(YIA) from the Biomedical Research Council (BMRC).
Supporting Information Available: Experimental details
and characterizations of compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
OL0275567
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Org. Lett., Vol. 5, No. 5, 2003