D. B. A. de Bont et al. / Bioorg. Med. Chem. 7 (1999) 1043±1047
1047
�
recorded on a VARIAN G300-spectrometer (75.5 MHz).
Fast atom bombardment (FAB) mass spectrometry was
carried out using a Jeol JMS SX/SX 102A four-sector
mass spectrometer, coupled with a HP-9000 data sys-
tem. All peptides and their corresponding peptidosulfo-
namides were fully characterized by H and C NMR
as well as mass spectrometry. Their purity was assessed
by HPLC and generally greater than 95%. If necessary,
the peptidosulfonamides were puri®ed by preparative
reverse phase HPLC. HPLC analysis was carried out on
a Gilson automated HPLC system 205 with a 233XL
autosampler and a 119 UV-Vis detector. Mass spectro-
metry was also used to identify the fragments of enzy-
matic cleavage.
containing 0.1% DMSO to a 10 mg mL 1 solution of
TAP substrate peptide 10 (52 mL). Aliquots (50 mL)
were taken at time intervals as described above for the
trypsin catalyzed hydrolysis of 10 and the reaction was
stopped by dilution with 1 H HCl solution (150 mL).
The amount of peptide 10 and TAP substrate sulfona-
mides 11±13, normalized to the internal standard (0.1%
DMSO), were determined by integration of the peak
areas of the appropriate signals. The percentage of
degradation and half-life of the C. subtilisin cata-
lyzed hydrolysis is shown in Figure 2 and Table 2,
respectively.
1
13
Acknowledgements
Pepsin catalyzed hydrolysis of Leu-enkephaline amide
Financial support from Solvay-Duphar is gratefully
acknowledged (D.B.A.B.). Mr. C. Versluis is thanked
for recording the mass spectra.
(
(
1), Leu-enkephaline sulfonamides 2±5, Leu-enkephaline
6), Leu-enkephaline sulfonamides 7±9, TAP peptide 20
and TAP peptidosulfonamides 11±13. 200 mL of a
�
1
10 mg mL solution of the peptide or peptidosulfona-
mide was diluted with 4.8 mL of a 50 mM formic acid
solution (pH 2.2). After incubation for 15 min at 37 C,
References
ꢀ
1
2
. Horwell, D. C. Bioorg. Med. Chem. 1996, 4, 1573.
. Adang, E. P.; Hermkens, P. H. H.; Linders, J. T. M.;
2
units of pepsin (12 mg) were added. Samples (200 mL)
were taken at 0, 15, 30, 45, 60, 90, 120, 180, 240, 300,
60, and 420 min, the reaction was stopped by dilution
Ottenheijm, H. J. C.; van Staveren, C. J. Recl. Trav. Chim.
Pays-Bas 1994, 113, 63.
3
with acetonitrile (600 mL), centrifuged for 5 min, the
supernatant was decanted and analyzed by HPLC using
a C-8 column and as eluent a gradient of 100% water
containing 0.1% TFA to 95% acetonitrile containing
3
4
6
5
. Liskamp, R. M. J. Recl. Trav. Chim. Pays-Bas 1994, 113, 1.
. Liskamp, R. M. J. Angew. Chem., Int. Ed. Engl. 1994, 33,
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. Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.;
0
.1% TFA. The relative amount of the remaining
Fodor, S. P. A.; Adams, C. L.; Sundaram, A.; Jacobs, J. W.;
Schultz, P. G. Science 1993, 26, 1303.
6. Miller, S. M.; Simon, R. J.; Ng, S.; Zuckermann, R. N.;
Kerr, J. M.; Moos, W. H. Bioorg. Med. Chem. Lett. 1994, 4,
peptide or peptidosulfonamide, normalized to an inter-
nal standard (formic acid) was determined by integra-
tion of the peak areas of the appropriate signals. The
percentage of degradation and half-life of the pepsin
catalyzed hydrolysis of the Leu-enkephalin peptides and
peptidosulfonamides is shown in Figure 1 and Table 1,
respectively.
2
7
657.
. Benkirane, N.; Guichard, G.; Briand, J. P.; Muller, S. J.
Biol. Chem. 1996, 271, 33218.
. Seebach, D.; Overhand, M.; Ku
B.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta
8
È
hnle, F. N. M.; Martinoni,
1
2
996, 79, 913; Hintermann, T.; Seebach, D. Chimia 1997, 50,
44.
Trypsin catalyzed hydrolysis of TAP substrate peptide
1
1
0 and TAP substrate sulfonamides 11±13. 52 mL of a
0 mg mL solution of peptide or peptidosulfonamide
9. de Bont, D. B. A.; Dijkstra, G. D. H.; den Hartog, J. A. J.;
Liskamp, R. M. J. Bioorg. Med. Chem. Lett. 1996, 6, 3035.
10. Salmon, S. E.; Lam, K. S.; Lebl, M.; Kandola, A.; Khat-
�
1
was diluted with 565 mL of a 100 mM Tris buer (pH
.8) containing 0.1% DMSO as an internal standard
and 25 mM CaCl . After addition of 32 mL of a trypsin
tri, P. S.; Wade, S.; Patek, M.; Kocis, P.; Krchnak, V.;
 Â
7
Thorpe, D.; Felder, S. Proc. Natl. Acad. Sci. USA 1993, 90,
11708.
11. We have introduced the term `peptidosulfonamide'
2
�
1
solution (0.43 units mL ) the mixture was gently shaken
at 37 C. Aliquots (50 mL) were taken at 0, 10, 20, 30, 45,
6
ꢀ
0, 90, 120, 180, 240, 300, 360, and 420 min, the reaction
(
Moree, W. J.; van der Marel, G. A.; Liskamp, R. M. J. J.
Org. Chem. 1995, 60, 5157) for peptides containing b-amino
sulfonamide residues. a-amino sulfonamides are not stable
and therefore peptides containing these residues are not stable,
see e.g.: Moree, W. J.; Van Gent, L. C.; Van der Marel, G. A.;
Liskamp, R. M. J. Tetrahedron 1993, 49, 1133; Merricks, D.;
Sammes, P. G.; Walker, E. R. H.; Henrick, K.; McPartlin, M.
M.; J. Chem. Soc. Perkin Trans. 1 1991, 2170.
was stopped by dilution with 1 H HCl solution (150 mL)
and analyzed by HPLC as described above for com-
pound 1. The amount of peptide 10 and peptidosulfo-
namides 11±13, normalized to the internal standard was
determined by integration of the peak areas of the
appropriate signals. The percentage of degradation and
half-life of the trypsin catalyzed hydrolysis is shown in
Figure 2 and Table 2, respectively.
12. Whitesides, G. M.; Wong, C. H. Enzymes in Synthetic
Organic Chemistry. Elsevier: Oxford, 1994; pp 41±130.
1
1
3. Meldal, M.; Breddam, K. Anal. Biochem. 1991, 195, 141.
4. Gromme, M.; van der Valk, R.; Sliedregt, K.; Vernie, L.;
mmerling, G.; Koopmann, J.-O.; Momburg,
F.; Neefjes, J. Eur. J. Immunol. 1997, 27, 898.
5. Eichler, J.; Houghten, R. A. Biochemistry 1993, 32, 11035.
Â
C. Subtilisin catalyzed hydrolysis of TAP substrate
peptide 10 and TAP substrate sulfonamides 11±13.
Degradation, catalyzed by C. subtilisin, was carried out
by adding a solution of C. subtilisin (32 mL, 0.05 units
Liskamp, R.; Ha
È
1
16. Kaspari, A.; Schierkorn, A.; Schutkowski, M. Int. J. Pept.
Prot. Res. 1996, 48, 486.
�
1
mL ) and HEPES buer (565 mL, 35 mM, pH 7.8)