1
7
1
d
.85%); d
.29 (2 H, d, J 8.1, Ph); d
33.81; d (282.23 MHz; d
-DMSO) −139.69; d
-DMSO) 2.41.
H
(300 MHz; d
6
-DMSO) 7.08 (2 H, d, J 8.1, Ph),
(75.9 MHz; d -DMSO) 126.79,
(96.29 MHz;
by the method of Lineweaver and Burk. Data were fitted to the
best straight line by the least-squares procedure.
C
6
F
6
B
Assay of inhibitors of trypsin. Trypsin Type IIs from bovine
pancreas was purchased from Sigma-Aldrich. The inhibition
constants were determined exactly as was done for chy-
motrypsin, except that the substrate was benzoyl-arginine-p-
nitroanilide dissolved in DMF.
6
Potassium 3-carboxyphenyltrifluoroborate.Yield 84%. (Found:
C, 36.55; H, 2.06%. Calc. for C BF KO : C, 36.87; H,
.21%); d (300 MHz; d -DMSO) 7.17 (1 H, t, J 7.35, Ph), 7.50
1 H, d, J 7.2, Ph), 7.61 (1 H, d, J 7.8, Ph), 7.94 (1 H, s,
Ph); d (75.9 MHz; d -DMSO) 126.96, 129.24, 133.26, 136.55,
69.34; d (282.23 MHz; d (96.29 MHz;
-DMSO) −139.97; d
-DMSO) 6.34.
7
H
5
3
2
2
(
H
6
C
6
Acknowledgements
1
d
F
6
B
The authors thank the Israeli Science Foundation, grant
6
6
63/99-2 and the Ministry of Science and Technology for
Potassium 4-fluorophenyltrifluoroborate. Yield 96%. (Found:
C, 35.33; H, 1.90%. Calc. for C BF K: C, 35.68; H, 2.00%);
(300 MHz; d -DMSO) 6.84 (2 H, t, J 8.55, Ph), 7.29
2 H, t, J 6.9, Ph); d (75.9 MHz; d -DMSO) 113.22, 113.46,
33.43, 133.53; d (282.23 MHz; d
-DMSO) −139.26, −119.07;
(96.29 MHz; d -DMSO) 4.13.
support of this work.
6
H
4
4
d
(
1
d
H
6
References
C
6
1
B. Walker and J. F. Lynas, Cell. Mol. Life Sci., 2001, 58, 596 and
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F
6
B
6
2
D. Leung, G. Abbenante and D. P. Fairlie, J. Med. Chem., 2000, 43,
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05.
Potassium 2-hydroxyphenyltrifluoroborate. Yield 88%. (Found:
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(300 MHz; d -DMSO) 6.48 (1 H, d, J 8.1, Ph), 6.60 (1 H,
t, J 6.6, Ph), 6.91 (1 H, t, J 7.65, Ph), 7.11 (Ph, d, J 6.6, Ph),
3
4
5
R. Babin and S. L. Bender, Chem. Rev., 1997, 97, 1359.
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H
5
3
H. U. Demuth, J. Enzyme Inhib., 1990, 3, 249.
d
H
6
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002, 102, 4639.
7
1
.32 (1H, bs, OH); d
27.70, 133.59, 160.01; d
(96.29 MHz; d -DMSO) 6.38.
C
(75.9 MHz; d
6
-DMSO) 114.02, 119.03,
-DMSO) −134.38;
6 P. E. J. Sanderson, Med. Res. Rev., 1999, 19, 179.
7 F. Al-Obeidi and J. A. Ostrem, Drug Discovery Today, 1998, 3, 223.
F
(282.23 MHz; d
6
8
9
M. R. Wiley and M. J. Fisher, Expert Opin. Ther. Pat., 1997, 7, 1265.
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d
B
6
Potassium phenyltrifluoroborate. Yield 90%. (Found: C,
9.02; H, 2.62%. Calc. for C BF K: C, 39.16; H, 2.74%).
(300 MHz; d -DMSO) 7.01 (3 H, m, Ph), 7.30 (2 H, d,
(75.9 MHz; d -DMSO) 125.65, 126.92, 131.95;
(282.23 MHz; d (96.29 MHz; d -DMSO)
-DMSO) −139.53; d
.08.
10 K. Tomoo, K. Satoh, Y. Tsuda, K. Wanaka, S. Okamoto, A. Hijikata-
Okunomiya, Y. Okada and T. Ishida, J. Biochem., 2001, 129, 455.
1 S. R. Presnell, G. S. Patil, C. Mura, K. M. Jude, J. M. Conley, J. A.
3
6
H
5
3
1
d
H
6
Bertrand, C.-M. Kam, J. C. Powers and L. D. Williams, Biochemistry,
J 6.6, Ph); d
d
F
3
C
6
1
998, 37, 17068.
6
B
6
1
2 (a) K. A. Koehler and G. E. Lienhard, Biochemistry, 1971, 10, 2477;
(b) R. V. Wolfenden, Acc. Chem. Res., 1972, 5, 10; (c) G. E. Lienhard,
Science, 1973, 180, 149; (d) R. V. Wolfenden and A. Radzicka, Curr.
Opin. Struct. Biol., 1991, 1, 780.
Fluorine NMR spectra
NMR samples were prepared by dissolving lyophilized enzyme
in 900 ll of 0.1 M KCl with about 10% D O added to provide the
1
1
1
3 E. Tsilikounas, C. A. Kettner and W. W. Bachovchin, Biochemistry,
1992, 31, 12839.
4 W. W. Bachovchin, W. Y. L. Wong, S. Farr-Jones, A. B. Shenvi and
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5 (a) T. Ohba, E. Ikeda and H. Takei, Bioorg. Med. Chem., 1996, 6,
2
field frequency lock signal. Enzyme concentration was typically
about 0.08 mM. Inhibitor boronic salt was dissolved in DMSO
1
875; (b) A. Kashima, Y. Inove, S. Sugio, I. Maeda, T. Nose and Y.
(
100 ll) and added directly to the NMR sample. The amount of
Shimoshigashi, Eur. J. Biochem., 1998, 255, 12; (c) M. F. Parisi and
R. H. Abeles, Biochemistry, 1992, 31, 9429.
the inhibitor added was 0.602 M, enough to saturate the enzyme.
1
1
6 D. H. Kinder and J. A. Katzenellenbogen, J. Med. Chem., 1985, 28,
1
917.
Biochemical assays
7 E. Vedejs, R. W. Chapman, S. C. Fields, S. Lin and M. R. Shrimpf,
Determination of inhibition constants (K
i
) to chymotrypsin.
J. Org. Chem., 1995, 60, 3020 and references therein.
18 S. Darses, G. Michaud and J.-P. Gen eˆ t, Eur. J. Org. Chem., 1999,
a-Chymotrypsin Type II from bovine pancreas was purchased
from Sigma-Aldrich (MW 25 000). The inhibitors were tested
by competitive assay against benzoyl-tyrosine-p-nitroanilide as
1
875.
1
9 H.-J. Frohn, H. Franke, P. Fritzen and V. V. Bardin, J. Organomet.
Chem., 2000, 598, 127.
◦
a substrate. The hydrolytic reaction was carried out at 37 C at
2
2
0 S. Darses and J.-P. Gen eˆ t, Eur. J. Org. Chem., 2003, 4313.
1 (a) J.-P. Tremblay-Morin, S. Raeppel and F. Gaudette, Tetrahedron
Lett., 2004, 45, 3471; (b) T. E. Barder and S. L. Buchwald, Org. Lett.,
2004, 16, 1523.
pH = 7.00, with 50 mM TRIS buffer and 0.02 M CaCl
2
. Inhibitor
stock solutions in DMSO were prepared with concentrations
ranging from 6.02 mM to 0.0602 mM. A stock enzyme solution
−
1
22 M. Dixon, Biochem. J., 1953, 55, 170.
of 0.2 mg ml was made in 1 mM HCl solution. A series
of substrate concentrations (dissolved in 36.6% DMSO and
2
2
2
3 D. G. Gorenstein and D. O. Shah, Biochemistry, 1982, 21, 4679.
4 D. O. Shah and D. G. Gorenstein, Biochemistry, 1983, 22, 6096.
5 B. D. Sykes and J. H. Weiner, in Magnetic Resonance In Biology, ed.
J. S. Cohen, Wiley, New York, 1980, vol. 1, p. 171.
6
3.4% methanol) ranging from 0.043 mM to 0.43 mM were
prepared. The assay was performed by adding 700 ll TRIS
buffer to a 1.5 ml Eppendorf, followed by 100 ll inhibitor and
26 J. T. Gerig, inBiochemical Magnetic resonance, ch. 5, eds. L. J. Berliner
1
00 ll enzyme. The mixture was incubated for 10 minutes at
and J. Reuben, Plenum Press, New York, 1978.
7 A. Saika and C. P. Slichter, J. Chem. Phys., 1954, 22, 26.
28 M. Karplus and T. P. Das, J. Chem. Phys., 1961, 34, 1683.
◦
C. Then, 100 ll of substrate was added to the reaction
2
37
◦
followed by incubation for 20 minutes at 37 C. The reaction
was stopped by adding 200 ll of 30% acetic acid solution.
The progress of the reaction was followed by monitoring the
appearance of the absorption band of p-nitroaniline at 410 nm.
2
9 M. Tsavalos, B. C. Nicholson and T. M. Spotswood, Aust. J. Chem.,
978, 31, 2179.
0 H. J. Knight, E. H. Williams and T. M. Spotswood, Aust. J. Chem.,
978, 31, 2187.
31 D. G. Gorenstein and D. O. Shah, Biochemistry, 1982, 21, 4679.
1
3
1
K
i
Values for reversible competitive inhibitors were estimated
9
4 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 4 1 – 9 4 4