Table 4 Proton NMR chemical shifts in D2O at pH 7 in the absence
J. N. Burstyn, Coord. Chem. Rev., 1998, 173, 133; (b) R. Krämer,
Coord. Chem. Rev., 1999, 182, 243; (c) G. M. Polzin and
J. N. Burstyn, in Metal Ions in Biological Systems, eds. A. Sigel
and H. Sigel, Marcel Dekker, New York, 2001, vol. 38, p. 103;
(d ) M. Komiyama, Metal Ions in Biological Systems, eds. A. Sigel
and H. Sigel, Marcel Dekker, New York, 2001, vol. 38, p. 25; (e)
S. A. Datwyler and C. F. Meares,Metal Ions in Biological Systems,
eds. A. Sigel and H. Sigel, Marcel Dekker, New York, 2001, vol. 38,
p. 213; ( f ) N. M. Milovic and N. M. Kostic, Metal Ions in Biological
Systems, eds. A. Sigel and H. Sigel, Marcel Dekker, New York, 2001,
vol. 38, p. 145.
3 (a) A. Radzicka and R. Wolfenden, J. Am. Chem. Soc., 1996, 118,
6105; (b) R. M. Smith and D. E. Hansen, J. Am. Chem. Soc., 1998,
120, 8910; (c) D. Kahne and W. C. Still, J. Am. Chem. Soc., 1988,
110, 7529.
4 (a) L. Meriwether and F. H. Westheimer, J. Am. Chem. Soc., 1956,
78, 5119; (b) H. L. Conley and R. B. Martin, J. Phys. Chem., 1965,
69, 2914.
and in the presence of 0.085 M ZnSO4
Substance
Chemical shift
alone
3.652
3.633
3.686
3.670
3.918
3.644
3.320
3.506
3.906
3.609
with ZnSO4
H2NCH2CONH2
3.551
3.654
3.713
3.660
3.931
3.639
3.276
3.508
3.906
3.609
H2NCH2CONHCH2COOH
H2NCH2CONHCH2COOH
H2NCH2CONHCH2CONHCH2COOH
H2NCH2CONHCH2CONHCH2COOH
H2NCH2CONHCH2CONHCH2COOH
H2NCH2COOH
H2NCH2CONHOH
H2NCH2CONHCH2CONHOH
H2NCH2CONHCH2CONHOH
5 (a) P. A. Sutton and D. A. Buckingham, Acc. Chem. Res., 1987, 20,
357; (b) K. W. Bentley and E. H. Creaser, Biochem. J., 1973, 135,
507.
Diketopiperazine
4.080
3.909
6 E. L. Hegg and J. N. Burstyn, J. Am. Chem. Soc., 1995, 117, 7015.
7 (a) L. Zhu and N. M. Kostic, Inorg. Chem., 1992, 31, 3994;
(b) L. Zhu and N. M. Kostic, J. Am. Chem. Soc., 1993, 115, 4566;
(c) T. N. Parac and M. N. Kostic, J. Am. Chem. Soc., 1996, 118, 51;
(d ) N. V. Kaminskaia and N. M. Kostic, Inorg. Chem., 2001, 40,
2368.
8 T. Takarada, M. Yashiro and M. Komiyama, Chem. Eur. J., 2000, 6,
3906.
9 (a) T. M. Rana and C. F. Meares, Proc. Natl. Acad. Sci. USA, 1991,
88, 10578; (b) T. M. Rana and C. F. Meares, J. Am. Chem. Soc., 1990,
112, 2457; (c) T. M. Rana and C. F. Meares, J. Am. Chem. Soc., 1991,
113, 1859.
acid used as a standard for identification of the reaction
products by NMR was prepared by reacting glycylglycine ethyl
ester with excess hydroxylamine in D2O. Reagent-grade
inorganic salts and hydoxylammonium hydrochloride from
Aldrich were used as supplied. All solutions were prepared
in purified (Milli-Q Reagent Water System) water or in D2O
(Aldrich) for NMR studies.
10 (a) F. Bergmann, Anal. Chem., 1952, 24, 1367; (b) V. Goldenberg and
P. E. Spoerri, Anal. Chem., 1958, 30, 1327.
Instrumentation
11 W. P. Jencks and M. Gilchrist, J. Am. Chem. Soc., 1964, 86, 5616.
12 (a) P. Bornstein and G. Balian, in Methods in Enzymology, eds.
C. H. W. Hirs and S. N. Timasheff, Academic Press, New York,
1977, Vol. 47, Part E, p. 132; (b) H. Park, S. Pyo, S. Hong and J. Kim,
Biotechnol. Lett., 2001, 23, 637.
13 R. Breslow, D. F. McClure, P. S. Brown and J. Eisenach, J. Am.
Chem. Soc., 1975, 97, 194.
Ultraviolet–Visible spectra were obtained with a Hewlett
Packard 8452A spectrophotometer. 1H NMR spectra were
recorded on 300 MHz Varian Unity INOVA spectrometer.
Methodology
Kinetics of the hydroxylaminolysis of glycinamide and pep-
tides were followed by colorimetric reaction of aliquots taken
periodically from the reaction mixture with Fe(NO3)3 in 0.3 M
nitric acid.10,11 The molar absorptivity at 540 nm produced by
glycine hydroxamic acid (650 MϪ1cmϪ1 respectively) was deter-
mined by using the standard solutions of glycine hydroxamic
acid of known concentrations. The hydroxylaminolysis of
acetamide was followed spectrophotometrically by appearance
of acetohydroxamic acid at 215 nm (molar absorptivity 744
MϪ1 cmϪ1).Typically 0.02–0.05 M solutions of amides or
peptides, 0.01–0.1 M metal salt and 0.5–1.5 M hydroxylamine
were employed. The reaction mixtures were adjusted to desired
pH by adding concentrated NaOH or HCl solutions and placed
into the thermostat.
14 M. A. Wells and T. C. Bruice, J. Am. Chem. Soc., 1977, 99, 5356.
15 B. K. Takasaki, J. H. Kim, E. Rubin and J. Chin, J. Am. Chem. Soc.,
1993, 115, 1157.
16 With the given above rate parameters at 37 ЊC the ratio rϩ/rϪ equals
1.1 and 6.4 at pH 5 and 4 respectively, and 1.2 and 8.4 at pH 7 and 8
respectively. Thus, in the range of pH 5–7 the ratio rϩ/rϪ is between
0.5 and 1, but outside this range rϩ/rϪ > 1 both in acidic and basic
solutions.
17 Since pH values were always measured at room temperature the rate
vs. pH profiles were analyzed by using pKa=6.0 for hydroxyl-
ammonium at all temperatures. When pKa was left as an adjustable
parameter in the equation (2), the fitting of the pH-profiles at
different temperatures gave the values 5.94, 6.10 and 6.0 at 37, 45
and 60 ЊC respectively. Thus using uncorrected pKa at different
temperatures seems justified and on this basis we used also
uncorrected pKa values for glycine derivatives employed as
substrates.
18 Obviously in the range of pH 5–7 the assumption of the rate-
determining addition step for the hydroxylaminolysis of acetamide
is rather rough and the calculated value of kAH (see Scheme 1b)
should be smaller than the real value of the addition rate constant
(k1 in Scheme 1a) by a factor of 2 or 3. The correct values of k1 at
different temperatures were calculated by using the equation (1)
from the results around pH 6 only and they are given as footnotes to
Table 1. For glycine derivatives as the substrates the assumption is
valid at all pH values.
Products of the hydroxylaminolysis of glycine derivatives
were identified by proton NMR in D2O. Chemical shifts of
starting materials and reaction products in D2O at pH 7 are
given in Table 4.
Acknowledgements
The work was supported by DGAPA-UNAM (Project IN
208901).
19 R. M. Smith and A. E. Martell, Critical Stability Constants, Plenum
Press, New York, 1975, Vol. 2, 1982, Vol.5.
20 D. L. Rabenstein and S. Libich, Inorg. Chem., 1972, 11, 2960.
21 K. K. Ghosh and S. Ghosh, J. Org. Chem., 1994, 59, 1369.
22 X. Hu and G. L. Boyer, Anal. Chem., 1996, 68, 1812.
23 M. T. B. Luiz, B. Szpoganicz, M. Rizzoto, M. G. Basallota and
A. E. Martel, Inorg. Chim. Acta, 1999, 287, 134.
References
1 K. B. Grant and S. Pattabhi, Anal.Biochem., 2001, 289, 196 and
references therein.
2 Recent reviews on artificial proteases/peptidases: (a) E. L. Hegg and
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 8 6 6 – 8 7 2
872