pH-Induced Change in the Rate-Determining Step
for the Hydrolysis of the Asp/Asn-Derived
Cyclic-Imide Intermediate in Protein Degradation
Minli Xie, David Vander Velde, Martha Morton,
Ronald T. Borchardt, and Richard L. Schowen*
Department of Pharmaceutical Chemistry and
NMR Laboratory, The UniVersity of Kansas
Lawrence, Kansas 66046
ReceiVed February 26, 1996
The ring-opening reaction of the cyclic imide shown in
Scheme 1, the key intermediate in the degradation of proteins
and peptides at Asp and Asn “hot spots,”
1
proceeds by rate-
limiting C-N bond fission at pH 1-4 and by rate-limiting attack
of water at higher pHs, as shown by
18
O-exchange into both
carbonyl groups of the cyclic imide below pH 4 but not at higher
pHs (Figure 1). The transition states for formation and
decomposition of the intermediate T
° are identical to those for
cyclic-imide formation at Asp residues in proteins. Thus protein
instability at pH > 5, deriving from cyclization at Asp residues
to form the cyclic imide, results from a relatively low free energy
of the transition state for the expulsion of water from the
tetrahedral intermediate T
° (Scheme 1), following rapid,
reversible C-N bond formation. The greater stability of
proteins at pH < 5 results from a relatively high free energy of
the transition state for C-N bond formation to generate T
°.
Similar considerations may apply to cyclization at Asn residues
in proteins.
Figure 1 shows the pH-rate profile for the
18
O-exchange and
hydrolysis reactions of the two cyclic imides shown in Scheme
1. In the region of pH 6-8, the filled triangles denote the rates
of loss of the cyclic imide at 50 °C determined by capillary
electrophoresis (CE). For pH 5.9-8.0, no
18
O-exchange into
the carbonyl groups of the cyclic imide was observed (k < 1.6
× 10
-8
s
-1
), which indicates that the attack of water on both
the R- and -carbonyl groups is rate determining. However, in
the region of pH 1-4, exchange was observed: the filled
symbols denote the rate constants for
18
O-exchange into the
R-carbonyl group (squares) and the -carbonyl group (circles)
of the cyclic imide, while only minimal hydrolysis was observed
at pH 3.9 (k ca. 8 × 10
-8
s
-1
), and none at pH 1.9 (k < 3 ×
10
-10
s
-1
). The results indicate that in the pH region of 1.9-
4, the rate-determining step of cyclic-imide breakdown is C-N
bond fission (leaving-group expulsion), while in the pH range
5.5-8, the rate-determining step is C-O bond formation (water
attack). A pH-induced change in the rate-determining step
occurs at pH 4-5.5.
During studies of hydrolysis of the cyclic imide, CE provided
a rapid, precise alternative to HPLC.
3
The
13
C-NMR method
of Van Etten and co-workers
4
was employed for the
18
O-
exchange studies. With a Bruker AM-500 NMR spectrometer
operating at 125.77 MHz for
13
C, the two carbonyl groups are
well resolved with the R-carbonyl signal at 180.7 ppm and the
-carbonyl signal at 179.9 ppm in H
2
18
O (60-80%) solution
with 20% D
2
O. The signal for an
18
O-labeled carbonyl has an
upfield shift of 0.04 ppm from the original
16
O-carbonyl signal,
permitting the concentration of
18
O-labeled cyclic imides to be
followed as a function of time (Figure 2). The relative amount
of
16
O- and
18
O-carbonyl groups can be determined from the
peak heights assuming that there is no isotope effect on the
NOEs
4
of the carbonyl carbon signals.
These findings are wholly consistent with the mechanism
proposed by Capasso et al.
2
on the basis of extensive kinetic
studies, a part of which are shown in Figure 1. The
18
O-
exchange results now reported in this work show unambiguously
that leaving-group expulsion is rate limiting below pH 4 and
water attack rate limiting above pH 5.5. The kinetic results
alone could not specify which step is rate limiting in the various
pH ranges. The solid lines in Figure 1 are plots of eq 1, the
kinetic law for the proposed model
2
extended to include an
uncatalyzed route of C-N bond fission at very low pH.
Hydrolysis is represented in Scheme 1 as proceeding by attack
of water at each of the carbonyl groups to form tetrahedral
intermediates (shown in the neutral forms T
R
° and T
°) with
total rate constants k
1R
and k
1
, followed by breakdown of the
(1) (a) Ahren, T. J., Manning, M. C., Eds. Stability of Protein Pharma-
ceuticals: Part A. Chemical and Physical Pathways of Protein Degradation;
Plenum Press: New York, 1992. (b) Aswad, D. W., Ed. Deamidation and
Isoaspartate Formation in Peptides and Proteins; CRC Press, Boca Raton,
FL, 1995.
(2) Capasso, S.; Kirby, A. J.; Salvadori, S.; Sica, F.; Zagari, A. J. Chem.
Soc., Perkin Trans. 2 1995, 437-442.
(3) (a) Nielsen, R. G.; Riggin, R. M.; Rickard, E. C. J. Chromatogr.
1989, 480, 393-401. (b) Wu, S.; Teshima, G.; Cacia, J.; Hancock, W. S.
J. Chromatogr. 1990, 516, 115-122.
(4) Cortes, S. J.; Mega, T. L.; Van Etten, R. L. J. Org. Chem. 1991, 56,
943-947 and references cited therein.
Figure 1. Rate constants of hydrolysis and
18
O-exchange for the cyclic
imides of Scheme 1 as a function of pH. Open symbols are from the
work of Capasso et al.
2
at 37 °C. Filled symbols are from this work at
37 °C (hydrolysis) and 50 °C (exchange). The solid lines are plots of
eq 1 with 10
7
k1W ) 4.85 (R), 1.66 ()s
-1
; k1B ) 140 (R), 100 ()M
-1
s
-1
; 10
8
k3W ) 5.86 (R), 1.30 ()s
-1
; k3B ) 5537 (R), 1544 ()M
-1
s
-1
, which are in acceptable agreement with values calculated by
Capasso et al.
2
on the basis of a rate law omitting k3W. The rate
constants shown for isotope exchange are, in terms of those in Scheme
1, given by k
1x k2x/2(k2x + k′3x), where x ) either R or .
Scheme 1
8955 J. Am. Chem. Soc. 1996, 118, 8955-8956
S0002-7863(96)00618-X CCC: $12.00 © 1996 American Chemical Society