Kinetics of Amide and Peptide Cleavage by Alkaline Hydrogen Peroxide

Article abstract of DOI:10.1021/ol035811x

(Equation Presented) The hydroperoxide anion cleaves unactivated amides and peptides although it is completely unreactive toward ethyl esters. The cleavage by HO₂^- proceeds faster than by OH^- and involves additional routes with general acid assistance by H ₂O₂ and general base assistance by OH^- and HO₂^-. Cleavage of polypeptides occurs at the N-terminal peptide bond.

Full text of DOI:10.1021/ol035811x

ORGANIC
LETTERS
2003
Vol. 5, No. 25
4831-4834
Kinetics of Amide and Peptide Cleavage
by Alkaline Hydrogen Peroxide
Baldomero Go´mez-Reyes and Anatoly K. Yatsimirsky*
Facultad de Qu´ımica, UniVersidad Nacional Auto´noma de Me´xico,
04510 D.F., Mexico
anatoli@serVidor.unam.mx
Received September 19, 2003
ABSTRACT
The hydroperoxide anion cleaves unactivated amides and peptides although it is completely unreactive toward ethyl esters. The cleavage by
HO - proceeds faster than by OH- and involves additional routes with general acid assistance by H O and general base assistance by OH-
2
2 2
and HO -. Cleavage of polypeptides occurs at the N-terminal peptide bond.
2
-
There is much current interest in the development of new
reagents and/or catalysts for the hydrolytic cleavage of
peptides and proteins.1-3 The most active system reported
so far is the iron chelate mediated cleavage achieved with
peptide-attached iron(II) complexes in combination with
H2O2 and ascorbic acid.4,5 The proposed reaction mechanism
involves the nucleophilic attack of the peptide bond by the
metal-bound HO2- anion producing the tetrahedral addition
intermediate, which undergoes further decomposition to the
final hydrolytic products. However, a serious problem with
this mechanism is that the HO2- anion is not expected to be
able to afford the nucleophilic substitution of unactivated
amide bonds. Indeed, the well-known R-nucleophilic char-
acter of HO2 , i.e., its strongly enhanced reactivity over that
expected from its basicity, is manifested only in reactions
with substrates possessing highly activated leaving groups,
such as aryl esters,6 phosphonates,7 or phosphates.8 Already
-
ethyl esters do not react with HO2 at all.9 The reason for
this lack of reactivity is that HO2 is not sufficiently basic
to throw out more basic ethoxide anion from the tetrahedral
intermediate. The situation should be apparently even worse
for amides and peptides possessing even more basic leaving
groups. This paper demonstrates, however, that the R-effect
is observed for HO2 in reactions with unactivated amides
and peptides and also gives preliminary results on the
selectivity of the peptide cleavage by HO2 .
-
-
-
The compounds studied as substrates (acetamide, glycin-
amide, N-acetylglycine, glycylglycine, and other peptides)
were purchased from Sigma and used without further
purification. Reagent-grade inorganic salts and hydrogen
peroxide from Aldrich were used as supplied. UV spectra
were obtained with a Hewlett-Packard 8452A spectropho-
(1) Grant, K.B.; Pattabhi, S. Anal.Biochem. 2001, 289, 196,.
(2) (a) Milovicˇ, N. M.; Kosticˇ, N. M. In Metal Ions in Biological Systems,
Vol. 38: Probing of Proteins by Metal Ions and Their Low-Molecular Weight
Complexes; Sigel, A., Sigel, H., Eds.; Dekker: New York, 2001; p 145.
(b) Komiyama, M. In Metal Ions in Biological Systems, Vol. 38: Probing
of Proteins by Metal Ions and Their Low-Molecular Weight Complexes;
Sigel, A., Sigel, H., Eds.; Dekker: New York, 2001; p 25. (c) Polzin, G.
M.; Burstyn, J. N. In Metal Ions in Biological Systems, Vol. 38: Probing of
Proteins by Metal Ions and Their Low-Molecular Weight Complexes; Sigel,
A., Sigel, H., Eds.; Dekker: New York, 2001; p 103.
1
tometer. H NMR spectra were recorded on a 300 MHz
Varian Unity INOVA spectrometer.
(3) (a) Hegg, E. L.; Burstyn, J. N. Coord.Chem.ReV. 1998, 173, 133. (b)
Kra¨mer, R. Coord. Chem. ReV. 1999, 182, 243
(6) Jencks, W. P.; Gilchrist, M. J. Am. Chem. Soc. 1968, 90, 2622.
(7) Behrman, E. J.; Bialla, M. J.; Braas, H. J.; Edwards, J. O.; Isaks, M.
J. Org. Chem. 1970, 35, 3069.
(8) Mej´ıa-Radillo, Y.; Yatsimirsky, A. K.; Foroudian, H. J.; Jillit, N.;
Bunton C. A. J. Phys. Org. Chem. 2000, 13, 505.
(9) Wiberg, K. B. J. Am. Chem. Soc. 1955, 77, 2519. See also: Klopman,
G.; Tsuda, K.; Louis, J. B.; Davis, R. E. Tetrahedron 1970, 26, 4549.
(4) (a) Rana, T. M.; Meares, C. F. Proc. Natl. Acad. Sci. U.S.A. 1991,
88, 10578. (b) Rana, T. M.; Meares, C. F. J. Am. Chem. Soc. 1990, 112,
2457. (c) Rana, T. M.; Meares, C. F. J. Am. Chem. Soc. 1991, 113, 1859.
(5) Datwyler, S. A.; Meares, C. F. In Metal Ions in Biological Systems,
Vol. 38: Probing of Proteins by Metal Ions and Their Low-Molecular Weight
Complexes; Sigel, A., Sigel, H., Eds.; Dekker: New York, 2001; p 213.
10.1021/ol035811x CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/13/2003

1
Kinetics of the cleavage reactions were followed by H
NMR at 37 °C in D2O containing 0.3-3% of H2O introduced
with H2O2 solution. Typically, 0.02-0.05 M solutions of
amides or peptides, 0.1-0.5 M NaOD, and 0.1-1.0 M H2O2
were employed.
Figure 1 illustrates a typical kinetic experiment with
GlyGly as a substrate. The signal at 3.0 ppm belongs to the
Figure 2. Observed first-order rate constants for the cleavage of
0.04 M AcNH2 vs total H2O2 concentration in the presence of 0.1
M NaOD (lower axis) and vs total NaOD concentration in the
presence of 0.2 M H2O2 (upper axis) at 37 °C. Solid lines are
theoretical profiles calculated in accordance with eq 2 and rate
constants given in Table 1.
Figure 1. 1H NMR spectra of 0.04 M GlyGly in D2O in the
presence of 0.5 M NaOD and 0.1 M H2O2 recorded after 13, 26,
38, 50, and 60 min and ca. 10 h at 37 °C.
spectrophotometric titration of H2O2 with NaOD in D2O as
described in ref 8. The value of K ) 276 ( 2 M-1 in D2O
agrees well with those reported in the literature for aqueous
solutions8,10 and indicates a small positive solvent isotope
effect of ca. 1.5.
glycinate anion, and its intensity grows with increase in the
incubation time. In this and other experiments, only signals
of the substrate and of the hydrolysis product(s) were
observed. Apparently, the peroxy acids expected as primary
reaction products are either too unstable under the reaction
conditions or give the NMR signals indistinguishable with
those of respective acids. The cleavage reactions were
followed up to ca. 10-20% substrate conversion, and the
initial rates were calculated from the plots of the product
concentration obtained by the integration of signals vs the
H2O2 + OH- / HO2
(1)
-
With this equilibrium constant, concentrations of free OH-,
-
HO2 , and H2O2 at each given mixture of known total NaOH
and H2O2 concentrations were calculated, and the results in
Figure 2 were analyzed in terms of the following kinetic
equation
-
-
kobs ) kOH[OH-] + kHO2[HO2 ] + kGA[HO2 ][H2O2] +
incubation time. The observed first-order rate constants (kobs
were then calculated by dividing the initial rates by the initial
substrate concentration.
)
kGBOH[HO2 ][OH-] (2)
-
Figure 2 shows the effect of added H2O2 on the rate of
acetamide cleavage in the presence of 0.1 M NaOD (solid
triangles) and the effect of added NaOD at a constant 0.2 M
H2O2 (open triangles). Mixing of H2O2 with NaOH leads to
a nearly quantitative deprotonation of H2O2. Therefore, an
where kOH and kHO2 are the second-order rate constants for
the cleavage of the substrate by OH- and HO2- respectively,
OH
kGA and kGB are the third-order rate constants for the
cleavage by HO2- assisted by general-acid and general-base
mechanisms, respectively. The kOH was measured in the
absence of H2O2 and appeared to be very close to the value
reported in the literature in H2O.11 Then the values of kobs
were corrected for the contribution of the alkaline hydrolysis
and the eq 2 was rearranged as follows:
-
increase in kobs on addition of H2O2 means that HO2 is a
stronger nucleophile than OH-. Additions of higher con-
centrations of H2O2 when it already is in excess over NaOH
does not produce more HO2- anions, but the reaction rate is
obviously going higher, thus indicating that the reaction with
-
HO2 is catalyzed by neutral H2O2. On the other hand,
-
(kobs-kOH[OH-])/[HO2 ] ) k2obs ) kHO2 + kGA[H2O2] +
additions of increasing amounts of NaOD in the presence
of a fixed concentration of H2O2 in excess of the latter also
lead to a significant acceleration, indicating that the reaction
kGBOH[OH-] (3)
-
with HO2 is catalyzed also by OH-.
To analyze the results quantitatively we determined the
Parameters of the eq 3 were then calculated by multiple
equilibrium constant K for the reaction 1 at 37 °C by
regression of k2obs as a linear function of two independent
4832
Org. Lett., Vol. 5, No. 25, 2003

Table 1. Rate Constants for Different Paths of the Amide and Peptide Cleavage by Alkaline Hydrogen Peroxide at 37 °Ca
OH
HO2
substrate
kOH, M-1 s-1
kHO , M-1 s-1
kGA, M-2 s-1
kGB
,
c M-2 s-1
kGB , M-2 s-1
2
AcNH2
GlyGly
1.2 × 10-4
3.8 × 10-5
2.4 × 10-4
6.8 × 10-5
3.4 × 10-4
3.9 × 10-4
2.1 × 10-3
4.8 × 10-4
1.9 × 10-4
a Relative error (10%.
variables [H2O2] and [OH-]. The resulting rate constants are
given in Table 1.
The cleavage of a simplest peptide substrate GlyGly was
studied in more details. Figure 3 shows the results obtained
between two anions, but most probably reflects the general-
base catalysis by the second hydroxide anion reported
previously for the hydrolysis of anilides.12,13 The analysis
of the results in terms of the eq 3 required also an additional
-
term proportional to the concentration of free HO2 , so the
results were fitted to the eq 5.
k2obs)kHO2 + kGA[H2O2] + kGBOH[OH-] + kGBHO2[HO2 ]
-
(5)
The numerical values of all rate constants for the cleavage
of GlyGly are given in Table 1. Lower values of these
constants, as compared to those for the cleavage of aceta-
mide, can be attributed to steric effects and the negative
charge of the peptide. The latter explains the decreased
relative contribution of the general-base-catalyzed path,
which involves an additional anionic species in comparison
with the general-acid path involving neutral hydrogen
peroxide.
Several other glycine derivatives were studied as substrates
under similar conditions for comparative purposes. The
results are collected in Table 2. As expected, glycinamide
Figure 3. Observed first-order rate constants for the cleavage of
0.04 M GlyGly vs total H2O2 concentration in the presence of 0.1,
0.3, and 0.5 M NaOD at 37 °C. Solid lines are theoretical profiles
calculated in accordance with eqs 4 and 5 and rate constants given
in Table 1.
Table 2. Selected First-Order Rate Constants for the Amide
and Peptide Cleavage by Alkaline Hydrogen Peroxide at 37 °Ca
substrate
k
obs, s-1
[NaOD]T, M
[H2O2]T, M
AcNH2
GlyNH2
3.9 × 10-5
2.5 × 10-4
1.9 × 10-5
4.4 × 10-4
8.0 × 10-5
6.3 × 10-5
4.7 × 10-5
0.1
0.1
0.3
0.3
0.3
0.3
0.3
0.6
0.6
0.8
0.6
0.8
0.7
0.6
by variations in total hydrogen peroxide concentration in the
presence of three different fixed total sodium hydroxide
concentrations. General tendencies are similar to those for
acetamide, but the reaction kinetics is more complicated in
this case. Under conditions employed, GlyGly is a mono-
anion and is ca. 1 order of magnitude less reactive than
acetamide.
The rate of the alkaline hydrolysis of GlyGly was not a
linear function of [OD-]: the second-order rate constant kOH
increased on increase in [OD-] in accordance with the
empirical eq 4
N-AcGly
N-AcGlyNH2
GlyGly
GlyGlyGlyGlyb
GlyGlyAlac
a
a Cleavage to N-AcGly and NH3. b Ceavage to Gly and GlyGlyGly.
c Cleavage to Gly and GlyAla.
and N-acetylglycine are more and less reactive than aceta-
mide, respectively, due to a positive inductive effect of the
R-amino group for the former and the negative charge for
the latter. In agreement with this, the hydrolysis of N-
acetylglycinamide proceeds with formation of N-acetylgly-
cine and NH3. The cleavage of GlyGly is faster than that of
N-acetylglycine due to the presence of R-amino group.
To see the exo/endo selectivity of the peptide cleavage a
tetra- and a tripeptide substrates were employed. In the case
kOH ) kOH1 + kOH2[OD-]
(4)
1
2
where kOH ) 3.8 × 10-5 M-1s-1 and kOH )5.1 × 10-5
M-2s-1. This may be partially due to a positive salt effect
produced by increased concentration of NaOD on the reaction
(10) McIsaac, J. E., Jr.; Subbaraman, L. R.; Subbaraman, J.; Mulhausen,
H. A.; Behrman, E. J. J. Org. Chem. 1972, 37, 1037.
(11) Packer, J.; Thomson, A. L.; Vaughan, J. J. Chem. Soc. 1955, 2601.
(12) Beichler, S. S.; Taft, R. W. J. Am. Chem. Soc. 1957, 79, 4927.
(13) Bender, M. L.; Thomas, R. J. J. Am. Chem. Soc. 1961, 83, 4183.
Org. Lett., Vol. 5, No. 25, 2003
4833

of tetraglycine during first 2 h only formation of Gly and
GlyGlyGly was observed with the rate constant similar to
that for the GlyGly cleavage (Table 2). Only after accumula-
tion of ca. 25% of GlyGlyGly did the appearance of GlyGly
became detectable. These results clearly indicate the cleavage
of one of the terminal peptide bonds. To distinguish between
N- and C-terminal bonds, a tripeptide with different terminal
amino acids GlyGlyAla was used. Only the cleavage of Gly-
Gly bond was observed with the rate constant again similar
to that for GlyGly (Table 2). Thus, the preferable reaction
path is the cleavage of N-terminal peptide bond.
of the system by using the kHO value for GlyGly as the rate
2
constant for the intermolecular reaction (kinter) and to obtain
EM ) kintra/kinter)1/(6.8 × 10-5) ) 1.4 × 104 M, a value
which is within the range typical for nucleophilic substitution
reactions.15 Thus, the results presented here indirectly confirm
the validity of the hydrolytic mechanism proposed for the
iron chelate mediated protein cleavage.
The HO2- anion shows a modest R-effect in the cleavage
of amide and peptide substartes: the ratio kHO2/kOH equals
ca. 2 for AcNH2 and GlyGly (Table 1). Since the rate-
determining step in the amide hydrolysis is the decomposition
of the tetrahedral addition intermediate to final products, this
ratio should be closer to the “equilibrium” R-effect observed
for nucleophile addition to carbonyl compounds 16 rather than
to the “kinetic” R-effect observed for the cleavage of
substrates with good leaving groups. In the case of hydrogen
peroxide both types of effects are similar and are of the order
of 102-103.16 However, for substrates with poor highly basic
leaving groups, e.g., with ethyl acetate, the low basicity of
HO2- makes it unreactive because the tetrahedral intermedi-
ate decomposes principally to the starting materials. Appar-
ently the protonation of the leaving RNH- anion, postulated
as a necessary step in the amide hydrolysis,13,17 reduces its
basicity sufficiently and makes possible the partition of the
tetrahedral intermediate toward the reaction products rather
than to starting materials.
There is a certain similarity in the kinetics of peptide
cleavage by HO2- and by NH2OH. Studies of the hydroxy-
laminolysis of similar substrates showed the same reaction
paths as indicated in the eq 5, the only difference being that
for NH2OH the general-acid path was always strongly
predominant over other reaction paths.14 This is explicable
by much the higher acidity of NH3OH+ (pKa ) 6.0) as
compared to H2O2 (pKa ) 11.5), which makes the former a
more powerful catalyst. On the other hand, the absolute
-
reactivity of HO2 assisted by H2O2 is substantially higher
than that of NH2OH assisted by NH3OH+: the third-order
rate constant for the latter with GlyGly anion equals 2.9 ×
14
10-4 M-2 s-1 at 60 °C while for the former kGA ) 3.9 ×
10-4 at 37 °C (Table 1).
No rate constants were reported for the iron chelate
mediated protein cleavage, but since the reaction is typically
complete in 10 s 4 one may expect the kobs value to be about
1 s-1. In accordance with the proposed mechanism (see
Introduction) this rate constant refers to the intramolecular
Acknowledgment. We authors are grateful to the DGAPA-
UNAM (Project IN 208901) for financial support.
OL035811X
-
attack (kintra) of the peptide bond by metal-bound HO2 .
Therefore, one may estimate the “effective molarity” (EM)15
(15) Kirby, A. J. AdV. Phys. Org. Chem. 1980, 17, 183.
(16) Sander, E. G.; Jencks, W. P. J. Am. Chem. Soc. 1968, 90, 6154.
(17) Jencks W. P. Catalysis in Chemistry and Enzymology; McGraw-
Hill: New York, 1969.
(14) Go´mez-Reyes, B.; Yatsimirsky, A. K. Org. Biomol. Chem. 2003,
1, 866.
4834
Org. Lett., Vol. 5, No. 25, 2003