CleaVage of Histidyl Peptides
J. Am. Chem. Soc., Vol. 118, No. 25, 1996 5947
Chart 1
value.31 It was always prepared fresh, and the reaction mixtures
were kept acidic in order to suppress or minimize the dimer-
ization shown in eq 1, which occurs at pH >4.0.
3
2
Stability of the Substrates. In the absence of cis-[Pd(en)-
2+
(
H2O)2] the peptide AcHis-Gly did not detectably hydrolyze
in the time that, in the presence of this catalyst, was sufficient
for complete hydrolysis. Monitoring over a long period of time
-
6
-1
yielded an estimate of 5 × 10 min for the rate constant of
the uncatalyzed reaction at pH 1.0. Clearly, the fast hydrolytic
cleavage of the peptide bond is caused by the palladium(II)
complex, not by the acidic solvent. Further evidence for this
conclusion will be given below.
aspects of acid-base equilibria and stereochemical aspects of
linkage isomerism. The results show what roles the weakly
acidic medium does and does not play in cleavage reactions
catalyzed by palladium(II) complexes, which of the several
interconverting complexes is the active one, and why these
reactions occur with turnover.
Binding of the Catalyst to the Substrates. As we recently
2
6
reported,
(
a reaction between equimolar amounts of cis-[Pd-
2+
en)(H2O)2] and AcHis-Gly spontaneously yields five com-
plexes, designated A through E in Chart 2. In this study we
clarify linkage isomerism in these complexes by methylating
N-1 and N-3 atoms of the imidazole ring. The atom numbering
and the methylated dipeptides are shown in Chart 1. The new
complexes in Chart 2 are designated with letters and subscripts
Experimental Procedures
Chemicals. Distilled water was demineralized and purified to a
resistance greater than 10 MΩ‚cm. The compounds D
NaOD, and K [PdCl ] were obtained from Aldrich Chemical Co.
Anhydrous AgClO was obtained from G. Frederic Smith Chemical
Co. Amino acids 1-methylhistidine (1-Me-His) and 3-methylhistidine
2 4
O, DClO ,
2
4
1
Me. The H NMR chemical shifts of the methylated substrates
4
and complexes, given in Table 1, differ only slightly from the
shifts of the corresponding unmethylated species.
(
3-Me-His) and the dipeptide His-Gly were obtained from Sigma
Chemical Co. All other chemicals were of reagent grade.
2+
Mixing of equimolar amounts of cis-[Pd(en)(H2O)2] and
Dipeptides 1-Me-AcHis-Gly and 3-Me-AcHis-Gly were synthe-
sized by a standard solid-state method, and their purity was checked
by HPLC; this was done by the staff of the Protein Facility. The
terminal amino group in amino acids and dipeptides was acetylated by
1
-Me-AcHis-Gly in solutions having 0.80 e pH e 5.0 results
in spontaneous, fast formation of the complexes AMe and DMe.
The unidentate coordination is favored at lower pH values, and
bidentate at higher, at which the amide nitrogen atom is
deprotonated. Mixing of equimolar amounts of cis-[Pd(en)-
29
2+
a standard procedure. The complex cis-[Pd(en)(H
2 2
O) ]
was prepared
by treating the corresponding dichloro complex with 2 equiv of AgClO
and removing AgCl by centrifugation, all in the dark.
Measurements. Proton NMR spectra of D O solutions containing
4
2
+
3
0
(H O) ] and 3-Me-AcHis-Gly in solutions having pH 1.2 or
2
2
3.0 yields complexes BMe and EMe as the major and the minor
2
DSS as internal reference were recorded at 300 and 500 MHz, with
Nicolet NT 300, Varian VXR 300, and Varian Unity 500 spectrometers.
Temperature was kept at 60 ( 0.5 °C. The pH values were measured
with a Fisher 925 instrument and a Phoenix Ag/AgCl reference electrode
and converted into hydrogen-ion concentrations. Neither the pH nor
product, respectively. These modes of coordination are
known.
3
3-37
Hydrolysis of the His-Gly Bond. When this reaction
1
occurs, free glycine is easily detected in H NMR spectra; see
Figure 1. Upon addition of glycine to the reaction mixture its
resonance is enhanced. Some of the liberated glycine reacts
with the catalyst to form a small amount of the bis(bidentate)
+
the [H ] values were corrected for the deuterium effect. Ultraviolet-
visible spectra were recorded with a IBM 9420 spectrophotometer.
Study of Hydrolysis. The following D
2
O solutions were mixed in
+
1
complex cis-[Pd(en)(Gly-N,O)] , easily detected by H NMR
spectroscopy; see Table 1. Indeed, this same complex is formed
upon mixing of equimolar amounts of cis-[Pd(en)(H2O)2]2 and
glycine. At the end of hydrolysis reaction palladium is
2
an NMR tube: 150 µL of 100 mM freshly-prepared cis-[Pd(en)-
2
+
(
H
2
O)
After addition of 150 µL of D
solution of DClO
2
]
, 150 µL of 100 mM peptide, and 50 µL of 100 mM DSS.
O the pH was adjusted with a 2.0 M
. The complete solution was 26-30 mM in the
+
2
4
peptide, and pH was in the range of 0.80-5.0. The pH value never
changed by more than 0.10 between the beginning and the end of the
experiment. Acquisition of H NMR spectra, at 60 ( 0.5 °C, began
as soon as possible, and 16 scans were taken each time. The error in
completely removed as the insoluble complex [Pd(ddtc) ], and
1
the H NMR spectra show only the two fragments of the cleaved
1
dipeptide. When hydrolysis occurs, it is a complete and “clean”
reaction.
integrating the resonances was estimated at ( 5%. A typical first-
Coordination Modes and Reactivity. The mixture of
complexes BMe and EMe did not show peptide hydrolysis except
for the slight “background” cleavage upon prolonged heating.
Clearly, neither of these complexes is reactive in hydrolysis.
Palladium(II) atom bound to the N-1 atom is too distant from
the His-Gly bond to cleave it by either of two mechanisms:
order plot of ln[C
0
/(C
0
t
- C )] versus time, based on concentrations of
glycine, consisted of 20 points spanning at least three half-lives. At
the end of hydrolysis 2 equiv of solid sodium diethyldithiocarbamate
2
+
(
Naddtc) were added for each equivalent of cis-[Pd(en)(H
2 2
O) ] , and
2
6
the precipitate of [Pd(ddtc) ] was removed by centrifugation.
2
Stability of Peptides without Palladium. A solution of AcHis-
Gly at pH 1.0 was prepared as described above except that the complex
2
+
(31) Hohmann, H.; Van Eldik, R. Inorg. Chim. Acta 1990, 174, 87.
cis-[Pd(en)(H
°
2
O)
2
]
was missing. The solution was kept at 60 ( 0.5
(
32) Siebert, A. F. M. Ph.D. Dissertation, University of Bochum,
Germany, 1995; p 166.
33) Appleton, T. G.; Pesch, F. J.; Wienken, M.; Menzer, S.; Lippert,
B. Inorg. Chem. 1992, 31, 4410.
34) Sundberg, R. J.; Martin, R. B. Chem. ReV. 1974, 74, 471.
(35) Rabenstein, D. L.; Isab, A. A.; Shoukry, M. M. Inorg. Chem. 1982,
21, 3234.
36) Remelli, M.; Munerato, C.; Pulidori, F. J. Chem. Soc., Dalton Trans.
1
C, and its H NMR spectra were recorded occasionally.
(
Results and Discussion
(
The Catalyst. The palladium(II) complex shown in Chart 1
had the UV absorption maximum at 340-345 nm, the correct
(
(
(
29) Wheeler, G. P.; Ingersol, A. W. J. Am. Chem. Soc. 1951, 73, 4604.
30) Mehal, G.; Van Eldik, R. Inorg. Chem. 1985, 24, 4165.
1994, 2049.
(37) Letter, J. E., Jr.; Jordan, R. B. Inorg. Chem. 1974, 13, 1152.