Regioselective hydrolysis of tryptophan-containing peptides promoted by palladium(II) complexes [10]

Full text of DOI:10.1021/ja9840246

J. Am. Chem. Soc. 1999, 121, 8663-8664
8663
Downfield shift of the indole N(1)-H proton resonance by 0.32
ppm rules out both the deprotonation of 1 and its tautomerization
to 2 upon coordination.13 Downfield shift of the C(3) resonance
by 41.6 ppm is characteristic of conversion of an aromatic atom
(in 1) to a tetrahedral atom (in 3).9,10,14 Downfield shifts of the
proton resonances of C(O)NH2 by 2.02 and 2.30 ppm and of R-CH
by 0.40 ppm are diagnostic of coordination of the C-terminal
amide oxygen (in 3).11,15,16 Without this auxiliary interaction indole
alone would not bind to palladium(II). The blue shift of the
palladium(II) d-d absorption bands upon tryptophan coordination
is consistent with the relative strengths of the ligand fields of the
carbanion at C(3), amide oxygen, and solvent (acetone and water,
as sol) ligands.17 Coordination of the N-terminal amide oxygen,
as in 4, was not observed, presumably because the seven-
membered ring in 4 is less favorable than the six-membered ring
in 3. The ethyl ester AcTrp-OEt does not detectably react with
cis-[Pd(en)(sol)2]2+ because the ester carbonyl oxygen is less
nucleophilic than the amide oxygen. NMR spectra show that
tryptophan-containing peptides AcTrp-NHX, for which NH2X
would be alanine, valine, and the valine methyl ester, react with
cis-[Pd(en)(sol)2]2+ to form complexes 3.11,18 The equilibrium
Regioselective Hydrolysis of Tryptophan-Containing
Peptides Promoted by Palladium(II) Complexes
Natalia V. Kaminskaia,† T. Wade Johnson,# and
Nenad M. Kostic´*
Department of Chemistry, Iowa State UniVersity
Ames, Iowa 50011-3111
ReceiVed NoVember 23, 1998
ReVised Manuscript ReceiVed July 5, 1999
Selective cleavage of peptides and proteins is an important
procedure in biochemistry and molecular biology. The half-life
for the uncatalyzed hydrolysis of amide bonds is 350-500 years
at room temperature and pH 4-8.1 Clearly, efficient methods of
cleavage are needed. Despite their great catalytic power and
selectivity to sequence, proteinases have some disadvantages.2
These enzymes cleave at numerous sites and yield relatively short
peptides that are ill-suited for automatic sequencing.2 Moreover,
proteinases tolerate only a narrow range of reaction conditions.
The only widely used chemical reagent for amide hydrolysis,
cyanogen bromide, is toxic and volatile and requires harsh reaction
conditions.2 Transition-metal complexes are promising reagents
for cleavage of amide bonds.3 Peptides4 and proteins5 can be
hydrolytically cleaved near histidine and methionine residues with
several palladium(II) aqua complexes, often with catalytic
turnover. Here, we report that palladium(II) complexes bind to
N-acetylated tryptophan-containing peptides AcTrp-Ala, AcTrp-
Val, and AcTrp-ValOMe in acetone solution and regioselectively
cleave them upon addition of an equivalent of water.
Binding of Tryptophan-Containing Peptides to Palladium-
(II). Coordination of indole (1) to metals in biological systems
is unprecedented. Few abiological metal complexes with tryp-
tophan are known.6-8 Pyridine-type nitrogen in the tautomer 3H-
indolenine (2) can coordinate to palladium(II).9,10
constants K for the binding of AcTrp-Ala to cis-[Pd(en)(sol)2]2+
in acetone-d6 in the presence of 0.30 and 2.0 M D2O are 44 ( 10
and 5 ( 1 M-1, respectively. Addition of 0.005 M NaOH to the
latter solution does not much affect this binding (K ) 13 ( 7
M-1). Water, itself a ligand, inhibits tryptophan coordination. In
(8) Robson, R. Inorg. Chim. Acta 1982, 57, 71.
(9) Yamauchi, O.; Takani, M.; Toyoda, K.; Masuda, H. Inorg. Chem. 1990,
29, 1856.
(10) Takani, M.; Masuda, H.; Yamauchi, O. Inorg. Chim. Acta 1995, 235,
367.
Upon mixing of N-acetyl-L-tryptophanamide (AcTrp-NH2) and
cis-[Pd(en)(sol)2]2+ in acetone solution, complex 3 appears.11,12
(11) (a) 1H NMR for AcTrp-NH2: N(1)H 10.08 br s, C(2)H 7.15 s; C(4)H
7.64 d; C(5)H 7.00 t, C(6)H 7.08 t; C(7)H 7.35 d, C(8)H 3.18 m; amide NH
6.90 br, 6.35 br, and 7.12 br; R-CH 4.65 m; CH3 1.85 s. 1H NMR for 3:
N(1)H 10.40 br; C(2)H 7.57 s; C(4)H 7.77 d; C(5)H 7.18 t; C(6)H 7.12 t;
C(7)H 7.54 d; C(8)H 3.70 m; amide NH 8.92 br, 8.65 br, and 7.3 br; R-CH
5.05 m; CH3 1.89 s. (b) 13C NMR for AcTrp-NH2: C(2) 124.2; C(3) 111.6;
C(4a) 128.8; C(4) 119.3; C(5) 119.3; C(6) 122.0; C(7) 112.0; C(7a) 137.2;
C(8) 23.0; R-C 54.3; C(O) 174.0; CH3 29.0; C(O)NH2 170.0. 13C NMR for 3:
C(2) 119.0; C(3) 70.0; C(4a) 128.9; C(4) 119.5; C(5) 126.7; C(6) 122.2;, C(7)
113.0; C(7a) 137.3; C(8) 17.5; R-C 56.0; C(O) 175.0; CH3 31.5; C(O)NH2
187.0. λmax is 333 and 325 nm for cis-[Pd(en)(sol)2]2+ and 3, respectively.
(12) Complex 3 is formed regardless of the mole ratio of Pd(II) to N-acetyl-
L-tryptophanamide. Concentration of 3 never exceeded the lowest initial
concentration of the substrates.
† Current address: Department of Chemistry, Massachusetts Institute of
Technology, Cambridge, MA 02139.
# Current address: Department of Biochemistry, Iowa State University,
Ames, IA 50011-3111.
(1) Radzicka, A.; Wolfenden, R. J. Am. Chem. Soc. 1996, 118, 6105.
(2) Croft, L. R. Handbook of Protein Sequence Analysis, 2nd ed.; Wiley:
Chichester, 1980.
(3) (a) Ermacora, M. R.; Ledman, D. W.; Fox, R. O. Nat. Struct. Biol.
1996, 3, 59. (b) Heyduk, T.; Heyduk, E.; Severino, K.; Tang, H.; Ebright, R.
H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10162. (c) Ghaim, J. B.; Greiner,
D. P.; Meares, C. F. Biochemistry 1995, 34, 11311. (d) Hegg, E. L.; Burstyn,
J. N. J. Am. Chem. Soc. 1995, 117, 7015. (e) Chin, J. Acc. Chem. Res. 1991,
24, 145. (f) Fife, T. H. Acc. Chem. Res. 1993, 26, 325.
(4) (a) Parac, T. N.; Kostic´, N. M. J. Am. Chem. Soc. 1996, 118, 51. (b)
Parac, T. N.; Kostic´, N. M. J. Am. Chem. Soc. 1996, 118, 5946. (c) Zhu, L.;
Kostic´, N. M. J. Am. Chem. Soc. 1993, 115, 4566. (d) Karet, G. B.; Kostic´,
N. M. Inorg. Chem. 1998, 37, 1021. (e) Zhu, L.; Kostic´, N. M. Inorg. Chem.
1992, 31, 3994. (f) Burgeson, I. E.; Kostic´, N. M. Inorg. Chem. 1991, 30,
4299.
(5) (a) Zhu, L.; Qin, L.; Parac, T. N.; Kostic´, N. M. J. Am. Chem. Soc.
1994, 116, 5218. (b) Zhu, L.; Bakhtiar, R.; Kostic´, N. M. J. Biol. Inorg. Chem.
1998, 3, 383.
(13) 2D 1H-15N HETCOR spectra ruled out the iminol species and
deprotonation of the amide nitrogen.
(14) In cis-[Pt(en)(C,O-AcTrp-NH2)]2+ the 13C resonance of C(3) is at 70.0
ppm.
(15) Fairlie, D. P.; Woon, T. C.; Wickramasinghe, W. A.; Willis, A. C.
Inorg. Chem. 1994, 33, 6425.
(16) Woon, T. C.; Fairlie, D. P. Inorg. Chem. 1992, 31, 4069.
(17) Storhoff, B. N.; Huntley, C. L., Jr. Coord. Chem. ReV. 1977, 23, 1.
(18) 1H NMR for AcTrp-Ala: N(1)H 10.10 br s; C(2)H 7.27 s; C(4)H
7.64 d; C(5)H 7.00 t; C(6)H 7.09 t; C(7)H 7.39 d; C(8)H 3.20 m; amide NH
8.12 br and 7.90 br; R-CH 4.75 q; R-CH 4.40 q; CH3 1.90 s; CH3 (Ala) 1.32
d. For cis-[Pd(en)(C,O-AcTrp-Ala)]2+: N(1)H 10.50 br; C(2)H 7.60 s; C(4)H
7.70 d; C(5)H 7.17 t; C(6)H 7.11 t; C(7)H 7.68 d; C(8)H 3.60 m; amide NH
8.12 br and 8.21 br, R-CH 5.30 m; R-CH 4.75 m; CH3 1.90 s; CH3 (Ala) 1.50
d. See also the Supporting Information.
(6) Fontana, A.; Toniolo, C. The Chemistry of Tryptophan in Peptides and
Proteins. In Progress in the Chemistry of Organic Natural Products; Herz,
W., Ed.; New York, 1976; pp 309-449.
(7) Corbeil, M. C.; Beauchamp, A. L. Can. J. Chem. 1988, 66, 2458.
10.1021/ja9840246 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/03/1999

8664 J. Am. Chem. Soc., Vol. 121, No. 37, 1999
Communications to the Editor
Table 1. Observed Rate Constants for the Hydrolysis of the Three
Tryptophanyl Dipeptides Promoted by the Four Palladium(II)
Complexesa
larger side chain (valine). Although the steric effect is small, it
may be parlayed into sequence-selectivity (preferential cleavage
next to a smaller leaving group).4b,c Similar rates for the valine
and its ester indicate that the bulk of the peptide terminus does
not affect the rate of cleavage.
103kobs, min-1
complexb
AcTrp-Ala AcTrp-Val AcTrp-ValOMe
Mechanism of Hydrolysis. Absence of aqua ligands in the
hydrolytically active complexes 3 rules out the internal attack of
Pd(II)-bound nucleophile at the amide carbon.4 Coordination of
the amide oxygen renders the amide carbon more electrophilic
and susceptible to external attack of solvent water, which causes
the hydrolytic cleavage on the C-terminal side of tryptophan (eq
1). There is no cleavage on the N-terminal side, presumably
because that amide bond is not activated by the palladium(II)
atom. Fitting the data in Figure S1 to the equations in the
Supporting Information yielded the rate constant for hydrolysis
kobs ) (k1k2[cis-Pd(en)(sol)22+])/(k1[cis-Pd(en)(sol)22+] + k-1) of
(2.4 ( 1.0) × 10-3 min-1 and the equilibrium constant K ) k1/
k-1 of 27 ( 6 that agrees with the result of binding studies. The
microscopic rate constants k2 in the presence of 0.30 and 2.0 M
D2O in acetone-d6 solution are (6.3 ( 1.0) × 10-4 and (4.6 (
1.0) × 10-3 min-1, respectively.21 That k2 increases 7.3-fold as
the concentration increases 6.7-fold is consistent with the external
attack of water at the coordinated AcTrp-Ala, as in eq 1. Initial
rate for alanine formation is a composite quantity: ν0 ) k2[3];
because k2 increases and [3] decreases as the water concentration
increases, the overall effect on ν0 is small.22
none
<0.0005
2.4 ( 0.2
0.21 ( 0.02
2.7 ( 0.3
<0.004
<0.0005
<0.0005
cis-[Pd(en)(sol)2]2+
cis-[Pd(Me4en)(sol)2]2+
cis-[Pd(dtcol)(sol)2]2+
[Pd(dien)(sol)]2+
0.99 ( 0.1
<0.01 ( 0.01
0.95 ( 0.10
1.2 ( 0.1
a At 323 K, in acetone-d6 that was made 0.0010 M in DClO4. Initial
concentrations of the dipeptides, palladium(II) complexes, and D2O were
0.025, 0.025, and 0.300 M, respectively. b en is ethylenediamine, Me4en
is N,N,N′,N′-tetramethylethylenediamine, dtcol is 1,5-ditiocyclooctan-
3-ol, and dien is diethylenetriamine.
aqueous solution this coordination is undetectable even in the
presence of a 100-fold excess of cis-[Pd(en)(sol)2]2+ 19
.
Hydrolysis of Tryptophan-Containing Peptides. As Table
1 shows, the “background” hydrolysis of the peptides is extremely
slow. In the presence of the palladium(II) complexes and 1 equiv
of water in an acetone-d6 solution, however, these peptides are
completely cleaved in less than a day. The active intermediates
are complexes 3, as Figure S1 in the Supporting Information
shows. As eq 1 shows, the products are the C-terminal amino
To our knowledge, this is the first report that coordination of
the tryptophan side chain is followed by hydrolysis of a nearby
peptide bond. Unlike methionine and histidine, which coordinate
to palladium(II) in aqueous as well as acetone solutions,4
tryptophan coordinates only in acetone solution. Tryptophan has
lower affinity to palladium(II) than methionine and histidine do.
Rate constants for palladium(II)-promoted hydrolysis next to these
residues show the same trend.23 Therefore, the overall selectivity
of cleavage, can be altered simply by changing the solvent or the
concentration of the palladium(II) promoter. Cleavage will occur
near histidine and methionine in aqueous solution, and near
histidine, methionine, and tryptophan in acetone solution, if the
promoter is present in excess. The fine selectivity, preference for
the C-terminal over the N-terminal amide bond of tryptophan, is
due to the stereochemistry of chelate rings. A prospect for cleaving
hydrophobic or membrane-bound proteins in nonaqueous solu-
tions, by palladium(II) complexes, has emerged.
acid and palladium(II) complex of N-acetyl-L-tryptophan. Acetic
acid is not detected. In control experiments with AcAla-Trp, which
lacks a C-terminal amide bond adjacent to the tryptophanyl
residue, neither amide bond is cleaved. Evidently, palladium(II)
regioselectively cleaves on the C-terminal side of tryptophan, not
on the N-terminal side. This useful regioselectivity is presumably
caused by the stereochemical preference for 3 over 4. In aqueous
solution coordination of the tryptophanyl residue to palladium-
(II) completely ceases, and the peptides do not hydrolyze.
The tridentate complex [Pd(dien)(sol)]2+ has only one labile
ligand and does not bind to the tryptophan-containing peptides
because they can act only as bidentate ligands.20 Consequently,
there is no hydrolytic cleavage.
Acknowledgment. We thank NSF for Grant No. CHE-816522.
Supporting Information Available: Syntheses of the palladium(II)
promoters and of the substrates; kinetic and other experimental procedures;
structures of the promoters; 13C NMR data for the complex cis-[Pd(dtcol)-
(C,O-AcTrp-NH2)]2+ 1H NMR data for the two dipeptides and their
;
palladium(II) complexes; Figure S1 showing the progress of hydrolysis;
and kinetic equations used to fit the data in Figure S1 (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
JA9840246
(21) Calculated from the initial rate of alanine formation and the known
initial concentration of 3.
The peptide containing the smaller side chain (alanine) is
cleaved more rapidly than the similar peptides containing the
(22) The initial rates in acetone-d6 that is 2.0 M in D2O and 0.0050 M in
NaOH, 2.0 M in D2O, and 0.30 M in D2O are (1.0 ( 0.5) × 10-4, (1.1 ( 0.2)
× 10-4, and (6.3 ( 1.0) × 10-6 M min-1, respectively, at 323 K. Initial con-
centrations of the dipeptides and palladium(II) complexes were 0.10 M each.
(23) In acetone solution in which there is an excess of cis-[Pd(en)(sol)2]2+
over a mixture of Met-, His-, and Trp-containing peptides, upon addition of
water only the Trp-containing peptide is cleaved.
(19) The K values are independent of the initial concentrations of AcTrp-
Ala and cis-[Pd(en)(sol)2]2+. We report average results of several experiments.
(20) Upon addition of [Pd(dien)(sol)]2+ to AcTrp-NHX the 1H NMR spectra
remain unchanged.