JBIC (1998) 3 :1–8 Q SBIC 1998

ORIGINAL ARTICLE

Xiaohua Chen 7 Longgen Zhu 7 Xiaozeng YouNenad M. Kostic

Steric effects, solvent effects, and turnover in hydrolytic cleavage of

peptides promoted by palladium(II) aqua complexes

Received: 13 June 1997 / Accepted: 24 September 1997

X. Chen 7 L. Zhu (Y) 7 X. YouState Key Laboratory of Coordination Chemistry,Nanjing University, Nanjing 210093, ChinaFax: c86-25-331-4502

N. M. KosticDepartment of Chemistry, Iowa State University, Ames,IA 50011, USA

Abstract Dipeptides and tripeptides AcMet-aaH con-taining N-acetyl methionine, in which the group aaH isGlyH, AlaH, ValH, or Gly-GlyH, undergo hydrolyticcleavage of the Met-aaH peptide bond in the presenceof the following complexes of palladium(II): cis-[Pd(en)(H2O)2]2c, cis-[Pd(tn)(H2O)2]2c, cis-[Pd(en)(CH3OH)2]2c, cis-[Pd(S,N-MetH)(H2O)2]2c,cis-[Pd(S,N-Met-GlyH)(H2O)2]2c, and cis-[Pd(S,N-Met-AlaH)(H2O)2]2c. These mononuclear complexesare precursors of binuclear palladium(II) complexescontaining the substrates AcMet-aaH as bridgingthioether ligands. The rate constant for cleavage ishigher when the bidentate ligand in the precursor com-plex is ethylenediamine (which is completely displaced)than S,N-methionine (of which only the amino group isdisplaced), because the number of aqua ligands availa-ble for cleavage is greater in the former than in the lat-ter case. The demonstrated dependence of the rate con-stant on the steric bulk (volume) of the leaving group,aaH, points the way toward achieving a degree of se-quence selectivity in cleavage of peptide bonds by pal-ladium(II) aqua complexes. One equivalent of cis-[Pd(en)(H2O)2]2c cleaves as many as ten equivalentsof AcMet-GlyH, but the rate constant decreases as themolar excess of the dipeptide over the catalyst in-creases. This demonstration of catalytic turnover pointsthe way to our ultimate goal – artificial metallopepti-dases.

Key words Peptides 7 Hydrolysis 7 Palladium 7Kinetics 7 Turnover

Introduction

Hydrolysis of unactivated amide bonds in peptides andproteins is an extremely slow reaction; the half-life inneutral solution is measured in hundreds of years [1].This hydrolysis, however, is an essential metabolicprocess and a desirable reaction in various bioanalyticalprocedures. The reagents that effect it, both in vivo andin vitro, usually are proteolytic enzymes [2, 3]. Only afew of them are routinely applicable in biochemistryand structural biology. They have great catalytic power,but their sequence selectivity can be changed only withdifficulty, and their size may limit their use as probes ofmolecular structures and interactions.

Synthetic chemical reagents are not likely to replaceproteolytic enzymes in the laboratory, but they holdsome potential advantages [3–5]. The rates and regio-selectivity of their reactions can, in principle, be con-trolled by design. Unlike enzymes, synthetic reagentsmay be active at various pH values and temperatures.Because these reagents are relatively small, they maybe useful in structural studies at the scale of individualchemical bonds and functional groups in proteins of in-terest. A particularly desirable property of any synthet-ic reagent is turnover, i.e., the ability of one molecule ofthe reagent to cleave multiple molecules of the sub-strate (peptide or protein).

Only one chemical reagent, cyanogen bromide, isbeing widely used for protein cleavage. It convenientlycleaves the peptide backbone on the carboxylic side ofmethionine residues, where proteolytic enzymes do notcleave. Cyanogen bromide, however, is very toxic, re-quires fairly harsh reaction conditions, and must be ap-plied in large excess over the substrate. Both the useand the safe disposal of this organic chemical are prob-lematic.

Metal complexes hold great promise as reagents forcleavage of amide bonds [6–8]. They fall into threeclasses. First, complexes of cobalt(III), which are inertin ligand-substitution reactions, were used in elegant

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kinetic and mechanistic studies of hydrolysis [6, 9, 10].But these complexes bind only to the N-terminal aminoacid, and therefore cleave only the first amide bond inthe sequence. Moreover, the cleavage is stoichiometric,not catalytic. Some complexes of copper(II) resemblethose of cobalt(III) [9, 11–13]. Second, iron complexeswith the ligand EDTA tethered to certain amino-acidside chains, in the presence of additional chemicals,cleave proximate amide bonds at rates that are high orlow and with yields that can be high or low, dependingon the substrate, conditions, and the method of tether-ing [14–23]. Use of untethered [Fe(EDTA)] complexeseliminates the synthetic work required for bioconjuga-tion [24–26].

A third class of complexes, introduced at Iowa StateUniversity [27–36] but used also elsewhere [37–40],spontaneously bind to substrates for cleavage uponsimple mixing of aqueous solutions. Initial studies withplatinum(II) complexes [27] gave way to recent studieswith complexes of palladium(II) [28–36] and copper(II)[37]. The palladium(II) aqua complexes anchor to theside chains of methionine and histidine residues inshort peptides and effect selective hydrolytic cleavageof the first amide bond “downstream” of the one in-volving the carboxylic group of the amino acid residuecoordinated to the palladium(II) atom. This regioselec-tivity, observed in all the experiments with short pep-tides published to date, is shown in Eq. 1. (The aminogroup in the leaving amino acid is actually protonatedin the acidic solutions used.) Half-lives of the reactionsinvolving the methione residue as the anchor, shown inEq. 1, range from less than 1.0 h to ca. 0.5 d. Theserates are sufficient for practical use of palladium(II)complexes in biochemistry. Indeed, they cleave pro-teins with explicable selectivity – cytochrome c at a sin-gle site [36] and myoglobin at thirteen identified sites.

Control experiments in the absence of palladium(II)compounds have ruled out simple acid catalysis of thehydrolysis, so-called background cleavage [27, 28].Acidic solution (1^pH^6) is needed to suppress de-protonation of aqua ligands and formation of oligonu-clear palladium(II) complexes with hydroxo bridging li-gands. Hydrolysis is due to the palladium(II) com-plexes, which can act in two ways. In one, coordinationof a carbonyl group to the metal atom activates the am-

ide bond toward external attack by a water moleculefrom the solvent. In the other, an aqua ligand from thecomplex internally attacks the scissile amide bond.

Kinetic experiments and NMR spectra consistentlyindicate that the active species in the hydrolysis of ol-igopeptides are binuclear complexes of the types I andII in Chart 1, in which the thioether group of the me-thionine side chain acts as a bridging and a terminal li-gand, respectively [28, 40]. In complexes of either typethe cleaving agent can be a terminal aqua ligand or awater molecule from the solvent.

In this article we report on the kinetics of the reac-tion in Eq. 1 involving several dipeptides in which theamino group of methionine is blocked by acetylation,the carboxylic group forms the scissile amide bond withthe leaving amino acid, and the thioether group is a li-gand in a binuclear palladium(II) complex. This studyimproves our understanding of hydrolytic cleavage ofpeptides and brings us a step closer to our ultimate goal– design of palladium(II) complexes as artificial metal-lopeptidases.

Materials and methods

Chemicals

Distilled water was further demineralized and purified. The deu-terium-containing solvents (D2O and CD3OD), K2[PdCl4],ethane-1,2-diamine (ethylenediamine, en), propane-1,3-diamine(triethylenediamine, tn), and 1,5-dithiacyclooctane-3-ol (dtco-OH) were obtained from Aldrich. Methionine (MetH), S-methyl-cysteine (CysMeH), methionyl glycine (Met-GlyH), methionyl al-anine (Met-AlaH), and methionyl valine (Met-ValH) were ob-tained from Sigma. All other chemicals were of reagent grade.

The terminal amino group in the dipeptides was acetylated bya standard procedure [28]. The N-acetylated tripeptide AcMet-Gly-GlyH was prepared by standard reactions [41, 42] in the fol-lowing sequence: Z-Cl ] Z-GlyH ] Z-Gly-GlyEt ] Gly-GlyEt7HCl ] AcMet-Gly-GlyEt ] AcMet-Gly-GlyH. The 1HNMR spectra of the products in D2O solutions showed the fol-lowing principal d values, in ppm: AcMet-GlyH, 2.04 (s, CH3CO),2. 11 (s, CH3S), and 3.99 (q, Gly CH2); AcMet-AlaH, 2.03 (s,CH3CO), 2.11 (s, CH3S), 1.44 (d, Ala CH3), and 4.39 (q, Ala CH);AcMet-ValH, 2.03 (s, CH3CO) 2.11 (s, CH3S), 0.96 (dd, Val CH3),and 4.27 (d, Val a-CH); and AcMet-Gly-GlyH, 2.02 (s, CH3CO),2.09 (s, CH3S), 3.92 (s, Gly CH2), and 4.01 (m, Gly CH2).

The following dichloro complexes were prepared by publishedmethods [43–48]: cis-[Pd(en)Cl2], cis-[Pd(tn)Cl2], cis-[Pd(S,N-MetH)Cl2], cis-[Pd(S,N-CysMetH)Cl2]7H2O, cis-[Pd(S,N-Met-GlyH)Cl2], and cis-[Pd(S,N-Met-AlaH)Cl2]. The amino acids anddipeptides are bidentate ligands, coordinated via the thioetherand amino groups. The corresponding diaqua complexes in Chart2 were obtained by stirring each precursor with 2.0 equivalents ofAgNO3 in a D2O solution of pH* 2 for 4 h at 35 7C and by remov-al of AgCl by centrifugation, all in the dark [27, 28, 49]. (Uncor-rected values of pH are designated pH*.) The ligands were ac-tually D2O, but the formulas will be written with H2O, for sim-plicity. When cis-[Pd(en)Cl2] was treated with AgNO3 in CD3ODas the solvent, the alcohol molecules may coordinate to palladi-um(II); again, the formula will be written simply with CH3OH.The aqua complexes were always prepared fresh and used as so-lutions. The 1H NMR spectra of D2O solutions showed the fol-lowing principal d values, in ppm: cis-[Pd(en)(H2O)2]2c, 2.63 (s,CH2); cis-[Pd(tn)(H2O)2]2c, 1.75 and 2.39 (both s, CH2); cis-

3

Chart 1 Hydrolytically-activecomplexes

Chart 2 Palladium(II) com-plexes

4

[Pd(S,N-MetH)(H2O)2]2c, 2.37 (s, CH3S); cis-[Pd(S,N-Met-GlyH)(H2O)2]2c, 2.31 (s, CH3S), 4.05 (s, Gly CH2), and 4.36 (q,CH); cis-[Pd(S,N-Met-AlaH)(H2O)2]2c, 2.35 (s, CH3S); 1.44 (d,Ala CH3); and 4.43 (q, Ala CH).

Measurements

Proton NMR spectra at 500 MHz of D2O solutions containing so-dium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) as an internalreference were recorded with a Bruker AM-500 spectrometer.The sample was kept at 40B0.5 7C. The pH was measured with anOrion 901 instrument and a Phoenix Ag/AgCl reference electro-de. The uncorrected values in D2O solutions and nominal valuesin CD3OD solutions are designated pH*. The corrected values inD2O are by 0.40 ppm greater.

Kinetics of hydrolysis

The palladium(II) aqua complexes were prepared fresh to minim-ize the formation of hydroxo-bridged polynuclear complexes. Thestock solutions had concentrations of 30–100 mM and pH* of 1.5–2.0. Equimolar amounts of the aqua complex and the peptide,both dissolved in D2O, were rapidly mixed in an NMR tube sothat the final concentration of each was 20.0 mM. The pH* valuewas adjusted with a 1.0 M solution of HClO4 in D2O at the begin-ning and was checked again at the end of the reaction. Acquisi-tion of the 1H NMR spectra began as soon as possible, and 16 or32 scans were taken at each time. The sample temperature was40B0.5 7C. As Fig. 1 shows, the reaction in Eq. 1 is easily fol-lowed by monitoring the growing 1H NMR resonance of the leav-ing amino acid (Gly, Ala, or Val) or dipeptide (Gly-Gly). Theirconcentrations (Ct) were determined on the basis of the signalarea and the known initial concentrations (C0) of the substrate(dipeptide or the tripeptide). The estimated errors in Ct areB5%. Plots of Pln[(CePCt)/Ce] versus time contained 13–20points each and were linear over at least three half-lives, with cor-relation coefficients of 0.994–0.999. This linearity is evidence thatthe hydrolysis reactions obey the first-order rate law, i.e., that thereaction rate is proportional to the concentration of the substrate.The rate constants are the slopes of these plots. The reactionswere run for six half-lives.

Results and discussion

Palladium(II) complexes that promote hydrolysis

The states of protonation and the net charges shown inChart 1 are for the species in acidic solutions used inthis study. Because these solutions remain clear wellafter the reactions of interest are completed, we assumethat the complexes are sufficiently stable for the experi-ments in which they were used. The complex formationby ligand-substitution reactions is conveniently moni-tored by 1H NMR spectroscopy [28, 40], but in the ab-sence of other characterization their structures must beconsidered uncertain. The CH3S resonances of terminaland doubly-bridging thioether ligands fall in two non-overlapping intervals, 2.26–2.39 and 2.44–2.55 ppm, re-spectively. The resonance of free methionine and of themethionyl dipeptides occurs at 2.11 ppm. BidentateS,N-coordination of methionine has been well estab-lished by X-ray crystallography [45–48], and the me-thionyl moiety of the dipeptides coordinates in thesame manner. The 1H NMR spectra of the dipeptide

complexes cis-[Pd(S,N-Met-GlyH)(H2O)2]2c (4.04, m,Gly CH2) and cis-[Pd(S,N-Met-AlaH)(H2O)2]2c (1.42,d, AlaCH3 and 4.42, q, Ala a-CH) in acidic D2O solu-tion showed no free glycine or alanine after severalmonths. Evidently, the dipeptide ligands in these com-plexes are stable with respect to the hydrolysis of theamide bond.

Hydrolysis of the dipeptides AcMet-aaH promoted bypalladium(II) complexes containing the dipeptidesMet-GlyH and Met-AlaH as ligands

When the mononuclear complexes cis-[Pd(S,N-Met-GlyH)(H2O)2]2c and cis-[Pd(S,N-Met-Al-aH)(H2O)2]2c were treated with the equimolaramounts of the acetylated methionyl dipeptides, binu-clear complexes having the structure III in Chart 1 wereformed. The incoming dipeptide AcMet-aaH coordi-nates only via its thioether group as a bridging ligand.The chelate ring involving the methionyl moiety ofMet-GlyH or Met-AlaH opens, presumably under thetrans effect of the new thioether ligand, and these di-peptides remain coordinated as unidentate terminal li-gands via the thioether group. Hydrolysis is easily fol-lowed by 1H NMR spectroscopy as in the previousstudies [27–34, 38–40]. A typical set of spectra in Fig. 1shows the disappearance of the substrate AcMet-aaHand concomitant appearance of the free amino acidaaH. The rate constants are given in Table 1.

When both the promoter and the substrate con-tained glycine (the first row in Table 1), the 1H NMRspectra were complicated by the overlap of the glycineresonances in the binuclear complex III. Although therate constants were determinable from the growth ofthe resonance of free glycine, it was unclear whetherthis free glycine comes from Met-GlyH (in the promot-er complex) or AcMet-GlyH (the added substrate).

The dilemma vanished in the other four experimentsreported in Table 1. When the C-terminal amino acidsaaH in the two methionyl dipeptides differed, it be-came clear than only the one in AcMet-aaH was liber-ated. The terminal dipeptide ligand, Met-aaH, in thecomplex III remained, while the bridging dipeptide li-gand was cleaved.

In our previous studies [27–30], we examined themechanism of hydrolytic cleavage of methionine-con-taining peptides by palladium(II) complexes [28–30].To our knowledge, this is the first study in which transi-tion-metal complexes with peptides are used to pro-mote cleavage of other peptides. Its interesting resultsraise the possibility of using the secondary structure ofthe complexes containing longer peptides to control theselectivity of cleavage of other peptides as substrates.This possibility will be the subject of our future re-search.

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Fig. 1 Proton NMR spectra in the a-CH (left) and CH3 (right)regions of a D2O solution made with equimolar amounts of cis-[Pd(S,N-Met-Ala)(H2O)2]2c and AcMet-Val at pH* 0.91 and40B0.5 7C, at the following times after mixing: (a) 15 min, (b)3.2 h, (c) 5.7 h, (d) 10.3 h, and (e) 35 h. This is a subset of a full setof spectra from which the rate constant was determined. The per-sistent resonances belong to the ligand Met-AlaH, the disappear-ing ones to the substrate AcMet-ValH, and the appearing ones tothe free valine

Table 1 Hydrolysis of the methionine-aaH bond in the substrates AcMet-aaH at pH* 0.92 and 40 7C

Promoter aaH inAcMet-aaH

Groupin aaH

1H NMR of aaH, ppm 103 kobs,minP1

In III Free

cis-[Pd(S,N-Met-GlyH)(H2O)2]2c GlyHAlaH

ValH

CH2

CH3

a-CHCH3

a-CH

4.04 m1.42 d4.42 q0.96 dd4.24 d

3.90 s1.58 d4.14 q1.06 dd3.97 d

7.84.0

2.0

cis-[Pd(S,N-Met-AlaH)(H2O)2]2c GlyHValH

CH2

CH3

a-CH

4.04 m0.96 dd4.24 d

3.90 s1.06 dd3.97 d

5.51.4

Table 2 Hydrolysis of the methionine-aaH bond in the substratesAcMet-aaH at 40 7C

Promoter aaH 103 kobs, minP1

cis-[Pd(en)(H2O)2]2c a

cis-[Pd(en)(CH3OH)2]2c

GlyHAlaHValHGlyHAlaHValH

20104.6

41207.3

a At pH* 0.94

Kinetic effect of the solvent

When D2O is replaced as a solvent by CD3OD, cleav-age of the dipeptide becomes faster, as Table 2 shows.It is unclear whether the bottom half of Table 2 per-tains to hydrolysis in methanol or to methanolysis. Thelatter possibility is consistent with the fact that metha-nol is more nucleophilic than water. The rate enhance-

ment bodes well for the application of the new palla-dium(II) reagents for cleavage of relatively hydrophob-ic peptides, which may be insoluble in water but solublein alcohols.

Kinetic effect of the bidentate ligand

Comparison of the first three rows of data in Tables 1and 2 shows that replacement of methionine by ethy-lenediamine in the mononuclear promoter complexcauses approximate doubling of the rate constant forcleavage. This effect can be explained in terms of thebinuclear reactive complexes III and IV, in which hy-drolysis of AcMet-aaH actually occurs. This dipeptide,effectively a thioether ligand, exerts a kinetic trans ef-fect, which is enhanced if the potential leaving groupcan also be protonated. Only the amino end of the S,N-chelated ligand (methionine) can be protonated, andconsequently only this end is displaced from palladi-um(II). Hence there are only two aqua ligands in thecomplex III. Both ends of the N,N-chelated ligand

(ethylenediamine) can be protonated, and thereforeboth of them are displaced, and free enH2

2c is detectedby its 1H NMR singlet at 3.37 ppm. Hence there arefour aqua ligands in the complex IV [28, 40]. Thegreater the number of terminal aqua ligands, the higherthe probability of internal attack on the substrate Ac-Met-aaH, which occupies bridging positions in bothcomplexes III and IV.

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Fig. 2 Dependence of the rate constant (from Tables 1 and 2) forhydrolytic cleavage of the dipeptide AcMet-aaH on the stericbulk of the a-CHR group in the leaving amino acid, aa. The rela-tive volume, DV, is the difference between the volumes (in Å3) ofthe a-CHR groups in a given amino acid and in glycine. The pro-moters and the corresponding linear fits are as follows: bottom:cis-[Pd(Met-Gly)(H2O)2]2c and lnkobsp4.5B0.023 DV, middle:cis-[Pd(en)(H2O)2]2c and lnkobsp3.6c0.025 DV, and top: cis-[Pd(en)(CH3OH)2]2c and lnkobsp2.8c0.029 DV

Steric effect of the leaving amino acid

As Tables 1 and 2 show, the rate constant for cleavagedepends to some extent on the C-terminal amino acid,aaH in AcMet-aaH, which is the leaving group. Sincethe strength (thermodynamic stability) and susceptibili-ty to hydrolysis (kinetic stability) of the Met-aaH amidebond should not depend significantly nor systematicallyon the identity of aaH, the small downward decrease inthe rate constant is attributable to the increase in thesteric bulk of the leaving amino acid. As in our pre-vious studies [29, 35], the steric bulk is quantitated asvolume calculated from van der Waals dimensions offunctional groups in amino acids and proteins. The re-lative volume, DV in Fig. 2, is the difference betweenthe volumes (in Å3) of the a-CHR groups in a givenamino acid and in glycine. The bulkier the leaving ami-no acid, the greater the shielding of the scissile bondfrom the palladium(II) complex and from the aqua li-gand that this complex delivers.

Although the slopes of the plots in Fig. 2 are rela-tively small, the existence of the steric effect is impor-tant in principle. Volume is characteristic of amino-acidresidues in peptides and proteins, and our results mayallow design of palladium(II) complexes that exibit adegree of sequence-selectivity in cleavage.

Regioselectivity of cleavage

The tripeptide AcMet-Gly-GlyH (whose glycyl CH2

groups give a 1H NMR multiplet at 4.02 ppm) in thepresence of four palladium(II) aqua complexes consis-tently yields the dipeptide Gly-GlyH (whose CH2

Table 3 Hydrolysis of the Met-GlyH bond in a tripeptide and adipeptide at 0.89^pH*^0.99 and 40 7C, promoted by differentcomplexes of palladium(II)

Promoter 103 kobs, minP1

AcMet-Gly-GlyH

AcMet-GlyH

cis-[Pd(en)(H2O)2]2c

cis-[Pd(tn)(H2O)2]2c

cis-[Pd(S,N-MetH)(H2O)2]2c

cis-[Pd(S,N-Met-GlyH)(H2O)2]2c

13233.53.6

20386.87.8

groups give 1H NMR singlets at 3.90 and 4.08 ppm) andnot glycine. Evidently, only the Met-Gly bond iscleaved. Although this regioselectivity is the same asthat for AcMet-GlyH, the rate constants are lower forthe tripeptide than for the dipeptide, as Table 3 shows.This finding is consistent with the analysis of steric ef-fects in the preceding subsection; the larger leavinggroup, Gly-GlyH, is cleaved off more slowly than thesmaller one, glycine.

Catalytic turnover in cleavage

Initial coordination of AcMet-GlyH to the palladi-um(II) aqua complexes was easily monitored by 1HNMR spectroscopy. The resonances in Fig. 3, in the or-der of decreasing chemical shifts (from left to right) areassigned as follows: 4.60 ppm, Met a-CH; 4.05 ppm,GlyH CH2; 3.37 ppm, enH2

2c; 3.00–3.10 ppm, Met, g-CH2; 2.51 ppm, Met CH3S; 2.34 and 2.27 ppm, Met b-CH2; and 2.05, CH3CO. Significantly, the resonance ofthe free thioether group, at 2.11 ppm, is completely ab-sent, even though there is an excess of the dipeptideover the palladium(II) complex. The resonance of thebridging thioether group, at 2.51 ppm, is present, and itbecomes broader as the molar excess of the dipeptideincreases. These facts are consistent with polynuclearpalladium(II) complexes containing methionyl residuesfrom the dipeptide as bridging ligands.

To test this hypothesis, we examined the 1H NMRspectra of two mixtures, in which AcMet-Gly was pres-ent in fivefold molar excess over cis-[Pd(tn)(H2O)2]2c

and over cis-[Pd(dtco-OH)(H2O)2]2c. The former mix-ture gave a spectrum virtually identical to that in Fig.3C, except that the resonance of enH2

2c was replacedby two resonances of tnH2

2c. Evidently, both ethylene-diamine and trimethylenediamine are released, and po-lynuclear complexes are formed. The latter mixture,however, contained both AcMet-GlyH and dtco-OH asligands to palladium(II). The dtco-OH ligand was notprotonated and released, and polynuclear complexeswere also formed. Our explanation of the spectra inFig. 3 is thus corroborated.

The rate constant decreases as the molar excess ofthe dipeptide over the promoter increases (Table 4).This fact does not contradict the first-order kinetic law

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Fig. 3 Proton NMR spectra of solutions in D2O at1.05^pH*^120 and 40 7C that initially contained 10 mM cis-[Pd(en)(H2O)2]2c and (a) 30 mM, (b) 40 mM, and (c) 50 mM Ac-Met-GlyH. The spectra were recorded approximately 10 min aftermixing of the promoter and the dipeptide

Table 4 Hydrolysis of the Met-GlyH bond in AcMet-GlyH in thepresence of subequivalent amounts of cis-[Pd(en)(H2O)2]2c at40 7C

Mole ratiodipeptide :catalysta

pH* 103 kobs, minP1

1 :12 :13 :14 :15 :1

10 :11 :0b

0.911.031.051.121.171.000.81

20101.70.210.100.0600.0066(8)

a Concentration of cis-[Pd(en)(H2O)2]2c was 10 mM, and theconcentration of AcMet-GlyH was varied from 10 to 100 mMb The control reaction was run for 1.5 half-lives

discussed above, but its analysis would require a sepa-rate study of the kinetics of catalysis. In each case, thepromoter effected complete cleavage of the dipeptide.The products were N-acetylmethionine and glycine,and the reactions followed the first-order kinetic law.These experiments showed that the palladium(II)-thioether bond is labile enough to permit catalytic turn-over in cleavage of methionine-containing peptides.Turnover in the cleavage of histidine-containing pep-tides has already been reported [33]. Although the

turnover number of ten is relatively small, the achieve-ment of catalysis is important. This result supports theprinciple that simple metal complexes can indeed act asartificial metallopeptidases. Their design remains ourultimate goal.

Acknowledgements We thank National Natural Science Founda-tion of China and Natural Science Foundation of Jiangsu Prov-ince, and the United States National Science Foundation (grantCHE-9404971 to N.M.K.) for financial support.

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