Combined use of platinum(II) complexes and palladium(II) complexes for selective cleavage of peptides and proteins

This study shows, for the first time, the advantages of combining two transition-metal complexes as selective proteolytic reagents. In this procedure, cis-[Pt(en)(H(2)O)(2)](2+) is followed by [Pd(H(2)O)(4)](2+). In the peptide AcAla-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala, the Pt(II) reagent cleaves the Met6-Ala7 peptide bond, whereas the Pd(II) reagent cleaves the Gly4-Gly5 bond. In the peptide AcVal-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala, the Pt(II) reagent cleaves the Met11-Ala12 peptide bond, whereas the Pd(II) reagent cleaves the Gly3-Gly4 bond. All cleavage reactions are regioselective and complete at pH 2.0 and 60 degrees C. Each metal ion binds to an anchoring side chain and then, as a Lewis acid, activates a proximal peptide bond toward hydrolysis by the solvent water. The selectivity in cleavage is a consequence of the selectivity in this initial anchoring. Both Pt(II) and Pd(II) reagents bind to the methionine side chain, whereas only the Pd(II) reagent binds to the histidine side chain under the reaction conditions. Consequently, only methionine residues direct the cleavage by the Pt(II) reagent, whereas both methionine and histidine residues direct the cleavage by the Pd(II) reagent. The Pt(II) reagent cleaves the first bond downstream from the anchor, i.e., the Met-Z bond. The Pd(II) reagent cleaves the second bond upstream from the anchor, i.e., the X-Y bond in the X-Y-Met-Z and in the X-Y-His-Z segments. The diethylenetriamine complex [Pt(dien)(H(2)O)](2+) cannot promote cleavage. Its prior binding to the Met11 residue in the second peptide prevents the Pd(II) reagents from binding to Met11 and cleaving the Gly9-Gly10 bond and directs the cleavage by the Pd(II) reagent exclusively at the Gly3-Gly4 bond. Our new method was tested on equine myoglobin, which contains 2 methionine residues and 11 histidine residues. The complete methionine-directed cleavage of the Met55-Lys56 and Met131-Thr132 bonds by the Pt(II) reagent produced three fragments, suitable for various biochemical applications because they are relatively long and contain amino and carboxylic terminal groups. The deliberately incomplete histidine-directed cleavage of the long fragments 1.55 and 56.131 at many sites by the Pd(II) reagent produced numerous short fragments, suitable for protein identification by mass spectrometry. The ability of combined Pt(II) and Pd(II) complexes to cleave proteins with explicable and adjustable selectivity and with good yields bodes well for their greater use in biochemical and bioanalytical practice.     

Palladium(II) complexes, as synthetic peptidases, regioselectively cleave the second peptide bond "upstream" from methionine and histidine side chains

Palladium(II) complexes promote hydrolysis of natural and synthetic oligopeptides with unprecedented regioselectivity; the only cleavage site is the second peptide bond upstream from a methionine or a histidine side chain, that is, the bond involving the amino group of the residue that precedes this side chain. We investigate this regioselectivity with four N-acetylated peptides as substrates: neurotransmitter methionine enkephalin (Ac-Tyr-Gly-Gly-Phe-Met) and synthetic peptides termed Met-peptide (Ac-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala), His-peptide (Ac-Val-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met(OX)-Ala-Ala-Arg-Ala), in which a Met is oxidized to sulfone, and HisMet-peptide (Ac-Val-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala). While maintaining protein-like properties, these substrates are suitable for quantitative study since their coordination to Pd(II) ion can be determined (by NMR spectroscopy), and the cleavage fragments can be separated (by HPLC methods) and identified (by MALDI mass spectrometry). The only peptide bonds cleaved were the Gly3-Phe4 bond in methionine enkephalin, Gly4-Gly5 bond in Met-peptide, Gly3-Gly4 in His-peptide, and Gly3-Gly4 and Gly9-Gly10 bonds in HisMet-peptide. We explain this consistent regioselectivity of cleavage by studying the modes of Met-peptide coordination to the Pd(II) ion in [Pd(H(2)O)(4)](2+) complex. In acidic solution, the rapid attachment of the Pd(II) complex to the methionine side chain is followed by the interaction of the Pd(II) ion with the peptide backbone upstream from the anchor. In the hydrolytically active complex, Met-peptide is coordinated to Pd(II) ion as a bidentate ligand - via sulfur atom in the methionine side chain and the first peptide nitrogen upstream from this anchor - so that the Pd(II) complex approaches the scissile peptide bond. Because the increased acidity favors this hydrolytically active complex, the rate of cleavage guided by either histidine or methionine anchor increased as pH was lowered from 4.5 to 0.5. The unwanted additional cleavage of the first peptide bond upstream from the anchor is suppressed if pH is kept above 1.2. Four Pd(II) complexes cleave Met-peptide with the same regioselectivity but at somewhat different rates. Complexes in which Pd(II) ion carries labile ligands, such as [Pd(H(2)O)(4)](2+) and [Pd(NH(3))(4)](2+), are more reactive than those containing anionic ligands, such as [PdCl(4)](2)(-), or a bidentate ligand, such as cis-[Pd(en)(H(2)O)(2)](2+). When both methionine and histidine residues are present in the same substrate, as in HisMet-peptide, 1 molar equivalent of the Pd(II) complex distributes itself evenly at both anchors and provides partial cleavage, whereas 2 molar equivalents of the promoter completely cleave the second peptide bond upstream from each of the anchors. The results of this study bode well for growing use of palladium(II) reagents in biochemical and bioanalytical practice.     

Palladium(II) complex as a sequence-specific peptidase: hydrolytic cleavage under mild conditions of X-Pro peptide bonds in X-Pro-Met and X-Pro-His segments

The X-Pro peptide bond (in which X represents any amino acid residue) in peptides and proteins is resistant to cleavage by most proteolytic enzymes. We show that [Pd(H(2)O)(4)](2+) ion can selectively hydrolyze this tertiary peptide bond within the X-Pro-Met and X-Pro-His sequence segments. The hydrolysis requires an equimolar amount of the Pd(II) reagent and occurs under mild conditions-at temperature as low as 20 degrees C (with half-life of 1.0 h at pH 2.0) and at pH as high as 7.0 (with half-life of 4.2 h at pH 7.0 and 40 degrees C). The secondary peptide bond, exemplified by X-Gly in the X-Gly-Met and X-Gly-His sequence segments, however, is cleaved only in weakly acidic solution (pH < 4.0) and more slowly (half-life is 4.2 h at pH 2.0 and 60 degrees C). We explain the sequence-specificity of X-Pro cleavage by NMR spectroscopic analysis of the coordination of the X-Pro-Met segment to the Pd(II) ion. We give indirect evidence for the mechanism of cleavage by analyzing the conformation of the scissile X-Pro peptide bond, and by comparing the rate constants for the cleavage of the tertiary X-Pro peptide bond, the tertiary X-Sar peptide bond (Sar is N-methyl glycine), and the typical secondary X-Gly peptide bond in a set of analogous oligopeptides. Methionine and histidine side chains provide the recognition by selectively binding (anchoring) the Pd(II) ion. The proline residue provides the enhanced activity because its tertiary X-Pro peptide bond favors the cleavage-enhancing binding of the Pd(II) ion to the peptide oxygen atom and prevents the cleavage-inhibiting binding of the Pd(II) ion upstream of the anchoring (histidine or methionine) residue. Cleavage can be switched from the residue-selective to the sequence-specific mode by simply adjusting the pH of the aqueous solution. In acidic solutions, any X-Y bond in X-Y-Met and X-Y-His segments is cleaved because the cleavage is directed by anchoring methionine and histidine residues. In mildly acidic and neutral solutions, only the X-Pro bond in X-Pro-Met and X-Pro-His sequences is cleaved because of an interplay between the anchoring residue and the proline residue preceding it. Because Pro-Met and Pro-His sequences are rare in proteins, this sequence-specific cleavage is potentially useful for the removal of the fusion tags from the bioengineered fusion proteins.     

Gas-phase investigation of Pd(II)-alanine complexes with small native and derivatized peptides containing histidine

We report here the generation of gas-phase complexes containing Pd(II), a ligand (deprotonated alanine, A-), and/or N-terminus derivatized peptides containing histidine as one of the amino acids. The species were produced by electrospray ionization, and their gas-phase reactions were investigated using ion-trap tandem mass spectrometry. Pd(II) forms a stable diaqua complex in the gas phase of the formula, [Pd(A-) (H(2)O)(2)]+, (where A- = deprotonated alanine) along with ternary complexes containing A- and peptide. The collision-induced dissociation (CID) patterns of the binary and ternary complexes were investigated, and the dissociation patterns for the ternary complexes suggest that: (a) the imidazole ring of the histidine side group may be the intrinsic binding site of the metal ion, and (b) the peptides fragment primarily by cleavage of the amide bond to the C-terminal side of the histidine residues. These observations are in accord with previous solution-state studies in which Pd(II) was shown to cause hydrolysis of an amide bond of a peptide at the same position.     

Hydrolysis of amide bond in histidine-containing peptides promoted by chelated amino acid palladium(II) complexes: dependence of hydrolytic pathway on the coordination modes of the peptides

Hydrolytic reactions between cis-[Pd( -Ala-N,O)Cl₂]⁻ and cis-[Pd( -Ala-N,O)(H₂O)₂]⁺, in which -Ala is alanine coordinated through N and O atoms, and N-acetylated peptides -histidylglycine (MeCO-His-Gly), glycyl- -histidine (MeCO-Gly-His), glycylglycyl- -histidine (MeCO-Gly-Gly-His) and glycyl- -histidylglycine (MeCO-Gly-His-Gly) were studied by ¹H NMR spectroscopy. All reactions were carried out in the pH range 2.0–2.5 and two different temperatures, 22 and 60°C. In the reactions of these two palladium(II) complexes with MeCO-His-Gly, complete hydrolysis of the amide bond involving carboxylic group of histidine occurs in less than 24 h. The cleavage is regioselective. With peptides containing free a carboxylic group of histidine, MeCO-Gly-His and MeCO-Gly-Gly-His, palladium(II) complex promote the cleavage of the MeCO–Gly and Gly–Gly amide bonds. No cleavage of the Gly–His amide bond was observed. The mechanism of these hydrolytic reactions involves release of -Ala ligand and aquation of the palladium(II) complex chelated to the substrate through the imidazole N-3 atom and deprotonated nitrogen atom of the amide bond involving amino group of histidine. This aqua complex represents a catalytically active form different from the initially added catalytically inactive complex. In the reactions of palladium(II) complexes with tripeptide MeCO-Gly-His-Gly, two amide bonds, MeCO–Gly and His–Gly, were cleaved. The mechanism of the cleavage of these amide bonds is correlated with two different palladium(II)–substrate catalytically active forms. These findings contribute to the better understanding of selective cleavage of peptides and proteins and must be taken into consideration in designing new reagents for this purpose.     

Transition-metal complexes as enzyme-like reagents for protein cleavage: complex cis-[Pt(en)(H2O)2]2+ as a new methionine-specific protease

Complex cis-[Pt(en)(H(2)O)(2)](2+) promotes selective hydrolytic cleavage of two proteins, horse cytochrome c and bovine beta-casein. The cleavage is completed in 24 h under relatively mild conditions, at about pH 2.5, and a temperature as low as 40 degrees C. The results of HPLC and TSDS PAGE separations, MALDI mass spectrometry, and Edman sequencing showed that cleavage occurred exclusively at the peptide bond involving the C-terminus of each methionine residue, both such residues in cytochrome c and all six such residues in beta-casein. While having the same selectivity as cyanogen bromide (CNBr), the most common chemical protease, cis-[Pt(en)(H(2)O)(2)](2+) has several advantages. It is nonvolatile, easy to handle, and recyclable. Its cleavage is residue-selective, the rest of the polypeptide backbone remains intact, and the other side chains remain unmodified. It is applied in approximately equimolar amounts with respect to methionine residues, creates free amino and carboxylic groups, and cleaves even the Met-Pro bond, which is resistant to CNBr and most proteolytic enzymes. Finally the complex also works in the presence of the denaturing reagent sodium dodecyl sulfate. Experiments with the synthetic peptides, AcAla-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed Met-peptide) and AcVal-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed HisMet-peptide) as substrates, revealed structural and mechanistic features of the proteolytic reactions. We explain why two similar complexes with similar metal ions, cis-[Pt(en)(H(2)O)(2)](2+) and cis-[Pd(en)(H(2)O)(2)](2+), differ in selectivity as proteolytic reagents. The selectivity of cleavage is governed by the selectivity of the cis-[Pt(en)(H(2)O)(2)](2+) binding to the methionine side chain. The proteolytic activity is governed by the modes of coordination, which control the approach of the anchored Pt(II) ion to the scissile peptide bond. The cleavage occurs with a small, but significant, catalytic turnover of more than 18 after 7 days. The ability of cis-[Pt(en)(H(2)O)(2)](2+) to cleave proteins at relatively few sites, with explicable selectivity and catalytic turnover, bodes well for its use in biochemical practice.     

Interaction of palladium(II) and platinum(II) complexes with microperoxidase-11 studied by electrospray mass spectrometry and MS/MS analysis

Interactions of cis-[Pd(en)(H(2)O)(2)](2+) (en, ethylenediamine) and cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) with microperoxidase-11 (MP-11) in a molar ratio of 1:1 or 2:1 at pH 1.4 were investigated via electrospray mass spectrometry and MS/MS analysis at room temperature and at 40 degrees C with an incubation time of 2 or 3 days. The composition of the Pd(II)- and Pt(II)-anchored MP-11 was confirmed on the basis of the precise molecular mass and the simulated isotope distribution pattern. MS/MS analysis revealed that the Pd(II) center anchored to the side chain of Cys7 as Pd(II) and MP-11 were mixed in an equimolar ratio and to side chains of Cys7 and Cys4 as Pd(II) and MP-11 mixed in a 2:1 molar ratio. When Pt(II) and MP-11 were mixed in a 2:1 molar ratio, Pt(II) first anchored to the side chain of Cys7, and then to the side chain of Cys4 with time. The initial coordination of Pd(II) and Pt(II) to the side chain of Cys7 is the essential step for the Pd(II)- and Pt(II)-promoted cleavage of the His8-Thr9 bond in MP-11. These results support the hypothesis that the Pd(II)-mediated cleavage of the His18-Thr19 bond in cytochorome c is due to the identical binding mode.     

Conjugate of palladium(II) complex and beta-cyclodextrin acts as a biomimetic peptidase

We combined the newly discovered ability of [Pd(H2O)4]2+ to residue-selectively hydrolyze X-Pro bonds in peptides at 6 相似文献   

Products of Cu(II)-catalyzed oxidation of alpha-synuclein fragments containing M1-D2 and H50 residues in the presence of hydrogen peroxide

Metal-catalyzed oxidation (MCO) of proteins is mainly a site-specific process in which one or a few amino acids at metal-binding sites on the protein are preferentially oxidized. The oxidation of proteins by MCO can lead to oxidation of amino acid residue side chains, cleavage of the peptide bonds and formation of covalent protein-protein cross-linked derivatives. In an attempt to elucidate the products of the copper(II)-catalyzed oxidation of the 29-56, M29-D30-56 and Ac-M29-D30-56 fragments of alpha-synuclein, high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. The peptide solution (0.50 mM) was incubated at 37 degrees C for 24 h with metal : peptide : hydrogen peroxide 1 : 1 : 4 molar ratio in phosphate buffer, pH 7.4. Oxidation targets for all studied peptides are the histidine residues coordinated to the metal ions. For the M29-D30-56 and Ac-M29-D30-56 peptides the oxidation of the methionine residue to methionine sulfoxide and sulfone is observed. The cleavage of the peptide bond M29-D30 for the M29-D30-56 peptide was detected as metal binding residues. The fragmentations of the M29-D30-56 peptide near the Lys residues were observed supporting the participation of this (Lys) residue in the coordination of the copper(II) ions.     

Platinum(II) complex as an artificial peptidase: selective cleavage of peptides and a protein by cis-[Pt(en)(H2O)2]2+ ion under ultraviolet and microwave irradiation

Two synthetic peptides were completely cleaved by the cis-[Pt(en)(H2O)2]2+ (en is ethylenediamine) complex at pH 2.5 under thermal heating at 60 degrees C in a selective way: only the amide bonds involving the carboxylic group of the methionine residue, i.e., the Met-Z bonds (where the residue Z has a noncoordinating side chain), were hydrolyzed. Under irradiation at 300 nm, the rate constants for these cleavage reactions were approximately doubled, but side reactions occurred. Under microwave irradiation, the rate constants were increased 2-3 times at 60 degrees C and ca. 7 times at 100 degrees C, and no side reactions were detected. Microwave irradiation similarly accelerated the complete and selective cleavage of Met-Z bonds in cytochrome c at 60 degrees C in comparison with this cleavage under thermal heating, again without detected side reactions. The microwave-assisted cleavage of peptides and proteins by the platinum(II) reagent holds promise in proteomics and other biotechnological applications.     

The effect of histidine oxidation on the dissociation patterns of peptide ions

Oxidative modifications to amino acid side chains can change the dissociation pathways of peptide ions, although these variations are most commonly observed when cysteine and methionine residues are oxidized. In this work we describe the very noticeable effect that oxidation of histidine residues can have on the dissociation patterns of peptide ions containing this residue. A common product ion spectral feature of doubly charged tryptic peptides is enhanced cleavage at the C-terminal side of histidine residues. This preferential cleavage arises as a result of the unique acid/base character of the imidazole side chain that initiates cleavage of a proximal peptide bond for ions in which the number of protons does not exceed the number of basic residues. We demonstrate here that this enhanced cleavage is eliminated when histidine is oxidized to 2-oxo-histidine because the proton affinity and nucleophilicity of the imidazole side chain are lowered. Furthermore, we find that oxidation of histidine to 2-oxo-histidine can cause the misassignment of oxidized residues when more than one oxidized isomer is simultaneously subjected to tandem mass spectrometry (MS/MS). These spectral misinterpretations can usually be avoided by using multiple stages of MS/MS (MS(n)) or by specially optimized liquid chromatographic separation conditions. When these approaches are not accessible or do not work, N-terminal derivatization with sulfobenzoic acid avoids the problem of mistakenly assigning oxidized residues.     

Synergic effect of two metal centers in catalytic hydrolysis of methionine-containing peptides promoted by dinuclear palladium(II) hexaazacyclooctadecane complex

The species obtained by the reaction of [Pd2([18]aneN6)Cl2](ClO4)2(where [18]aneN6 is 1,4,7,10,13,16-hexaazacyclooctadecane) with AgBF4 have been determined by electrospray ionization mass spectrometry (ESI-MS) to be an equilibrium mixture of three major types of dinuclear Pd(II) complex cations, [Pd2(mu-O)([18]aneN6)]2+, [Pd2(mu-OH)([18]aneN6)]3+ and [Pd2(H2O)(OH)([18]aneN6)](3+), in aqueous solution. The hydroxo-group-bridged one, [Pd2(mu-OH)([18]aneN6)]3+, is a dominant species, whose crystal structure has been obtained. The crystal structure of [Pd2(mu-OH)([18]aneN6)](ClO4)3 shows that each Pd(II) ion in the dinuclear complex is tetra-coordinated by three nitrogen atoms and one hydroxo group bridge in a distorted square configuration. The two Pd(II) ions are 3.09 A apart from each other. The dinuclear Pd(II) complex cations [Pd2(mu-OH)([18]aneN6)]3+ and [Pd2(H2O)(OH)([18]aneN6)]3+ can efficiently catalyze hydrolysis of the amide bond involving the carbonyl group of methionine in methionine-containing peptides with turnover number of larger than 20. In these hydrolytic reactions, the two Pd(II) ions are synergic; one Pd(II) ion anchors to the side chain of methionine and the other one delivers hydroxo group or aqua ligand to carbonyl carbon of methionine, or acts as a Lewis acid to activate the carbonyl group of methionine, resulting in cleavage of Met-X bond. The binding constant of dinuclear Pd(II) complex cations with AcMet-Gly and AcMet were determined by 1H NMR titration to be 282 +/- 2 M(-1) and 366 +/- 4 M(-1), respectively. The relatively low binding constants enable the catalytic cycle and the possible catalytic mechanism is proposed. This is the first artificial mimic of metallopeptidases with two metal active centers.     

Side chain dependence of intensity and wavenumber position of amide I' in IR and visible Raman spectra of XA and AX dipeptides

A series of AX and XA dipeptides in D2O have been investigated by FTIR, isotropic, and anisotropic Raman spectroscopy at acidic, neutral, and alkaline pD, to probe the influence of amino acid side chains on the amide I' band. We obtained a set of spectral parameters for each peptide, including intensities, wavenumbers, half-widths, and dipole moments, and found that these amide I' parameters are indeed dependent on the side chain. Side chains with similar characteristic properties were found to have similar effects on the amide I'. For example, dipeptides with aliphatic side chains were found to exhibit a downshift of the amide I' wavenumber, while those containing polar side chains experienced an increase in wavenumber. The N-terminal charge causes a substantial upshift of amide I', whereas the C-terminal charge causes a moderate decrease of the transition dipole moment. Density functional theory (DFT) calculations on the investigated dipeptides in vacuo yielded different correlations between theoretically and experimentally obtained wavenumbers for aliphatic/aromatic and polar/charged side chains, respectively. This might be indicative of a role of the hydration shell in transferring side chain-backbone interactions. For Raman bands, we found a correlation between amide I' depolarization ratio and wavenumber which reflects that some side chains (valine, histidine) have a significant influence on the Raman tensor. Altogether, the obtained data are of utmost importance for utilizing amide I as a tool for secondary structure analysis of polypeptides and proteins and providing an experimental basis for theoretical modeling of this important backbone mode. This is demonstrated by a rather accurate modeling for the amide I' band profiles of the IR, isotropic Raman, and anisotropic Raman spectra of the beta-amyloid fragment Abeta(1-82).     

Solid-phase and solution-phase syntheses of oligomeric guanidines bearing peptide side chains

[reaction: see text] Synthetic strategies for preparing N,N'-bridged oligomeric guanidines bearing peptide side chains both on solid support and in solution are presented. Monomers are prepared from common alpha-amino acids and therefore contain conventionally protected peptide side chains. The side chains include alkyl, aromatic, hydroxyl, amino, carboxylic acid, and amide functional groups. Oligomer elongation utilizes acid-sensitive sulfonyl activated thiourea through the formation of carbodiimide intermediate. With proper preparation of monomers, synthesis of oligomer can be performed in two directions (equivalent to N to C terminal or C to N terminal in a peptide sequence) with excellent efficiency.     

New selectivity in peptide hydrolysis by metal complexes. Platinum(II) complexes promote cleavage of peptides next to the tryptophan residue

Tryptophan-containing N-acetylated peptides AcTrp-Gly, AcTrp-Ala, AcTrp-Val, and AcTrp-ValOMe bind to platinum(II) and undergo selective hydrolytic cleavage of the C-terminal amide bond; the N-terminal amide bond remains intact. In acetone solution, bidentate coordination of the tryptophanyl residue via the C(3) atom of indole and the amide oxygen atom produces complexes of spiro stereochemistry, which are characterized by (1)H, (13)C, and (195)Pt NMR spectroscopy, and also by UV-vis, IR, and mass spectroscopy. Upon addition of 1 molar equiv of water, these complexes undergo hydrolytic cleavage. This reaction is as much as 10(4)-10(5) times faster in the presence of platinum(II) complexes than in their absence. The hydrolysis is conveniently monitored by (1)H NMR spectroscopy. We report the kinetics and mechanism for this reaction between cis-[Pt(en)(sol)(2)](2+), in which the solvent ligand is water or acetone, and AcTrp-Ala. The platinum(II) ion as a Lewis acid activates the oxygen-bound amide group toward nucleophilic attack of solvent water. The reaction is unimolecular with respect to the metal-peptide complex. Because the tryptophanyl fragment AcTrp remains coordinated to platinum(II) after cleavage of the amide bond, the cleavage is not catalytic. Added ligand, such as DMSO and pyridine, displaces AcTrp from the platinum(II) complex and regenerates the promoter. This is the first report of cleavage of peptide bonds next to tryptophanyl residues by metal complexes and one of the very few reports of organometallic complexes involving metal ions and peptide ligands. Because these complexes form in nonaqueous solvents, a prospect for cleavage of membrane-bound and other hydrophobic proteins with new regioselectivity has emerged.     

Characterization of amino acid side chain losses in electron capture dissociation

We have used electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry to characterize amino acid side chain losses observed during electron capture dissociation (ECD) of ten 7- to 14-mer peptides. Side-chain cleavages were observed for arginine, histidine, asparagine or glutamine, methionine, and lysine residues. All peptides containing an arginine, histidine, asparagine or glutamine showed the losses associated with that residue. Methionine side-chain loss was observed for doubly-protonated bombesin. Lysine side-chain loss was observed for triply-protonated dynorphin A fragment 1-13 but not for the doubly-protonated ion. The proximity of arginine to a methoxy C-terminal group significantly enhances the extent of side-chain fragmentation. Fragment ions associated with side-chain losses were comparable in abundance to those resulting from backbone cleavage in all cases. In the ECD spectrum of one peptide, the major product was due to fragmentation within an arginine side chain. Our results suggest that cleavages within side chains should be taken into account in analysis of ECD mass spectral data. Losses from arginine, histidine, and asparigine/glutamine can be used to ascertain their presence, as in the analysis of unknown peptides, particularly those with non-linear structures.     

Regioselective cleavage of myoglobin with copper(II) compounds at neutral pH

Selective hydrolytic cleavage of myoglobin was studied with CuCl2, Cu(ClO4)2, Cu(AC)2, and binuclear Cu(II) complexes of 3,6,9,16,19,22-hexaaza-6,19-bis (2-hydroxyethyl)-tricyclo- [22,2,2,2(11,14)]-triaconta-1,11,13,24,27,29-hexaene (1) and 3,6,9,16,19,22-hexaaza-tricyclo-[22,2,2,2(11,14)]-triaconta- 1,11,13,24,27,29-hexaene (2). The sites of cleavage were precisely determined by LC-ESIMS and further confirmed by an MS/MS method through fragmentation from both the N-terminal and C-terminal. The peptide bonds of Gln91-Ser92 and Ala94-Thr95 were remarkably cleaved by Cu(II) anchored to the side chain of the His93 residue. The data presented in this study show that Cu(II)-mediated cleavage of myoglobin is able to proceed at neutral pH, more selectively than Pd(II)-mediated cleavage, and buffer solution of phosphate and NH4HCO3 accelerates the cleavage reaction.     

炔基苯甲酰胺衍生物的液相合成

液相合成兼容了固相合成可快速简便地对产物进行分离纯化以及溶液相合成可 在均相条件下进行反应和用常规手段对反应中间体进行分析检测的优点，用聚乙二 醇(PEG)4000作为可溶性聚合物支持体，通过醚键将[1，3，5]氯三嗪连接在PEG上 制备成PEG支持的新型的可溶性聚合物试剂I．该试剂在N-甲基吗啉的作用下与对碘 苯甲酸反应生成相应的活性酯中间体Ⅱ，该中间体继而在Pd(Ⅱ)／Cu(Ⅰ)共催化下 与端炔发生Sonogashira偶联反应得到另一中间体Ⅲ，在伯胺或仲胺的存在下进行 胺解反应得到炔基苯甲酰胺衍生物Ⅳ．由于PEG具有柔韧的长链而不会对链端连接 的反应中心的活性产生影响，因此该反应在均相体系中进行，不仅反应条件温和， 产率良好，而且反应还具有良好的选择性．实验发现，芳香胺和非芳香胺均可获得 良好的反应结果，但芳香胺所需的反应时间较长；伯胺和仲胺都是良好的胺解试剂 ，但使用仲胺时产率较低；N原子亲核试剂比O原子亲核试剂具有更大的反应性．此 外，PEG 4000支持的[1，3，5]氯三嗪试剂没有强烈的刺激性，可长期储存使用．     

Synthesis and Characterization of the 2‐Methylaminopyridine‐functionalized Polystyrene Resin‐supported Pd(II) Catalyst for the Mizoroki–Heck and Sonogashira Reactions in Water

A new polystyrene‐anchored Pd(II) pyridine complex is synthesized and characterized. This Pd(II) pyridine complex behaves as a very efficient heterogeneous catalyst in the Heck reaction of methyl acrylate with aryl halides and the Sonogashira reaction of terminal alkynes with aryl halides in water. Furthermore, the catalyst shows good thermal stability and recyclability. This polymer‐supported Pd(II) catalyst could easily be recovered by simple filtration of the reaction mixture and reused for more than five consecutive trials without a significant loss in its catalytic activity.     

Heterogeneous Suzuki and copper-free Sonogashira cross-coupling reactions catalyzed by a reusable palladium(II) complex in water medium

A new polystyrene anchored Pd(II) azo complex has been synthesized and characterized. The present Pd(II) azo complex behaves as a very efficient heterogeneous catalyst in the Suzuki coupling and Sonogashira coupling reaction in water medium. Aryl halides, coupled with phenylboronic acids (Suzuki-Miyaura reaction) or terminal alkyne (Sonogashira reaction), smoothly afford the corresponding cross-coupling products in excellent yields (83-100% yield for Suzuki reaction and 68-96% yield for Sonogashira reaction of aryl halides) under phosphine-free reaction conditions in the presence of polystyrene anchored Pd(II) azo complex catalyst in water medium. Furthermore, the catalyst has shown good thermal stability and recyclability. This polymer-supported Pd(II) catalyst could be easily recovered by simple filtration of the reaction mixture and reused for more than six consecutive trials without a significant loss of its catalytic activity.