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PubMed2002

Crystal structure of the dinuclear zinc aminopeptidase PepV from Lactobacillus delbrueckii unravels its preference for dipeptides.

PepV from Lactobacillus delbrueckii, a dinuclear zinc peptidase, has been characterized as an unspecific amino dipeptidase. The crystal structure of PepV in complex with the phosphinic inhibitor AspPsi[PO(2)CH(2)]AlaOH, a dipeptide substrate mimetic, reveals a "catalytic domain" and a "lid domain," which together form an internal active site cavity that traps the inhibitor. The catalytic domain is topologically similar to catalytic domains from amino- and carboxypeptidases. However, the lid domain is unique among the related enzymes. In contrast to the other related exopeptidases, PepV recognizes and fixes the dipeptide backbone, while the side chains are not specifically probed and can vary, rendering it a nonspecific dipeptidase. The cocrystallized inhibitor illustrates the two roles of the two catalytic zinc ions, namely stabilization of the tetrahedral intermediate and activation of the catalytic water molecule.

Amino Acid Sequence↗
PubMed2002

Familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase are susceptible to disulfide reduction.

We observed that 14 biologically metallated mutants of copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis all exhibited aberrantly accelerated mobility during partially denaturing PAGE and increased sensitivity to proteolytic digestion compared with wild type SOD1. Decreased metal binding site occupancy and exposure to the disulfide-reducing agents dithiothreitol, Tris(2-carboxyethyl)phosphine (TCEP), or reduced glutathione increased the fraction of anomalously migrating mutant SOD1 proteins. Furthermore, the incubation of mutant SOD1s with TCEP increased the accessibility to iodoacetamide of cysteine residues that normally participate in the formation of the intrasubunit disulfide bond (Cys-57 to Cys-146) or are buried within the core of the beta-barrel (Cys-6). SOD1 enzymes in spinal cord lysates from G85R and G93A mutant but not wild type SOD1 transgenic mice also exhibited abnormal vulnerability to TCEP, which exposed normally inaccessible cysteine residues to modification by maleimide conjugated to polyethylene glycol. These results implicate SOD1 destabilization under cellular disulfide-reducing conditions at physiological pH and temperature as a shared property that may be relevant to amyotrophic lateral sclerosis mutant neurotoxicity.

Amino Acid Substitution↗
PubMed1996

A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A simple methodology has been developed to characterize the number and location of free cysteine and cystine groups in peptides and proteins, using chemical modification and matrix-assisted laser desorption/ ionization time-of flight mass spectrometry (MALDITOF MS). This new approach employs a specific reaction between free sulfhydryls and 2-nitro-5-thiocyanobenzoic acid (NTCB) to selectively cyanylate cysteine thiols. The N-terminal peptide bond of the modified cysteinyl residue can then be cleaved under alkaline conditions to form an amino-terminal peptide and a series of 2-iminothiazolidine-4-carboxylyl peptides which can be mapped to the sequence by MALDI-MS. The number and location of cysteines can be deduced from mass analysis of the peptide mixture resulting from NTCB chemical processing of the original protein of known sequence under nonreducing conditions. Similar experiments are then performed following disulfide bond reduction to further characterize both cysteine and cystine groups. Experimental conditions are described for protein disulfide bond reduction, sulfhydryl cyanylation, and cleavage reactions performed both in solution and on Zetabind membranes.

Cysteine↗
PubMed1992

Peptide mapping of complex proteins at the low-picomole level with capillary electrophoretic separations.

A variety of different peptide-mapping schemes are presented, with emphasis on the development of procedures which can be done with limited quantities (i.e. 5 pmol) of protein. Results are obtained from model proteins which contain disulfide bonds, which must be broken prior to fragmentation of the protein. A reaction involving the simultaneous use of tributylphosphine and 2-methylaziridine to reduce and alkylate the disulfide bonds is employed, due to favorable attributes of these reagents for the scaled-down procedure. The traditional performic acid oxidation reaction to cleave cystine groups is also successfully used with low-picomole quantities of protein. Three different protein digestion reagents are used: trypsin, chymotrypsin, and cyanogen bromide. Each reagent produces a unique mixture of peptides. Capillary electrophoresis is used to separate the peptides, offering high separation efficiencies, short analysis times, and compatibility with small sample sizes. In addition to the conventional use of UV detection for underivatized peptides, laser-induced fluorescence detection is employed in conjunction with an arginine-selective derivatization reaction. This latter procedure for derivatization and detection offers an alternative peptide-mapping mode, in which only the arginine-containing peptides are detected, and is useful in simplifying the peptide maps of large proteins.

Alkylation↗
PubMedDate not supplied

Determination of traces of iron in indium phosphide by electrothermal atomic absorption spectrometry combined with solvent extraction.

An electrothermal atomic absorption (ETAAS) method for the determination of traces of iron (0.1-1.0 microgram g-1) in Fe-doped indium phosphide (InP) has been developed. In order to overcome the indium matrix-effect and to achieve a useful detection limit, a preliminary solvent-extraction of Fe(III) with acetylacetone (HAA) is necessary. After sample dissolution with hydrochloric acid (1 + 1) the digest is evaporated to dryness, Fe(II) is oxidized to Fe(III) with nitric acid, the residue is dissolved in 0.01 mol L-1 HCl and the iron is extracted at pH 2.0 with 0.5 mol L-1 HAA in toluene. The organic phase is injected into the graphite furnace and the iron is directly evaluated by external organic standard calibration. The limit of detection (3SB) resulting from further in-situ preconcentration is 0.03 microgram g-1. When the method was applied to the analysis of real samples containing 0.2-0.7 microgram g-1 Fe, the RSD was in the range 8-21%. Results were compared with those independently obtained on the decomposed sample solution with inductively coupled atomic emission spectrometry (ICP-AES). The detection limit of the ICP-AES method, that needs matrix-matched standards, is 0.20 microgram g-1.

Environmental Monitoring↗
PubMed2006

A highly active palladium catalyst for intermolecular hydroamination. Factors that control reactivity and additions of functionalized anilines to dienes and vinylarenes.

We report a catalyst for intermolecular hydroamination of vinylarenes that is substantially more active for this process than catalysts published previously. With this more reactive catalyst, we demonstrate that additions of amines to vinylarenes and dienes occur in the presence of potentially reactive functional groups, such as ketones with enolizable hydrogens, free alcohols, free carboxylic acids, free amides, nitriles, and esters. The catalyst for these reactions is generated from [Pd(eta(3)-allyl)Cl](2) (with or without added AgOTf) or [Pd(CH(3)CN)(4)](BF(4))(2) and Xantphos (9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene), which generates complexes with large P-Pd-P bite angles. Studies on the rate of the C-N bond-forming step that occurs by attack of amine on an eta(3)-phenethyl and an eta(3)-allyl complex were conducted to determine the effect of the bite angle on the rate of this nucleophilic attack. Studies on model eta(3)-benzyl complexes containing various bisphosphines showed that the nucleophilic attack was faster for complexes containing larger P-Pd-P bite angles. Studies of substituted unsymmetrical and unsubstituted symmetrical model eta(3)-allyl complexes showed that nucleophilic attack on complexes ligated by Xantphos was faster than on complexes bearing ligands with smaller bite angles and that nucleophilic attack on unsymmetrical allyl complexes with larger bite angle ligands was faster than on unsymmetrical allyl complexes with smaller bite angle ligands. However, monitoring of catalytic reactions of dienes by (31)P NMR spectroscopy showed that the concentration of active catalyst was the major factor that controlled rates for reactions of symmetrical dienes catalyzed by complexes of phosphines with smaller bite angles. The identity of the counterion also affected the rate of attack: reactions of allylpalladium complexes with chloride counterion occurred faster than reactions of allylpalladium complexes with triflate or tetrafluoroborate counterion. As is often observed, the dynamics of the allyl and benzyl complexes also depended on the identity of the counterion.

Alkenes↗
PubMed2002

Characterizing closely spaced, complex disulfide bond patterns in peptides and proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Identifying the Cys residues involved in disulfide linkages of peptides and proteins that contain complex disulfide bond patterns is a significant analytical challenge. This is especially true when the Cys residues involved in the disulfide bonds are closely spaced in the primary sequence. Peptides and proteins that contain free Cys residues located near disulfide bonds present the additional problem of disulfide shuffling via the thiol-disulfide exchange reaction. In this paper, we report a convenient method to identify complex disulfide patterns in peptides and proteins using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with partial reduction by tris(2-carboxyethyl)phosphine (TCEP). The method was validated using well-characterized peptides and proteins including endothelin, insulin, alpha-conotoxin SI and immunoglobulin G (IgG2a, mouse). Peptide or protein digests were treated with TCEP in the presence of an alkylation reagent, maleimide-biotin (M-biotin) or N-ethylmaleimide (NEM), followed by complete reduction with dithiothreitol and alkylation by iodoacetamide (IAM). Subsequently, peptides that contained alkylated Cys were analyzed by capillary LC/ESI-MS/MS to determine which Cys residues were modified with M-biotin/NEM or IAM. The presence of the alkylating reagent (M-biotin or NEM) during TCEP reduction was found to minimize the occurrence of the thiol-disulfide exchange reaction. A critical feature of the method is the stepwise reduction of the disulfide bonds and the orderly, sequential use of specific alkylating reagents.

Alkylation↗
PubMed1991

Determination of 2-mercaptopropionylglycine and its metabolite, 2-mercaptopropionic acid, in plasma by ion-pair reversed-phase high-performance liquid chromatography with post-column derivatization.

A simple and fast high-performance liquid chromatographic method was developed for the simultaneous measurement of 2-mercaptopropionylglycine (Tiopronine) and its metabolite (2-mercaptopropionic acid) in human plasma after the administration of a pharmaceutical dosage form (Acadione). The sample treatment before high-performance liquid chromatographic analysis consisted of the reduction of the corresponding disulphides by tri-n-butylphosphine and protein precipitation with ethanol. Separation was achieved by ion-pair high-performance liquid chromatography on a reversed-phase column (LiChrospher RP 18e) with cetrimonium bromide as counter ion and detection by fluorimetry after post-column derivatization with a selective thiol reagent, i.e. pyrenemaleimide. The high frequency of the analyzed samples and validation results make the method suitable for pharmacokinetic studies, and this was demonstrated by the first results obtained after the administration of an oral dose of 500 mg of Tiopronine to two healthy subjects.

Chemical Precipitation↗
PubMedDate not supplied

A novel approach to the high-specific-activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP = diphosphine ligand).

A new labeling approach for incorporating bioactive peptides into a technetium-99m coordination complex is described. This method exploits the chemical properties of the novel metal-nitrido fragment [99mTc(N)(PXP)]2+, composed of a terminal Tc[triple bond] N multiple bond bound to an ancillary diphosphine ligand (PXP). It will be shown that this basic, molecular building block easily forms in solution as the dichloride derivative [99mTc(N)(PXP)Cl2], and that this latter complex selectively reacts with monoanionic and dianionic, bidentate ligands (YZ) having soft, pi-donor coordinating atoms to afford asymmetrical nitrido heterocomplexes of the type [99mTc(N)(PXP)(YZ)]0/+ without removal of the basic motif [99mTc(N)(PXP)]2+. The reactions of the amino acid cysteine was studied in detail. It was found that cysteine readily coordinates to the metal fragment [99mTc(N)(PXP)]2+ either through the [NH2, S-] pair of donor atoms or, alternatively, through the [O-, S-] pair, to yield the corresponding asymmetrical complexes in very high specific activity. Thus, these results were conveniently employed to devise a new, efficient procedure for labeling short peptide sequences having a terminal cysteine group available for coordination to the [99mTc(N)(PXP)]2+ fragment. Examples of the application of this novel approach to the labeling of the short peptide ligand H-Arg-Gly-Asp-Cys-OH (H(2)1) and of the peptidomimetic derivative H-Cys-Val-2-Nal-Met-OH (H2) will be discussed.

Amines↗
PubMed1988

Afferents to the rat red nucleus studied by means of D-[3H]aspartate, [3H]choline and non-selective tracers.

Following injection of horseradish peroxidase-labeled wheat germ agglutinin or of rhodamine-labeled microspheres as non-selective tracers into the rat red nucleus, the origins of the corticorubral and cerebellorubral pathways, as well as a considerable number of other brain structures including dorsal raphé nucleus, zona incerta and several hypothalamic nuclei showed retrogradely labeled perikarya. Labeling patterns obtained with horseradish peroxidase-labeled wheat germ agglutinin compared well with those observed following application of rhodamine-labeled microspheres which produced injection sites restricted to the small nucleus. In these latter cases, counterstaining with phosphine allowed a better definition of anatomical structures. After D-[3H]aspartate application, retrogradely labeled perikarya were observed in cerebral cortex (layer V), zona incerta, dorsal raphé nucleus and in several other structures also labeled by non-selective tracers. Following application of [3H]choline and using an improved autoradiographic method, perikaryal labeling was massive within nucleus interpositus, while it was absent in dorsal raphé nucleus, cerebral cortex and zona incerta. Retrograde tracing experiments with D-[3H]aspartate and [3H]choline revealed that these transmitter related compounds are selective markers for two subsets of afferents to the red nucleus. The transmitter specificity of the selective labeling with [3H]choline in the cerebellorubral pathway is supported only in part by the results obtained with other methods. The selective labeling with D-[3H]aspartate in the corticorubral pathway, on the other hand, is consistent with its transmitter specificity.

Animals↗
PubMed2001

High-nuclearity close-packed palladium-nickel carbonyl phosphine clusters: heteropalladium.

[Pd(16)Ni(4)(CO)(22)(PPh(3))(4)](2)(-) (1) and [Pd(33)Ni(9)(CO)(41)(PPh(3))(6)](4)(-) (2) were obtained as the two major products from the reduction of PdCl(2)(PPh(3))(2) with [Ni(6)(CO)(12)](2)(-). Their crystal structures as [PPh(4)](+) salts were unambiguously determined from CCD X-ray crystallographic analyses; the resulting stoichiometries were ascertained from elemental analyses. Infrared, multinuclear (1)H, (31)P[(1)H] NMR, UV-vis, CV, variable-temperature magnetic susceptibility, and ESI FT/ICR mass spectrometric measurements were performed. The Pd(16)Ni(4) core of 1 ideally conforms to a ccp nu(3) tetrahedron of pseudo-T(d)() (4 3m) symmetry. Its geometry normal to each tetrahedral Pd(7)Ni(3) face (i.e., along each of the four 3-fold axes) may be viewed as a four-layer stacking of 20 metal atoms in a ccp [a(Ni(1)) b(Pd(3)) c(Pd(6)) a(Pd(7)Ni(3))] sequence. A comparative analysis of the different ligand connectivities about the analogous metal-core geometries in 1 and the previously reported [Os(20)(CO)(40)](2)(-) has stereochemical implications pertaining to the different possible modes of carbon monoxide attachment to ccp metal(111) surfaces. The unique geometry of the Pd(33)Ni(9) core of 2, which has pseudo-D(3)(h)() (6 2m) symmetry, consists of five equilateral triangular layers that are stacked in a hcp [a(Pd(7)Ni(3)) b(Pd(6)) a(Pd(7)Ni(3)) b(Pd(6)) a(Pd(7)Ni(3))] sequence. Variable-temperature magnetic susceptibility measurements indicated both 1 and 2 to be diamagnetic over the entire temperature range from 5.0 to 300 K. Neutral Pd(12)(CO)(12)(PPh(3))(6) (3) and [Pd(29)(CO)(28)(PPh(3))(7)](2)(-) (4) as the [PPh(4)](+) salt were obtained as minor decomposition products from protonation reactions of 1 and 2, respectively, with acetic acid. Compound 3 of pseudo-D(3)(d)() (3 2/m) symmetry represents the second highly deformed hexacapped octahedral member of the previously established homopalladium family of clusters containing uncapped, monocapped, bicapped, and tetracapped Pd(6) octahedra. The unprecedented centered 28-atom polyhedron for the Pd(29) core of 4 of pseudo-C(3)(v)() (3m) symmetry may be described as a four-layer stacking of 29 metal atoms in a mixed hcp/ccp [a(Pd(1)) b(Pd(3)) a(Pd(10)) c(Pd(15))] sequence.

Journal Article↗
PubMed2006

Development of selective inhibitors and substrate of matrix metalloproteinase-12.

Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO2-CH2)-Xaa'-Yaa'-Zaa'-NH2 have been prepared and screened against 10 matrix metalloproteinases (MMPs). We identified two phosphinic peptides with Ki values of 0.19 and 4.4 nM toward MMP-12 (macrophage elastase) that are more than 2-3 orders of magnitude less potent toward the other MMPs tested. These highly selective MMP-12 inhibitors contain a Glu-Glu motif in their Yaa'-Zaa' positions. Incorporation of this Glu-Glu motif into the sequence of a nonspecific fluorogenic peptide cleaved by MMPs provides a highly selective substrate for MMP-12. A model of one of these inhibitors interacting with MMP-12 suggests that the selectivity observed might be due, in part, to the presence of two unique polar residues in MMP-12, Thr239 and Lys177. These MMP-12-selective inhibitors may have important therapeutic applications to diseases in which MMP-12 has been suggested to play a key role, such as in emphysema, atherosclerosis, and aortic abdominal aneurysm.

Drug Design↗
PubMed2002

New diphosphine ligands containing ethyleneglycol and amino alcohol spacers for the rhodium-catalyzed carbonylation of methanol.

The new diphosphine ligands Ph(2)PC(6)H(4)C(O)X(CH(2))(2)OC(O)C(6)H(4)PPh(2) (1: X=NH; 2: X=NPh; 3: X=O) and Ph(2)PC(6)H(4)C(O)O(CH(2))(2)O(CH(2))(2)OC(O)C(6)H(4)PPh(2) (5) as well as the monophosphine ligand Ph(2)PC(6)H(4)C(O)X(CH(2))(2)OH (4) have been prepared from 2-diphenylphosphinobenzoic acid and the corresponding amino alcohols or diols. Coordination of the diphosphine ligands to rhodium, iridium, and platinum resulted in the formation of the square-planar complexes [(Pbond;P)Rh(CO)Cl] (6: Pbond;P=1; 7: Pbond;P=2; 8: Pbond;P=3), [(Pbond;P)Rh(CO)Cl](2) (9: Pbond;P=5), [(P-P)Ir(cod)Cl] (10: Pbond;P=1; 11: Pbond;P=2; 12: Pbond;P=3), [(Pbond;P)Ir(CO)Cl] (13: Pbond;P=1; 14: Pbond;P=2; 15: Pbond;P=3), and [(Pbond;P)PtI(2)] (18: Pbond;P=2). In all complexes, the diphosphine ligands are trans coordinated to the metal center, thanks to the large spacer groups, which allow the two phosphorus atoms to occupy opposite positions in the square-planar coordination geometry. The trans coordination is demonstrated unambiguously by the single-crystal X-ray structure analysis of complex 18. In the case of the diphosphine ligand 5, the spacer group is so large that dinuclear complexes with ligand 5 in bridging positions are formed, maintaining the trans coordination of the P atoms on each metal center, as shown by the crystal structure analysis of 9. The monophosphine ligand 4 reacts with [[Ir(cod)Cl](2)] (cod=cyclooctadiene) to give the simple derivative [(4)Ir(cod)Cl] (16) which is converted into the carbonyl complex [(4)Ir(CO)(2)Cl] (17) with carbon monoxide. The crystal structure analysis of 16 also reveals a square-planar coordination geometry in which the phosphine ligand occupies a position cis with respect to the chloro ligand. The diphosphine ligands 1, 2, 3, and 5 have been tested as cocatalysts in combination with the catalyst precursors [[Rh(CO)(2)Cl](2)] and [[Ir(cod)Cl](2)] or [H(2)IrCl(6)] for the carbonylation of methanol at 170 degrees C and 22 bar CO. The best results (TON 800 after 15 min) are obtained for the combination 2/[[Rh(CO)(2)Cl](2)]. After the catalytic reaction, complex 7 is identified in the reaction mixture and can be isolated; it is active for further runs without loss of catalytic activity.

Journal Article↗
PubMed1999

Characterization of the Batl (Bacteroides aerotolerance) operon in Bacteroides fragilis: isolation of a B. fragilis mutant with reduced aerotolerance and impaired growth in in vivo model systems.

YT135.2.8, a Tn4400' insertion mutant of Bacteroides fragilis strain TM4000, grows poorly when used to infect Monika or Chinese hamster ovary (CHO) cell monolayers and is outcompeted by wild-type strains in mixed infections. YT135.2.8 also shows defects in the rat granuloma pouch model system in monoculture and is completely outcompeted by the wild-type strain in a mixed infection. In addition, this mutant shows defects in a new model system consisting of CHO suspension cell columns. All of these defects may be explained by the finding that YT135.2.8 shows decreased tolerance to exposure to atmospheric oxygen (less aerotolerant). The monolayer growth defect (MGD) of YT135.2.8 can be influenced significantly by the presence of sulphur-containing reducing agents (cysteine, dithiothreitol, thiodiglycol) or the non-sulphur reducing agent Tris-(2-carboxylethyl)phosphine (TCEP). The defects in YT135.2.8 can be complemented by a 6.6 kb fragment of the B. fragilis chromosome. DNA sequencing of this fragment and of the regions flanking the Tn4400' insertion in the B. fragilis chromosome revealed the presence of five open reading frames, corresponding to genes bat (Bacteroides aerotolerance) A, B, C, D, E, which form the Batl operon; Tn4400' inserted within batD. All of the hypothetical proteins possess one or more membrane-spanning domains. BatA and BatB show high similarity to each other but, like BatD, they show no match to sequences of known function in the databases. BatC and BatE contain 2-4 repeated sequences similar to the tetratricopeptide repeats (TPRs) seen in many eukaryotic proteins. The function of TPR sequences in protein interactions in other systems leads to the suggestion that the Bat proteins form a complex. The Batl complex may be involved in the generation or export of reducing power equivalents to the periplasm of the B. fragilis cell.

Amino Acid Sequence↗
PubMed2003

High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine.

Increased interest in the analysis of aminothiols in body fluids during the last years results in a request for high-throughput analytical methods for their determination. We report here a novel, high-throughput method for the determination of total concentrations of biogenous aminothiols - homocysteine, cysteine, glutathione, cysteinylglycine, gamma-glutamylcysteine, and of penicilamine, mercaptopropionylglycine, and cysteamine, three compounds used to treat disorders of aminothiol metabolism in plasma and urine. Samples were reduced with tris(carboxyethyl)phosphine and labeled with 5-(bromomethyl)fluorescein. Capillary electrophoretic separations were performed in 60 mmol/L borate - 15 mmol/L sodium dodecyl sulfate - 2-amino-2-methyl-1-propanol, pH 10.0, with laser-induced fluorescence detection. Analysis time was less than 2 min. The assay is linear (r > 0.999) up to 500 micromol/L. Reproducibilities of migration times (coefficient of variation, CV) were < 0.5%. Interassay repeatabilities (CV, n = 10) were 5.08% and 6.09% for 5 micromol/L addition of homocysteine and 0.60% and 3.78% for 100 micromol/L addition of cysteine in plasma and urine, respectively. Recovery values were within 94-106% and sensitivity was better than 0.19 micromol/L for all analyzed compounds. Results agreed well with a standard high-performance liquid chromatography (HPLC) method. The diagnostic usefulness of the method has been proven on 79 samples of cystinuric patients and 12 samples of homocystinuric patients. We report here a novel method for the determination of aminothiols in body fluids by capillary electrophoresis (CE). Determination is fast and sensitive enough for diagnostic purposes.

Adolescent↗
PubMed2005

Inhibitory effects of the guanine moiety on Suzuki couplings of unprotected halonucleosides in aqueous media.

In the Suzuki arylations of unprotected halonucleosides in aqueous media, 8-bromo-2'-deoxyguanosine (8BrdG) couplings were slower to reach completion than the corresponding 8-bromo-2'-deoxyadenosine (8BrdA) couplings. The guanine moiety has an acidic proton, which under our Suzuki conditions (pH congruent with 10) may be deprotonated to give an anion that can coordinate to palladium. The possibility that guanine coordination was responsible for the observed slower rates was explored using additive experiments in which nonhalogenated nucleosides were added to the Suzuki coupling reaction of 8BrdA or 4-bromotoluene and PhB(OH)2 and the reaction progress monitored by HPLC or GC. Adding dG slowed these reactions, and an induction period was observed. The addition of dA or 1-methyl-2'-deoxyguanosine (1MedG) to these couplings did not affect the rate of conversion to product. Guanine coordination was further explored using 13C and 31P NMR spectroscopy, which implies that guanine is coordinating to palladium through N-1 or O-6, or both. Furthermore, the presence of dG inhibited the formation of the active palladium(0) catalytic species, which may account for both the observed induction period and the sluggishness of reactions where guanine is involved.

Anions↗
PubMed2003

Rhodium-catalyzed disulfide exchange reaction.

A system of RhH(PPh3)4, trifluoromethanesulfonic acid, and (p-tol)3P catalyzes the disulfide exchange reaction. Treatment of two symmetrical dialkyl disulfides with the catalyst provides an equilibrium mixture of three disulfides within 15 min in refluxing acetone. The catalyst is active after reaching the equilibrium, and addition of a disulfide to the mixture changes the ratio of the products. The use of 4 mol equiv excess of one of the disulfides provides the unsymmetrical disulfide in a yield exceeding 80%. Disulfide-containing peptides also undergo an exchange reaction. The reactions of diaryl disulfides and dialkyl disulfides are even faster, and reach equilibrium within 5 min at room temperature in the presence of the rhodium complex and 1,2-bis(diphenylphosphino)ethane (dppe). This exchange reaction is considerably affected by the substituents on the disulfides. Treatment of diphenyl disulfide, di(p-tolyl) disulfide, and bis(sec-butyl) disulfide yields phenyl p-tolyl disulfide at room temperature with unchanged bis(sec-butyl) disulfide; random disproportionation occurs at reflux. The rhodium catalysis can be used for the exchange reaction of disulfides and diselenides giving selenosulfides as well as disulfides and ditellurides giving tellurinosulfides.

Catalysis↗
PubMed1993

Disulfide structures of highly bridged peptides: a new strategy for analysis.

A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide chain be split between close or adjacent Cys residues. The water-soluble tris-(2-carboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra- and interchain disulfides; -Cys.Cys- pair), endothelin and apamin (two disulfides; -Cys.x.Cys- pair), conotoxin GI and isomers (two disulfides; -Cys.Cys- pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys- pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.

Alkylation↗