Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 14 de 14
Filter
Add more filters










Publication year range
1.
J Am Chem Soc ; 131(12): 4174-5, 2009 Apr 01.
Article in English | MEDLINE | ID: mdl-19265423

ABSTRACT

The palladium-catalyzed oxidative coupling of pyrido- and benzotriazol-1-yloxyquinazolines and -thienopyrimidines with aryl boronic acids in the presence of Pd(PPh(3))(4) and Cs(2)CO(3) under oxygen in DME containing 0.4-0.8% water for the preparation of heteroaryl ethers is described. These transformations of triazol-1-yloxy reagents demonstrate excellent O-chemoselective control under mild conditions and good yields. Mechanistic studies based on (18)O labeling indicate that phenols as intermediates in S(N)Ar reactions with ethers are formed in oxidative and nonoxidative pathways.


Subject(s)
Boronic Acids/chemistry , Chemistry, Organic/methods , Ethers/chemistry , Palladium/chemistry , Quinazolines/chemistry , Catalysis , Models, Chemical , Molecular Structure , Nitrogen/chemistry , Oxygen/chemistry , Phenol/chemistry , Pyrimidines/chemistry , Sulfur/chemistry , Time Factors
2.
J Med Chem ; 52(8): 2181-4, 2009 Apr 23.
Article in English | MEDLINE | ID: mdl-19309081

ABSTRACT

The naturally occurring pyranonaphthoquinone (PNQ) antibiotic lactoquinomycin and related aglycones were found to be selective inhibitors of the serine-threonine kinase AKT. A set of synthetic PNQs were prepared and a minimum active feature set and preliminary SAR were determined. PNQ lactones inhibit the proliferation of human tumor cell lines containing constitutively activated AKT and show expected effects on cellular biomarkers. Biochemical data are presented supporting a proposed bioreductive alkylation mechanism of action.


Subject(s)
Antineoplastic Agents/chemical synthesis , Cysteine/metabolism , Lactones/chemical synthesis , Oncogene Protein v-akt/antagonists & inhibitors , Pyrans/chemical synthesis , Alkylation , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biomarkers/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Lactones/chemistry , Lactones/pharmacology , Naphthoquinones/chemical synthesis , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Pyrans/chemistry , Pyrans/pharmacology , Stereoisomerism , Structure-Activity Relationship
3.
J Org Chem ; 72(26): 10194-210, 2007 Dec 21.
Article in English | MEDLINE | ID: mdl-18044930

ABSTRACT

An efficient "one-step" synthesis of cyclic amidines and guanidines has been developed. Treatment of cyclic amides and ureas with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), base, and nitrogen nucleophiles leads to the formation of the corresponding cyclic amidines and guanidines, typically in good to excellent yields. This method has also been used to prepare heteroaryl ethers and thioethers using phenol and thiophenol nucleophiles. Time course NMR and HPLC-MS studies have facilitated explicit characterization of the proposed intermediates (the phosphonium salt and HOBt adduct); the data reveal a stepwise reaction pathway.


Subject(s)
Amides/chemistry , Amidines/chemical synthesis , Guanidines/chemical synthesis , Organophosphorus Compounds/chemistry , Urea/chemistry , Amidines/chemistry , Amination , Guanidines/chemistry , Molecular Structure , Solvents/chemistry , Stereoisomerism , Time Factors
4.
J Org Chem ; 72(16): 6016-21, 2007 Aug 03.
Article in English | MEDLINE | ID: mdl-17628103

ABSTRACT

Dimethyl sulfoxide (DMSO) efficiently causes the reductive elimination of 3-aryl 2,3-dibromopropanoates to cinnamates with good yield. With 3-phenyl 2,3-dihalopropanoates, debromination is the major pathway providing 3-phenylacrylate derivatives in high yields, whereas dehydrobromination is a competing pathway with thiophene derivatives. 1H NMR, 81Br NMR, and MS techniques indicated the formation of brominated-DMSO, MeBr, and HBr as byproducts in this transformation with no evidence for the formation of Br2. The dual role of DMSO as a nucleophile and bromine scavenger accounts for the products formed in this reaction.


Subject(s)
Chemistry, Organic/methods , Dimethyl Sulfoxide/chemistry , Propionates/chemistry , Bromine/chemistry , Magnetic Resonance Spectroscopy/methods , Mass Spectrometry , Models, Chemical , Pyridines/chemistry
5.
Anal Biochem ; 360(1): 14-22, 2007 Jan 01.
Article in English | MEDLINE | ID: mdl-17107653

ABSTRACT

Many bacterial surface proteins containing an LPXTG motif are anchored to the cell wall peptidoglycan by catalysis with the thiol transpeptidase sortase. The transpeptidation and hydrolysis reactions of sortase have been proposed to proceed through a common acyl enzyme intermediate. The reactions of Staphylococcus aureus sortase with fluorogenic substrate Abz-LPETG-Dnp in the presence or absence of triglycine were characterized in this study to gain additional insight into the kinetic mechanism of sortase. We report here the development of a reverse-phase HPLC assay to identify and characterize sortase reaction intermediates. The HPLC results provide for the first time clear evidence for the formation of a kinetically competent acyl enzyme intermediate during the overall transpeptidation reaction. The results also suggest that sortase undergoes an unexpected intramolecular acyl transfer reaction in the absence of a nucleophile. The significance of this type of HPLC assay as a tool to study enzyme mechanism is discussed.


Subject(s)
Aminoacyltransferases/metabolism , Chromatography, High Pressure Liquid/methods , Cysteine Endopeptidases/metabolism , Staphylococcus aureus/enzymology , Base Sequence , DNA Primers , Enzyme Stability , Kinetics , Mutagenesis, Site-Directed , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
6.
J Bacteriol ; 187(19): 6651-8, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16166526

ABSTRACT

A close homologue of mecA, the determinant of broad-spectrum beta-lactam resistance in Staphylococcus aureus was recently identified as a native gene in the animal commensal species Staphylococcus sciuri. Introduction of the mecA homologue from a methicillin-resistant strain of S. sciuri into a susceptible strain of S. aureus caused an increase in drug resistance and allowed continued growth and cell wall synthesis of the bacteria in the presence of high concentrations of antibiotic. We determined the muropeptide composition of the S. sciuri cell wall by using a combination of high-performance liquid chromatography, mass spectrometric analysis, and Edman degradation. Several major differences between the cell walls of S. aureus and S. sciuri were noted. The pentapeptide branches in S. sciuri were composed of one alanine and four glycine residues in contrast to the pentaglycine units in S. aureus. The S. sciuri wall but not the wall of S. aureus contained tri- and tetrapeptide units, suggesting the presence of dd- and ld-carboxypeptidase activity. Most interestingly, S. aureus carrying the S. sciuri mecA and growing in methicillin-containing medium produced a cell wall typical of S. aureus and not S. sciuri, in spite of the fact that wall synthesis under these conditions had an absolute dependence on the heterologous S. sciuri gene product. The protein product of the S. sciuri mecA can efficiently participate in cell wall biosynthesis and build a cell wall using the cell wall precursors characteristic of the S. aureus host.


Subject(s)
Bacterial Proteins/genetics , Methicillin Resistance/genetics , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Cell Wall/chemistry , Cell Wall/metabolism , Methicillin/metabolism , Mutation , Penicillin-Binding Proteins , Peptidoglycan/chemistry , Peptidoglycan/metabolism , Species Specificity , Staphylococcus/drug effects , Staphylococcus/genetics , Transduction, Genetic
7.
Antimicrob Agents Chemother ; 48(12): 4566-73, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15561827

ABSTRACT

A combination of biochemical and genetic experiments were performed in order to better understand the mechanism of expression of high-level vancomycin resistance in Staphylococcus aureus. The transcription of pbp2 of the highly vancomycin- and oxacillin-resistant strain COLVA200 and its mutant derivative with inactivated mecA were put under the control of an inducible promoter, and the dependence of oxacillin and vancomycin resistance and cell wall composition on the concentration of the isopropyl-beta-D-thiogalactopyranoside inducer was determined. The results indicate that mecA--the genetic determinant of oxacillin resistance--while essential for oxacillin resistance, is not involved with the expression of vancomycin resistance. Penicillin binding protein 2A, the protein product of mecA, appears to be unable to utilize the depsipeptide cell wall precursor produced in the vancomycin-resistant cells for transpeptidation. The key penicillin binding protein essential for vancomycin resistance and for the synthesis of the abnormally structured cell walls characteristic of vancomycin-resistant S. aureus (A. Severin, K. Tabei, F. Tenover, M. Chung, N. Clarke, and A. Tomasz, J. Biol. Chem. 279:3398-3407, 2004) is penicillin binding protein 2.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Carbon-Oxygen Ligases/biosynthesis , Carbon-Oxygen Ligases/genetics , Cell Wall/metabolism , Enterococcus/genetics , Penicillin-Binding Proteins/metabolism , Staphylococcus aureus/genetics , Vancomycin Resistance/genetics , Chromatography, High Pressure Liquid , Cytoplasm/metabolism , Microbial Sensitivity Tests , Oxacillin/pharmacology , Penicillin Resistance/genetics , Penicillin-Binding Proteins/genetics , Penicillins/pharmacology , Peptidoglycan/isolation & purification , Peptidoglycan/metabolism , Staphylococcus aureus/metabolism , Staphylococcus aureus/ultrastructure , Uridine Diphosphate/metabolism
8.
Microb Drug Resist ; 10(2): 77-82, 2004.
Article in English | MEDLINE | ID: mdl-15256021

ABSTRACT

The peptidoglycan of Bacillus cereus RSVF1, a close relative of Bacillus anthracis, has several distinguishing features: the overwhelming majority of cross-linked muropeptides are dimers, higher oligomers are only present in minute quantities; and virtually all muropeptides lack the N-acetyl group from glucosamine residues, thus explaining resistance of the cell walls to lysozyme.


Subject(s)
Bacillus anthracis/chemistry , Bacillus cereus/chemistry , Cell Wall/chemistry , Peptidoglycan/chemistry , Acetylation , Amino Acid Sequence , Chromatography, High Pressure Liquid , Dimerization , Oligopeptides/chemistry , Peptidoglycan/isolation & purification
9.
J Med Chem ; 47(14): 3674-88, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15214794

ABSTRACT

The reactions of 6-methylidene penems 4-7 with beta-lactamases (TEM-1, SHV-1, Amp-C) were characterized by electrospray ionization mass spectrometry (ESI-MS). The kinetics of the reactions were monitored, demonstrating that only one penem molecule reacts to form an acyl-enzyme complex. For penem 5, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized dihydro[1,4]thiazepine 10, confirming the rearrangement of the penem ring system to a seven-membered dihydro[1,4]thiazepine structure. Gas-phase ESI-MS/MS fragmentation data were rationalized due to tautomerization between imine and enamine substructures. ESI-MS/MS analysis of the T-6 trypsin-digested fragments of TEM-1 and SHV-1 demonstrated that the penems were only attached to Ser-70 of these class A beta-lactamases and that the penem ring structures were rearranged to seven-membered dihydro[1,4]thiazepines.


Subject(s)
beta-Lactamases/chemistry , beta-Lactams/chemistry , Bacterial Proteins/chemistry , Binding Sites , Hydrolysis , Spectrometry, Mass, Electrospray Ionization , beta-Lactamase Inhibitors , beta-Lactams/chemical synthesis
10.
J Biol Chem ; 279(5): 3398-407, 2004 Jan 30.
Article in English | MEDLINE | ID: mdl-14613936

ABSTRACT

Recently, for the first time in the history of this bacterial species, methicillin-resistant Staphylococcus aureus (MRSA) carrying the enterococcal vanA gene complex and expressing high level resistance to vancomycin was identified in clinical specimens (CDC (2002) MMWR 51, 565-567). The purpose of our studies was to understand how vanA is expressed in the heterologous background of S. aureus and how it interacts with the mecA-based resistance mechanism, which is also present in these strains and is targeted on cell wall biosynthesis. The vanA-containing staphylococcal plasmid was transferred from the clinical vancomycin-resistant S. aureus (VRSA) strain HIP11714 (CDC (2002) MMWR 51, 565-567) to the methicillin-resistant S. aureus (MRSA) strain COL for which extensive genetic and biochemical information is available on staphylococcal cell wall biochemistry and drug resistance mechanisms. The transconjugant named COLVA showed high and homogeneous resistance to both oxacillin and vancomycin. COLVA grown in vancomycin-containing medium produced an abnormal peptidoglycan: all pentapeptides were replaced by tetrapeptides, and the peptidoglycan contained at least 22 novel muropeptide species that frequently showed a deficit or complete absence of pentaglycine branches. The UDP-MurNAc-pentapeptide, the major component of the cell wall precursor pool in vancomycin-sensitive cells was replaced by UDP-MurNAc-depsipeptide and UDP-MurNAc-tetrapeptide. Transposon inactivation of the beta-lactam resistance gene mecA caused complete loss of beta-lactam resistance but had no effect on the expression of vancomycin resistance. The two major antibiotic resistance mechanisms encoded by mecA and vanA residing in the same S. aureus appear to use different sets of enzymes for the assembly of cell walls.


Subject(s)
Anti-Bacterial Agents/chemistry , Bacterial Proteins/genetics , Carbon-Oxygen Ligases/genetics , Drug Resistance, Microbial , Drug Resistance , Enterococcus/metabolism , Oxacillin/chemistry , Staphylococcus aureus/metabolism , Vancomycin/chemistry , Anti-Bacterial Agents/pharmacology , Cell Wall/chemistry , Cell Wall/metabolism , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Lactams/chemistry , Mass Spectrometry , Models, Chemical , Operon , Oxacillin/pharmacology , Peptides/chemistry , Peptidoglycan/chemistry , Time Factors , Vancomycin/pharmacology
11.
Biochemistry ; 42(38): 11307-15, 2003 Sep 30.
Article in English | MEDLINE | ID: mdl-14503881

ABSTRACT

Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. We have also demonstrated in this study that the nucleophile binding site of S. aureus sortase SrtA is specific for diglycine. While S1' and S2' sites of the enzyme both prefer a glycine residue, the S1' site is exclusively selective for glycine. Lengthening of the polyglycine acceptor nucleophile beyond diglycine does not further enhance the binding and catalysis.


Subject(s)
Aminoacyltransferases/chemistry , Aminoacyltransferases/metabolism , Staphylococcus aureus/enzymology , Amino Acid Sequence , Bacterial Proteins , Binding Sites , Cysteine Endopeptidases , Fluorescence Resonance Energy Transfer , Glycine/analogs & derivatives , Glycine/metabolism , Hydrolysis , Kinetics , Oligopeptides/chemistry , Oligopeptides/metabolism , Spectrometry, Mass, Electrospray Ionization , Substrate Specificity
12.
J Biol Chem ; 277(48): 45935-41, 2002 Nov 29.
Article in English | MEDLINE | ID: mdl-12324463

ABSTRACT

The enzymatic synthesis of the complete l-alanyl(1)-l-alanine(2) side chain of the peptidoglycan precursors of Enterococcus faecalis was obtained in vitro using purified enzymes. The pathway involved alanyl-tRNA synthetase and two ligases, BppA1 and BppA2, that specifically transfer alanine from Ala-tRNA to the first and second positions of the side chain, respectively. The structure of the UDP-N-acetylmuramoyl-l-Ala-gamma-d-Glu-l-Lys(N(epsilon)-l-Ala(1)-l-Ala(2))-d-Ala-d-Ala product of BppA1 and BppA2 was confirmed by mass spectrometry (MS) and MS/MS analyses. The peptidoglycan structure of the wild-type E. faecalis strain JH2-2 was determined by tandem reverse-phase high-pressure liquid chromatography-MS revealing that most muropeptides contained two l-alanyl residues in the cross-bridges and in the free N-terminal ends. Deletion of the bppA2 gene was associated with production of muropeptides containing a single alanyl residue at these positions. The relative abundance of monomers, dimers, trimers, and tetramers in the peptidoglycan of the bppA2 mutant indicated that precursors containing an incomplete side chain were efficiently used by the dd-transpeptidases in the cross-linking reaction. However, the bppA2 deletion impaired expression of intrinsic beta-lactam resistance suggesting that the low affinity penicillin-binding protein 5 did not function optimally with precursors substituted by a single alanine.


Subject(s)
Alanine/chemistry , Dipeptides/chemical synthesis , Enterococcus faecalis/metabolism , Peptidoglycan/chemistry
13.
Anal Chem ; 74(15): 3877-86, 2002 Aug 01.
Article in English | MEDLINE | ID: mdl-12175179

ABSTRACT

Two new applications using multiple ion isolations in the cell of a Fourier transform-ion cyclotron resonance mass spectrometer equipped with an electrospray ionization source are described. A procedure that uses multiple ion isolations of an analyte and calibrants for internal calibration at each stage in a MSn experiment, under high-resolution exact-mass conditions, for structural characterization/elucidation of angiotensin I and rapamycin is illustrated. Fragment ion mass accuracies < 1.0 ppm are demonstrated and routinely achieved. Purification of a mixture is illustrated by isolating multiple charge states of a protein-drug complex from residual protein for further MSn studies to elucidate the site of covalent drug bonding using IRMPD for a mixture of epidermal growth factor receptor (EGFr) protein and EGFr-drug complex. The procedure developed for multiple ion isolations is referred to as multi-CHEF, multiple correlated harmonic excitation fields, in which tailored waveforms are used to notch out multiple mass regions of a spectrum with minimal off-resonance excitation.


Subject(s)
Pharmaceutical Preparations/analysis , Proteins/metabolism , Angiotensin I/analysis , Angiotensin I/pharmacokinetics , Animals , Binding Sites , Bradykinin/analysis , Bradykinin/metabolism , Calibration , Cyclotrons , ErbB Receptors/analysis , ErbB Receptors/metabolism , Fourier Analysis , Humans , Ions , Mass Spectrometry , Melitten/analysis , Melitten/metabolism , Molecular Weight , Pharmacokinetics , Proteins/analysis , Sirolimus/analysis , Sirolimus/pharmacokinetics
14.
J Bacteriol ; 184(8): 2141-7, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11914345

ABSTRACT

Previous studies suggested that a Gly-containing branch of cell wall precursor [C(55)-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.


Subject(s)
Aminoacyltransferases/physiology , Cell Wall/metabolism , Peptidoglycan/biosynthesis , Streptomyces/metabolism , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Uridine Diphosphate N-Acetylmuramic Acid/metabolism , Bacterial Proteins , Cysteine Endopeptidases , Membrane Proteins/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...