Keiko Tabei

Further Evidence that a Cell Wall Precursor [C55-MurNAc-(Peptide)-GlcNAc] Serves as an Acceptor in a Sorting Reaction

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.

Oxidative Palladium Catalysis in SNAr Reactions Leading to Heteroaryl Ethers from Pyridotriazol-1-yloxy Heterocycles with Aryl Boronic Acids

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.

Rapid methods for screening low molecular mass compounds non‐covalently bound to proteins using size exclusion and mass spectrometry applied to inhibitors of human cytomegalovirus protease

General and rapid methods were developed for determining the extent of non-covalent binding between small molecules and proteins, using the model system of human cytomegalovirus protease and several drug candidates which inhibit the protease by non-covalently binding to it. The assay was performed by off-line coupling of size-exclusion methods with mass spectrometry in the following manner. The protease and inhibitor were incubated together under native conditions and then subjected to separation based on size, by use of a spin column (gel permeation chromatography) and/or a microconcentrator (ultrafiltration). The spin column selectively passed the high molecular mass (M(r)) protease and trapped low M(r) molecules. Alternatively, the microconcentrator passed low M(r) molecules and retained the protease. If the inhibitor bound non-covalently to the protease, both the inhibitor and protease passed through the spin column (or were retained by the microconcentrator). Electrospray ionization mass spectrometry was used to assay the spin column eluate (or the microconcentrator retentate) and to characterize the amounts of protease and inhibitor based on known standards. An advantage of these techniques is that a mixture containing inhibitors can be analyzed in the presence of the protease, and inhibitors with the greatest binding affinity can be identified. Non-covalent binding specificity was demonstrated using spin columns by comparing the binding affinity of inhibitors using several mutants of cytomegalovirus protease. The techniques described are applicable to the rapid screening of compound libraries for selecting substances which bind non-covalently to a known protein.

Comparative mass spectrometric analyses of Photofrin oligomers by fast atom bombardment mass spectrometry, UV and IR matrix-assisted laser desorption/ionization mass spectrometry, electrospray ionization mass spectrometry and laser desorption/jet-cooling photoionization mass spectrometry.

Photofrin (porfimer sodium) is a porphyrin derivative used in the treatment of a variety of cancers by photodynamic therapy. This oligomer complex and a variety of porphyrin monomers, dimers and trimers were analyzed with five different mass spectral ionization techniques: fast atom bombardment, UV and IR matrix-assisted laser desorption/ionization, electrospray ionization, and laser desorption/jet-cooling photoionization. All five approaches resulted in very similar oligomer distributions with an average oligomer length of 2.7 +/- 0.1 porphyrin units. In addition to the Photofrin analysis, this study provides a side-by-side comparison of the spectra for the five different mass spectrometric techniques.

Evaluation of a dual electrospray ionization/atmospheric pressure chemical ionization source at low flow rates (∼50 µL/min) for the analysis of both highly and weakly polar compounds

The atmospheric pressure ionization (API) source for a commercial mass spectrometer was modified to operate as a dual source in both the electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) techniques by simultaneously utilizing the electrospray probe and the corona discharge needle. A switching box was designed to operate in either manual or programmable modes to permit rapid switching between ionization techniques without changing sources, probes, or breaking vacuum. The source can be operated using the following ionization techniques: ESI only, APCI only, ESI/APCI simultaneously, and ESI/APCI alternatingly. The optimum operating conditions for these ionization techniques were similar to the manufacturer’s original specifications except that the APCI flow rate was lower (∼50 µL/min versus 1000 µL/min) and externally heated nebulizing gas was found to be desirable. A four-component mixture, introduced by flow injection, was used to demonstrate the versatility of the dual ESI/APCI source.

Polysulfated Carbohydrates Analyzed as Ion‐paired Complexes with Basic Peptides and Proteins Using Electrospray Negative Ionization Mass Spectrometry

Electrospray ionization mass spectrometry was used in the negative ion mode to analyze complexes of sucrose octasulfate, sucrose heptasulfate and sulfated alpha-, beta- and gamma-cyclodextrins with synthetically prepared basic peptides, the basic protein ubiquitin and polyamines. The spectra presented demonstrate that complexes with these basic molecules facilitate the analysis of these polysulfated oligosaccharides. Stable (1:1) complexes result from the ion pairing between the protonated basic arginine and lysine residues of the peptide and the anionic sulfate groups of the polysulfated oligosaccharides. Fragmentation of the polysulfated oligosaccharides resulting in the loss of SO3 could be suppressed by controlling the experimental conditions, such as the nozzle-skimmer voltage, used to obtain the spectra. In the absence of fragmentation, it was possible to obtain data on the purity of sucrose octasulfate and sucrose heptasulfate as well as the distribution of the sulfated cyclodextrins. The confounding presence of sodium counter-ions is also eliminated using this method. Complete chemical sulfation of oligosaccharides is difficult to achieve. Thus, data on sample purity are essential for the characterization of sulfated oligosaccharides used as pharmaceutical agents.

The Structure of the Cell Wall Peptidoglycan of Bacillus cereus RSVF1, a Strain Closely Related to Bacillus anthracis

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.

INHIBITION OF HUMAN CYTOMEGALOVIRUS UL80 PROTEASE BY SPECIFIC INTRAMOLECULAR DISULFIDE BOND FORMATION

A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.

Biopolymer sequencing using a triple quadrupole mass spectrometer in the ESI nozzle-skimmer/precursor ion MS/MS mode

A variety of model biopolymers, including oligonucleotides, oligosaccharides and a synthetic pharmaceutical agent, were sequenced using a triple quadrupole mass spectrometer equipped with an electrospray source and operated in a scan mode referred to as pseudo-MS3. This scan mode consists of three steps: (1) in-source collision-induced dissociation (CID) in the nozzle-skimmer (NS) region, (2) scanning of the fragment ions into the collision cell for further CID, and (3) passing of the secondary fragment ions through the final mass filter at a preselected mass, generally corresponding to the mass of a terminal sequence ion for the biopolymer. The mass spectra are recorded in the precursor ion MS/MS mode where ion selection and detection occur at the third stage of the triple quadrupole but the scan function is determined by the first stage. The advantages and limitations in using this pseudo-MS3 NS/precursor ion MS/MS scan mode for biopolymer sequencing are discussed.

High-level β-lactam resistance and cell wall synthesis catalyzed by the mecA homologue of Staphylococcus sciuri introduced into Staphylococcus aureus

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.