Peter L. Bartner

Microwave‐enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes

Accelerated proteolytic cleavage of proteins under controlled microwave irradiation has been achieved. Selective peptide fragmentation by endoproteases trypsin or lysine C led to smaller peptides that were analyzed by matrix‐assisted laser desorption ionization (MALDI) or liquid chromatography‐electrospray ionization (LC‐ESI) techniques. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon α‐2b. Most important, using this novel approach digestion of proteins occurs in minutes, in contrast to the hours required by conventional methods.

Electrospray ionization mass spectrometry for the study of non-covalent complexes : an emerging technology

The detection of non-covalent complexes in the mass range 19,000-34,000 Da, using electrospray ionization mass spectrometry (ESI-MS), is reviewed. The examples discussed include (1) a protein-ligand interaction (ras-GDP), (2) an inhibitor-protein-ligand interaction (SCH 54292/SCH 54341-ras-GDP), (3) a protein-protein interaction (gamma-IFN homodimer) and (4) a protein-metal complex [HCV (1-181)-Zn]. In each case, the ESI-MS method is capable of releasing the intact non-covalent complex from its native solution state into the gas phase in the form of multiply-charge ions. The molecular masses of these complexes were determined with a mass accuracy of better than 0.01%, which is far superior to the traditional methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation chromatography. The method provides the researcher with a quick, reliable and reproducible method for probing difficult biological problems. The key to success in the study of non-covalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions; special care must be taken with the source orifice potential and the solution pH and organic co-solvents must be avoided. This paper also illustrates the usefulness of ESI-MS for addressing biological problems leading to the discovery of new therapeutics; the approach involves the rapid screening of potential drug candidates, such as weakly bound inhibitors.

Synthesis of heterocyclic compounds using radical reactions

A generalised radical reaction has been used to synthesise heterocyclic compounds which could serve as ligands for drug discovery. Attempt also have been made to rationalise the formation of oxidation products formed during TBTH reaction.

Microwave enhanced Akabori reaction for peptide analysis

The Akabori reaction, devised in 1952 for the identification of C-terminus amino acids, involves the heating of a linear peptide in the presence of anhydrous hydrazine in a sealed tube for several hours. We report here a modified Akabori reaction that rapidly identifies the C-terminus amino acid in a polypeptide including its amino acid sequence information at both the C-terminus and the N-terminus. This modified methodology demonstrates the fundamentals of microwave chemistry applied to bioanalytical problems. In this modified process, hydrazinolysis has been accelerated by the application of microwave irradiation. In our reaction, the linear peptide and hydrazine solution, contained in a loosely covered conical flask, was exposed to a few minutes of irradiation using an unmodified domestic microwave oven. While the classical Akabori reaction required several hours, the microwave assisted reaction takes just minutes. If dimethyl sulfoxide is added to dilute the reaction mixture, the process is retarded enough to allow aliquots of the reaction mixture to be drawn every few minutes over a period of about an hour in order to study the progress of hydrazinolysis. Reaction products were monitored by mass spectrometry—primarily FAB-MS. In addition to providing sequence information, the microwave enhanced Akabori reaction quickly detects the presence of arginine (Arg) by converting each Arg to ornithine (Orn). Furthermore, certain amino acids, containing β-SH, CO2H, and CONH2 groups in their side chain, are susceptible to modification by hydrazine, thereby, providing rapid confirmation of the presence of these amino acid residues. In these preliminary studies, the following oligopeptides were analyzed to demonstrate the effectiveness of our approach; the dipeptide (Trp-Phe), the tripeptide (Tyr-Gly-Gly), the tetrapeptide (Pro-Phe-Gly-Lys), the heptapeptide (Ala-Pro-Arg-Leu-Arg-Phe-Tyr), and a N-terminal blocked tripeptide (N-acetyl-Met-Leu-Phe).

High-resolution LC/MS for analysis of minor components in complex mixtures: negative ion ESI for identification of impurities and degradation products of a novel oligosaccharide antibiotic

High-resolution mass spectrometry has been routinely used for structural confirmation and identification; however, it has mostly been applied to relatively pure samples. Exact mass measurement of minor components such as impurities, degradation products or metabolites in complex mixtures has been difficult without prior separation and isolation. Here we report the utilization of on-line liquid chromatography in combination with high-resolution mass spectrometry for the identification of impurities and base degradation products of Sch 27899, a member of the everninomicin class of antibiotics. Nine Sch 27899-related impurities and degradation products were detected by negative ion electrospray ionization using a magnetic sector mass spectrometer. Exact mass measurements were obtained at a resolution of 5000 using polyethylene glycol (PEG) sulfates as internal standards. Corresponding elemental compositions were determined within a 2 ppm error tolerance and structures were proposed for all components.

Rapid cyclopeptide analysis by microwave enhanced Akabori reaction

Abstract The application of microwave assisted modification of the Akabori hydrazinolysis reaction has been found to cleave cyclic oligopeptides in a selective fashion to produce mainly the hydrazide of a specific linear peptide. This ring cleavage requires a few minutes of reaction in a domestic microwave oven. The linear peptide hydrazide can be analyzed by ESI-MS/MS, FAB-MS, and FAB-MS/MS methods for the determination of the amino acid sequence.

Multiple-stage mass spectrometric analysis of complex oligosaccharide antibiotics (everninomicins) in a quadrupole ion trap

Electrospray ionization (ESI) quadrupole ion-trap tandem mass spectrometry (MS/MS) was utilized to characterize a class of complex oligosaccharide antibiotics (everninomicins) that include SCH 27899, everninomicin-D, amino everninomicin (SCH 27900), and SCH 49088 (containing a hydroxylamino-ether sugar). The addition of sodium chloride (∼1 μg/mL) facilitates the formation of abundant metal complex ions, and this was used because protonation does not readily occur for most of these compounds. The multiple-stage mass analysis (MSn) of the sodiated species provides an important series of fragment ions that are specific for sugar sequence and for some sugar-ring opening. These data suggest a general charge-remote fragmentation pattern with the sodium cation residing in a specific, central location of the sugar chain and fragmentation occurring to trim the end of the molecule. For protonated everninomicin (SCH 27900), however, the proton appears to be mobile during the collisional activation process, opening different fragmentation pathways depending on the proton location. The use of water and acetonitrile with 0.1% acetic acid as the solvent in ESI-MS promotes rapid hydrolysis of the central ortho ester, resulting in the formation of abundant sodiated products that are hydrated. These product ions of the hydrated molecules are likely formed by the same charge-remote fragmentation processes as those that occur for the unhydrolyzed precursor.

The megalomicins. Part 7. A structural revision by carbon-13 nuclear magnetic resonance and X-ray crystallography. Synthesis and conformational analysis of 3-dimethylamino- and 3-azido-D- and -L-hexopyranosides, and the crystal structure of 4″-O-(4-lodobenzoyl)megalomicin A

An X-ray crystallographic study on 4″-O-(4-iodobenzoyl)megalomicin A has led to the revision of the structures of the megalomicins and the XK-41 antibiotics. Crystals are orthorhombic, space group P212121 with a= 12.669(2), b= 19.501 (6), c= 25.741 (9)A, and Z= 4. The structure was solved by the heavy-atom technique, and 1 812 observed reflections led to a final R of 0.095. The novel amino-sugar previously thought to be D-rhodosamine has been shown to have the L-configuration and is therefore renamed L-megosamine. It has also been shown to be glycosidically attached to the tertiary 6-hydroxy group. The 13C n.m.r. and circular dichroism (c.d.) parameters of these macrolides are described. The syntheses of methyl α- and β-D-rhodosaminide, methyl α- and β-D-megosaminide, methyl α- and β-L-megosaminide, methyl α- and β-D-angolosaminide, and methyl 2,3,6-trideoxy-3(dimethylamino)-α-D-xylo-hexopyranoside are described and their conformations and 13C n.m.r. parameters are discussed. Methyl α-D- and -L-amicetoside, methyl α-D- and -L-cineruloside and other model 4-oxopyranosides and pyrans have been synthesized. Their c.d. properties have been determined and they have been shown to exhibit Anti-Octant behaviour.

Structure elucidation of everninomicin-6, a new oligosaccharide antibiotic, by chemical degradation and FAB-MS methods

The structural characterization of a new oligosaccharide antibiotic, Everninomicin-6 (EV-6), is described. Detailed fast-atom bombardment mass spectrometry (FAB-MS) studies along with NMR and chemical degradation methods were conducted to elucidate the structure of EV-6. The effects of the use of various matrices, including salt addition, on the quality of the FAB-MS were explored. The use of 3-nitro benzyl alcohol, dimethyl sulfoxide (DMSO), and NaCl produced the best results: an intense sodiated molecular ion plus structurely informative fragmentation. FAB-MS yields information providing the complete sugar sequence information for everninomicins, which is quite valuable to the elucidation of the structure of this complex oligosaccharide antibiotic. In addition, the results of accurate mass work with the molecular ion are consistent with the assigned structure. The use of electrospray ionization mass spectrometry (ESI-MS) and ESI-MS/MS for the study of EV-6 was investigated and was found to produce an abundant molecular ion with limited structural information. These results revealed that EV-6 resembled EV-D quite closely except for the absence of the nitrosugar and the replacement on ring g of the -CH2OCH3 group with a -CH2OH group.

SCH 23831, a novel macrolide from Micromonospora rosaria

Abstract A novel macrolid elaborated by Micromonospora rosaria , SCH 23831, was assigned structure 2 on the basis of spectroscopic data. Several derivatives are also discussed.