Jeffrey M. Manthorpe

Synthesis and Biological Studies of 35‐Deoxy Amphotericin B Methyl Ester

The use of molecular editing in the elucidation of the mechanism of action of amphotericin B is presented. A modular strategy for the synthesis of amphotericin B and its designed ana- logues is developed, which relies on an efficient gram-scale synthesis of various subunits of amphotericin B. A novel method for the coupling of the mycosa- mine to the aglycone was identified. The implementation of the approach has enabled the preparation of 35- deoxy amphotericin B methyl ester. In- vestigation of the antifungal activity and efflux-inducing ability of this am- photericin B congener provided new clues to the role of the 35-hydroxy group and is consistent with the in- volvement of double barrel ion chan- nels in causing electrolyte efflux.

Synthesis and Biophysical Studies on 35-Deoxy Amphotericin B Methyl Ester

The use of molecular editing in the elucidation of the mechanism of action of amphotericin B is presented. A modular strategy for the synthesis of amphotericin B and its designed analogues is developed, which relies on an efficient gram-scale synthesis of various subunits of amphotericin B. A novel method for the coupling of the mycosamine to the aglycone was identified. The implementation of the approach has enabled the preparation of 35-deoxy amphotericin B methyl ester. Investigation of the antifungal activity and efflux-inducing ability of this amphotericin B congener provided new clues to the role of the 35-hydroxy group and is consistent with the involvement of double barrel ion channels in causing electrolyte efflux.

Trimethylation Enhancement Using Diazomethane (TrEnDi) II: Rapid In-Solution Concomitant Quaternization of Glycerophospholipid Amino Groups and Methylation of Phosphate Groups via Reaction with Diazomethane Significantly Enhances Sensitivity in Mass Spectrometry Analyses via a Fixed, Permanent Positive Charge

A novel mass spectrometry (MS)-based lipidomics strategy that exposes glycerophospholipids to an ethereal solution of diazomethane and acid, derivatizing them to contain a net fixed, permanent positive charge, is described. The sensitivity of modified lipids to MS detection is enhanced via improved ionization characteristics as well as consolidation of ion dissociation to form one or two strong, characteristic polar headgroup fragments. Our strategy has been optimized to enable a priori prediction of ion fragmentation patterns for four subclasses of modified glycerophospholipid species. Our method enables analyte ionization regardless of proton affinity, thereby decreasing ion suppression and permitting predictable precursor ion-based quantitation with improved sensitivity in comparison to MS-based methods that are currently used on unmodified lipid precursors.

Trimethylation Enhancement using Diazomethane (TrEnDi): Rapid On-Column Quaternization of Peptide Amino Groups via Reaction with Diazomethane Significantly Enhances Sensitivity in Mass Spectrometry Analyses via a Fixed, Permanent Positive Charge

Defining cellular processes relies heavily on elucidating the temporal dynamics of proteins. To this end, mass spectrometry (MS) is an extremely valuable tool; different MS-based quantitative proteomics strategies have emerged to map protein dynamics over the course of stimuli. Herein, we disclose our novel MS-based quantitative proteomics strategy with unique analytical characteristics. By passing ethereal diazomethane over peptides on strong cation exchange resin within a microfluidic device, peptides react to contain fixed, permanent positive charges. Modified peptides display improved ionization characteristics and dissociate via tandem mass spectrometry (MS(2)) to form strong a2 fragment ion peaks. Process optimization and determination of reactive functional groups enabled a priori prediction of MS(2) fragmentation patterns for modified peptides. The strategy was tested on digested bovine serum albumin (BSA) and successfully quantified a peptide that was not observable prior to modification. Our method ionizes peptides regardless of proton affinity, thus decreasing ion suppression and permitting predictable multiple reaction monitoring (MRM)-based quantitation with improved sensitivity.

[2+2] Photoadditions with chiral 2,5-cyclohexadienone synthons

Abstract Three chiral 2,5-cyclohexadienone synthons bearing different chiral auxiliaries were examined in [2+2] photoadditions with cyclopentene. Regeneration of the ‘masked’ double bond in the adducts resulted in the preparation of optically active 5-4-6 adducts. The enantiomeric purity of each adduct was found to be >95% using comparative 13 C NMR analysis of the appropriate ketals. The asymmetry induced in the cycloaddition step of our methodology indicated that the facial selectivity was directly correlated to the degree of steric bulk of the chiral auxiliary on the synthon.

Trimethylation Enhancement Using 13C-Diazomethane (13C-TrEnDi): Increased Sensitivity and Selectivity of Phosphatidylethanolamine, Phosphatidylcholine, and Phosphatidylserine Lipids Derived from Complex Biological Samples

Significant sensitivity enhancements in the tandem mass spectrometry-based analysis of complex mixtures of several phospholipid classes has been achieved via (13)C-TrEnDi. (13)C-TrEnDi-modified phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) lipids extracted from HeLa cells demonstrated greater sensitivity via precursor ion scans (PISs) than their unmodified counterparts. Sphingomyelin (SM) species exhibited neither an increased nor decreased sensitivity following modification. The use of isotopically labeled diazomethane enabled the distinction of modified PE and modified PC species that would yield isobaric species with unlabeled diazomethane. (13)C-TrEnDi created a PE-exclusive PIS of m/z 202.1, two PS-exclusive PISs of m/z 148.1 and m/z 261.1, and a PIS of m/z 199.1 for PC species (observed at odd m/z values) and SM species (observed at even m/z values). The standardized average area increase after TrEnDi modification was 10.72-fold for PE species, 2.36-fold for PC, and 1.05-fold for SM species. The sensitivity increase of PS species was not quantifiable, as there were no unmodified PS species identified prior to derivatization. (13)C-TrEnDi allowed for the identification of 4 PE and 7 PS species as well as the identification and quantitation of an additional 4 PE and 4 PS species that were below the limit of detection (LoD) prior to modification. (13)C-TrEnDi also pushed 24 PE and 6 PC lipids over the limit of quantitation (LoQ) that prior to modification were above the LoD only.

Enhanced selectivity of a molecularly imprinted polymer toward the target molecule via esterification of non-specific binding sites with diazomethane.

Diazomethane (CH2N2) was used to methylate the non‐specific binding sites after molecularly imprinted polymer particles were prepared using methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross‐linker and bisphenol A (BPA) as the template. After diazomethane treatment and subsequent removal of BPA by triethylamine, the treated molecularly imprinted polymer (TMIP) particles were tested for binding selectivity toward BPA and other organic compounds by capillary electrophoresis with ultraviolet detection. Even in the presence of compounds that were positively charged, neutral or negatively charged in the background electrolyte, BPA was selectively bound with the highest efficiency. A significant decrease in the affinity for metformin (MF, a positively charged compound), along with 13C nuclear magnetic resonance spectra and electrophoretic mobility data, provided strong evidence for the elimination of non‐specific –COOH binding sites in the TMIP particles. Only 8% of MF and 16% of diclofenac sodium salt (a negatively charged compound) remained as non‐specific bindings because of hydrophobic interactions. Further comparison with poly(methyl methacrylate) revealed the true merits of the TMIP, which exhibited minimal non‐specific bindings while preserving a high level of specific binding owing to molecular recognition. Copyright

Selectivity Enhancement in Molecularly Imprinted Polymers for Binding of Bisphenol A

Bisphenol A (BPA) is an estrogen-mimicking chemical that can be selectively detected in water using a chemical sensor based on molecularly imprinted polymers (MIPs). However, the utility of BPA-MIPs in sensor applications is limited by the presence of non-specific binding sites. This study explored a dual approach to eliminating these sites: optimizing the molar ratio of the template (bisphenol A) to functional monomer (methacrylic acid) to cross-linker (ethylene glycol dimethacrylate), and esterifying the carboxylic acid residues outside of specific binding sites by treatment with diazomethane. The binding selectivity of treated MIPs and non-treated MIPs for BPA and several potential interferents was compared by capillary electrophoresis with ultraviolet detection. Baclofen, diclofenac and metformin were demonstrated to be good model interferents to test all MIPs for selective binding of BPA. Treated MIPs demonstrated a significant decrease in binding of the interferents while offering high selectivity toward BPA. These results demonstrate that conventional optimization of the molar ratio, together with advanced esterification of non-specific binding sites, effectively minimizes the residual binding of interferents with MIPs to facilitate BPA sensing.

Efficient, scalable and economical preparation of tris(deuterium)- and 13C-labelled N-methyl-N-nitroso-p-toluenesulfonamide (Diazald®) and their conversion to labelled diazomethane.

A method for the preparation of multi-gramme quantities of N-methyl-d3-N-nitroso-p-toluenesulfonamide (Diazald-d3) and N-methyl-(13)C-N-nitroso-p-toluenesulfonamide (Diazald-(13)C) and their conversion to diazomethane-d2 and diazomethane-(13) C, respectively, is presented. This approach uses robust and reliable chemistry, and critically, employs readily commercially available and inexpensive methanol as the label source. Several reactions of labelled diazomethane are also reported, including alkene cyclopropanation, phenol methylation and α-diazoketone formation, as well as deuterium scrambling in the preparation of diazomethane-d2 and subsequent methyl esterification of benzoic acid.

Mild, Rapid, and Inexpensive Microwave-Assisted Synthesis of Allylic and Propargylic Esters

Abstract A variety of allylic and propargylic esters were rapidly prepared via microwave heating of their corresponding mixed anhydride derived from pivaloyl chloride. The reaction conditions were modified to account for the sterics of the alcohol and the electronics of the carboxylic acid. Supplemental materials are available for this article. Go to the publishers online edition of Synthetic Communications® to view the free supplemental file. GRAPHICAL ABSTRACT