Mechanistic Investigation of the Reactions between Cyclohexane Carboxaldehyde and Ureido Groups

Model reactions have been performed to explore the reactivity of a variety of ureido groups with cyclohexane carboxaldehyde. The reaction mechanism between ureido and aldehyde functionalities is more complicated than expected. A new heterocyclic product was identified, which is very stable and solvent resistant. The final product profile is reactant and solvent dependent. In the cases of urea, alkyl urea, and benzyl urea, the reaction pathway goes from hemiaminal to aminal, to enamine, and finally to the heterocyclic product with nearly 100% yield. For other investigated ureido groups, the reaction stopped at the enamine product, and products are a mixture of hemiaminal, aminal, and enamine. All reaction steps are reversible except for the last step. The structure of the unique cyclic product was determined combining NMR and LC–MS analysis, and the reaction pathway was verified by kinetics studies

Organic Acid Quantitation by NeuCode Methylamidation

We have developed a multiplexed quantitative analysis method for carboxylic acids by liquid chromatography high resolution mass spectrometry. The method employs neutron encoded (NeuCode) methylamine labels (¹³C or ¹⁵N enriched) that are affixed to carboxylic acid functional groups to enable duplex quantitation via mass defect measurement. This work presents the first application of NeuCode quantitation to small molecules. We have applied this technique to detect adulteration of olive oil by quantitative analysis of fatty acid methyl amide derivatives, and the quantitative accuracy of the NeuCode analysis was validated by GC/MS. Currently, the method enables duplex quantitation and is expandable to at least 6-plex analysis

Segmentation of Precursor Mass Range Using “Tiling” Approach Increases Peptide Identifications for MS¹‑Based Label-Free Quantification

Label-free quantification is a powerful tool for the measurement of protein abundances by mass spectrometric methods. To maximize quantifiable identifications, MS¹-based methods must balance the collection of survey scans and fragmentation spectra while maintaining reproducible extracted ion chromatograms (XIC). Here we present a method which increases the depth of proteome coverage over replicate data-dependent experiments without the requirement of additional instrument time or sample prefractionation. Sampling depth is increased by restricting precursor selection to a fraction of the full MS¹ mass range for each replicate; collectively, the <i>m</i>/<i>z</i> segments of all replicates encompass the full MS¹ range. Although selection windows are narrowed, full MS¹ spectra are obtained throughout the method, enabling the collection of full mass range MS¹ chromatograms such that label-free quantitation can be performed for any peptide in any experiment. We term this approach “binning” or “tiling” depending on the type of <i>m</i>/<i>z</i> window utilized. By combining the data obtained from each segment, we find that this approach increases the number of quantifiable yeast peptides and proteins by 31% and 52%, respectively, when compared to normal data-dependent experiments performed in replicate

Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by <i>Rhodopseudomonas palustris</i>

Lignocellulosic biomass hydrolysates hold great potential as a feedstock for microbial biofuel production, due to their high concentration of fermentable sugars. Present at lower concentrations are a suite of aromatic compounds that can inhibit fermentation by biofuel-producing microbes. We have developed a microbial-mediated strategy for removing these aromatic compounds, using the purple nonsulfur bacterium Rhodopseudomonas palustris. When grown photoheterotrophically in an anaerobic environment, R. palustris removes most of the aromatics from ammonia fiber expansion (AFEX) treated corn stover hydrolysate (ACSH), while leaving the sugars mostly intact. We show that R. palustris can metabolize a host of aromatic substrates in ACSH that have either been previously described as unable to support growth, such as methoxylated aromatics, and those that have not yet been tested, such as aromatic amides. Removing the aromatics from ACSH with R. palustris, allowed growth of a second microbe that could not grow in the untreated ACSH. By using defined mutants, we show that most of these aromatic compounds are metabolized by the benzoyl-CoA pathway. We also show that loss of enzymes in the benzoyl-CoA pathway prevents total degradation of the aromatics in the hydrolysate, and instead allows for biological transformation of this suite of aromatics into selected aromatic compounds potentially recoverable as an additional bioproduct

Lignin Conversion to Low-Molecular-Weight Aromatics via an Aerobic Oxidation-Hydrolysis Sequence: Comparison of Different Lignin Sources

Diverse lignin samples have been subjected to a catalytic aerobic oxidation process, followed by formic-acid-induced hydrolytic depolymerization. The yield of monomeric aromatic compounds varies depending on the lignin plant source and pretreatment method. The best results are obtained from poplar lignin isolated via a acidolysis pretreatment method, which gives 42 wt% yield of low-molecular-weight aromatics. Use of other pretreatment methods and/or use of maple and maize lignins afford yields of aromatics ranging from 3 to 31 wt%. These results establish useful references for the development of improved oxidation/depolymerization protocols