Ultrafast amidation of esters using lithium amides under aerobic ambient temperature conditions in sustainable solvents.

Lithium amides constitute one of the most commonly used classes of reagents in synthetic chemistry. However, despite having many applications, their use is handicapped by the requirement of low temperatures, in order to control their reactivity, as well as the need for dry organic solvents and protective inert atmosphere protocols to prevent their fast decomposition. Advancing the development of air- and moisture-compatible polar organometallic chemistry, the chemoselective and ultrafast amidation of esters mediated by lithium amides is reported. Establishing a novel sustainable access to carboxamides, this has been accomplished via direct C-O bond cleavage of a range of esters using glycerol or 2-MeTHF as a solvent, in air. High yields and good selectivity are observed while operating at ambient temperature, without the need for transition-metal mediation, and the protocol extends to transamidation processes. Pre-coordination of the organic substrate to the reactive lithium amide as a key step in the amidation processes has been assessed, enabling the structural elucidation of the coordination adduct [{Li(NPh2)(O[double bond, length as m-dash]CPh(NMe2))}2] (8) when toluene is employed as a solvent. No evidence for formation of a complex of this type has been found when using donor THF as a solvent. Structural and spectroscopic insights into the constitution of selected lithium amides in 2-MeTHF are provided that support the involvement of small kinetically activated aggregates that can react rapidly with the organic substrates, favouring the C-O bond cleavage/C-N bond formation processes over competing hydrolysis/degradation of the lithium amides by moisture or air.

Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.

The European Medicines Agency received recently the first marketing authorization application for a biosimilar monoclonal antibody (mAb) and adopted the final guidelines on biosimilar mAbs and Fc-fusion proteins. The agency requires high similarity between biosimilar and reference products for approval. Specifically, the amino acid sequences must be identical. The glycosylation pattern of the antibody is also often considered to be a very important quality attribute due to its strong effect on quality, safety, immunogenicity, pharmacokinetics and potency. Here, we describe a case study of cetuximab, which has been marketed since 2004. Biosimilar versions of the product are now in the pipelines of numerous therapeutic antibody biosimilar developers. We applied a combination of intact, middle-down, middle-up and bottom-up electrospray ionization and matrix assisted laser desorption ionization mass spectrometry techniques to characterize the amino acid sequence and major post-translational modifications of the marketed cetuximab product, with special emphasis on glycosylation. Our results revealed a sequence error in the reported sequence of the light chain in databases and in publications, thus highlighting the potency of mass spectrometry to establish correct antibody sequences. We were also able to achieve a comprehensive identification of cetuximab's glycoforms and glycosylation profile assessment on both Fab and Fc domains. Taken together, the reported approaches and data form a solid framework for the comparability of antibodies and their biosimilar candidates that could be further applied to routine structural assessments of these and other antibody-based products.

Effects of coancestry on accuracy of individual assignments to population of origin: examples using Great Lakes lake trout (Salvelinus namaycush).

Methods for assigning individuals to population of origin are widely used in ecological genetics, resources management, and forensics. Characteristics of genetic data obtained from putative source populations that enhance accuracy of assignment are well established. How non-independence within and among unknown individuals to be classified [i.e., gene correlations within individual (inbreeding) and gene correlations among individuals within group (coancestry)] affect assignment accuracy is poorly understood. We used empirical data for six microsatellite loci and offspring from full-sib crosses of hatchery strains of lake trout (Salvelinus namaycush; Salmonidae) representing known levels of coancestry (mean theta = 0.006 and 0.06) within families to investigate how gene correlations can affect assignment. Additional simulations were conducted to further investigating the influence of allelic diversity (2, 6 or 10 alleles per locus) and inbreeding (F = 0.00, 0.05, and 0.15) on assignment accuracy for cases of low and high inter-population variance in allele frequency (mean F (st) = 0.01 and 0.1, respectively). Inbreeding had no effect on accuracy of assignments. In contrast, variance in assignment accuracy across replicated simulations, and for each empirical case study increased with increasing coancestry, reflecting non-independence of probabilities of correct assignment among members of kin groups. Empirical estimates of assignment error rates should be interpreted with caution if appreciable levels of coancestry are suspected. Additional emphasis should be placed on sampling designs (spatially and temporally) that define or minimize the potential for sampling related individuals.

Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis.

Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500 fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9 min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30 min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) in combination with LC/MS/MS using 4 min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of post-translational modifications (PTMs). Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS-MS/MS switching and scanning capabilities.