Multicomponent Reactions and Their Libraries - a New Approach to Preparative Organic Chemistry

Classical chemical syntheses from n starting materials usually require sequences of at least n-I preparation steps, including separation and purification of the intermediates. A perfect alternative for the rapid syntheses of a large variety of agrichemically and pharmaceutically relevant products are one-pot syntheses by multicomponent reactions (MCR)on the basis of isocyanides. Four to seven different types of components mixed in a reaction vessel undergo the transformation to one molecule. Due to the last irreversible step that involves the isocyanides, a stable product results in quantitative yields. Using more than one representative of each type of starting materials (i.e. different isocyanides, amines, etc.) in the same vessel, all possible combinations will lead to a molecular library with hundreds and thousands of products formed according to a given reaction scheme. The design of such syntheses and the handling of the results require adequate mathematics and computer tools

MCR XVII. Three Types of MCRs and the Libraries – Their Chemistry of Natural Events and Preparative Chemistry

The one-pot Multicomponent Reactions (MCRs)1 convert more than two different components into their products with at least two new chemical bonds, and the products contain all educts or at least some parts of them. Many chemical reactions have several, but not all, aspects of the MCRs. Three different basic types (I–III) and two subclasses (A and B) of MCRs can take place. Chemistry had started in the nature of our world roughly 4.6 billion years ago, including MCRs of the types I and II, forming libraries of many different products. A little later, the living cells came into existence, and their biochemical MCRs of all three types started. In their various local parts their biochemical products are selectively formed by their enzyme-assisted procedures, but many of their MCRs belong to type III. The preparative chemistry of MCRs started in the middle of the last century, when the first equilibrating but isolateable 3CR products of type IB were formed. The pre-final reactions of type I form compounds, which react further and form their final products irreversibly by MCRs of type II. The type IIA products are usually heterocycles, whereas those of type IIB are generally products of isocyanides. The U-4CR of type IIB was introduced and this led to a new preparative MCR chemistry. Their educts and intermediate products equilibrate (type IA) and undergo irreversible CII → CIV &alpha,-additions of the isocyanides, followed by a variety of rearrangements into their final products (type IIB). In recent years, unions of higher numbers of components were introduced, forming even more diverse types of products. The MCR libraries were proposed in 1961, and since 1995 this chemistry has become an essential part of the chemical research in industrial search for new desirable products. This methodology requires much less work than all previous methods and proceeds many orders of magnitude faster

Correlations between milk and plasma levels of amino and carboxylic acids in dairy cows.

The objective of this study was to investigate the relationship between the concentrations of 19 amino acids, glucose, and seven carboxylic acids in the blood and milk of dairy cows and their correlations with established markers of ketosis. To that end, blood plasma and milk specimens were collected throughout lactation in two breeds of dairy cows of different milk yield. Plasma concentrations of glucose, pyruvate, lactate, α-aminobutyrate, β-hydroxybutyrate (BHBA), and most amino acids, except for glutamate and aspartate, were on average 9.9-fold higher than their respective milk levels. In contrast, glutamate, aspartate, and the Krebs cycle intermediates succinate, fumarate, malate, and citrate were on average 9.1-fold higher in milk than in plasma. For most metabolites, with the exception of BHBA and threonine, no significant correlations were observed between their levels in plasma and milk. Additionally, milk levels of acetone showed significant direct relationships with the glycine-to-alanine ratio and the BHBA concentration in plasma. The marked decline in plasma concentrations of glucose, pyruvate, lactate, and alanine in cows with plasma BHBA levels above the diagnostic cutoff point for subclinical ketosis suggests that these animals fail to meet their glucose demand and, as a consequence, rely increasingly on ketone bodies as a source of energy. The concomitant increase in plasma glycine may reflect not only the excessive depletion of protein reserves but also a potential deficiency of vitamin B6

Tools and applications for one- and two-dimensional gas chromatography – time-of-flight mass spectrometry-based metabolomics

The study of cellular metabolite profiles and changes therein due to genetic and environmental influences is termed metabolomics. A major, yet to be realized goal is the detection and quantification of all metabolites in a single analysis. In this work, a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) method was designed and validated that assembles the entire available analytical information from each sample in one data matrix for subsequent statistical evaluation. For the purpose of data merging we developed and implemented the retention time correction and data alignment tool INCA (Integrative Normalization and Comparative Alignment). The INCA module capitalized on the characteristic fragmentation behavior of silylated metabolites upon EI ionization by using the integral of the m/z 73 ion trace of the trimethylsilyl (TMS) group as quantitative measure for all features. The method was applied to reveal differences in metabolite composition between (i) an Escherichia coli wild type and a double-mutant strain lacking the transhydrogenases UdhA and PntAB and (ii) serum and plasma. Subsequently, we evaluated the performance of the Statistical Compare alignment function introduced later by LECO for GC×GC-TOFMS data and compared it to INCA. GC×GC-TOFMS was comprehensively evaluated against various 1D-GC-MS techniques using a set of 43 metabolite standards from different chemical classes and metabolic pathways. GC×GC-TOFMS proved to be the most powerful method with a linear range of more than three orders of magnitude, LLOQs in the sub-micromolar and LODs in the nanomolar range. GC×GC-TOFMS was also employed for absolute quantification of amino acid enantiomers (AAEs) as their methyl chloroformate derivatives and results were compared to those of a previously established 1D-GC-qMS method with single ion monitoring. The coupling of a gamma-cyclodextrin (Rt-gammaDEXsa) with an amino acid selective (ZB-AAA) column resulted in enhanced peak resolution. Twenty AAEs including the critcal peak pair L-leucine/D-isoleucine, which exhibited equal fragmentation behavior upon EI ionization in 1D-GC-MS, could be baseline separated. Except for methionine enantiomers, distinctly improved LLOQs were obtained. The method was applied to the analysis of AAE serum concentrations in patients suffering from liver cirrhosis and showed significantly increased D-AA concentrations and slightly decreased L-AA levels compared to a control group

Metabolic fingerprinting using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry

Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF-MS) is applied to the comparative metabolic fingerprinting of physiological fluids. Stable isotope-labeled internal standards plus norvaline serve as extraction standards and are added to the blanks, controls and patient samples prior to protein precipitation with methanol. The extracts are evaporated to complete dryness and derivatized in two steps using methoximation with methoxylamine hydrochloride (MeOx) and silylation with N-methyl-N-trimethylsily-trifluoroacetamide (MSTFA). Between derivatization steps a second internal standard containing odd-numbered, saturated straight chain fatty acids is added for quality control and to normalize retention time shifts. After GC × GC-TOF-MS analysis raw data are processed, aligned, and combined in one data matrix for subsequent statistical evaluation. Both a custom-made and the NIST 05 library are used to preliminarily identify significant metabolites. For verification purposes, commercial standards are run individually. Absolute quantification of selected metabolites is achieved by using a multi-point calibration curve and isotope-labeled internal standards

Comprehensive two-dimensional gas chromatography in metabolomics

One of the major objectives in metabolomics is the identification of subtle changes in metabolite profiles as affected by genetic or environmental factors. Comprehensive two-dimensional gas chromatography (GC × GC) hyphenated to a fast-acquisition mass spectrometer is a well-established analytical technique to study the composition of complex samples due to its enhanced separation capacity, sensitivity, peak resolution, and reproducibility. This review reports applications of GC × GC to metabolomics studies of sample of different types (biofluid, cells, tissue, bacteria, yeast, plants), and discusses its advantages and limitations