Mass spectrometry carboxylic acid derivatives

Mass Spectrometry A prominent peak m the mass spectra of most carboxylic acid derivatives corresponds to an acyhum ion derived by cleavage of the bond to the car bonyl group... 

Mass spectrometry is one of the major techniques in the interdisciplinary field of proteomics. It provides a rapid, sensitive and reliable means of protein identification and structural determination, allowing for development in this newly baptised but yet classical field of biochemistry and biomedicine. The use of electrospray ionisation in conjunction with a tandem mass spectrometer (MS/MS) provides essential amino acid sequence information from the m/z values of the so-called b andy ions formed from cleavage of the amide bond of a protonated peptide. This reaction requires proton catalysis, and the mechanism is of interest in the present context, since it is closely related to the processes occurring in other protonated carboxylic acid derivatives. 

We don t expect you to be satisfied with the bland statement that tetrahedral intermediates are formed in these reactions of course, you wonder how we know that this is true. The first evidence for tetrahedral intermediates in the substitution reactions of carboxylic acid derivatives was provided by Bender in 1951. He made carboxylic acid derivatives RCOX that had been labelled with an isotope of oxygen, 0. This is a non-radioactive isotope that is detected by mass spectrometry. He then reacted these derivatives with water to make labelled carboxylic acids. By any reasonable mechanism, the products would have one i 0 atom from the labelled starting material. Because the proton on a carboxylic acid migrates rapidly from one oxygen to another, both oxygens are labelled equally. 

Esters are carboxylic acid derivatives, and the spectrum of ethyl acetate is shown in Figure 14.19D. The carbonyl absorption does not distinguish this compound from an aldehyde or a ketone, but there is the C-O absorption at about 1200 cm 1. Because this is in the fingerprint region, however, its position can be difficult to identify. This is clearly the case for methyl pentanoate, where the C-0 absorption can easily be missed or misidentified. Based only on the infrared, it may be difficult to distinguish an ester from an aliphatic aldehyde or ketone. If the formula is known, however (from mass spectrometry), the identification is easier because an ester has two oxygen atoms, whereas the aldehyde or ketone has only one. 

Note Trimethylsilylether (TMS) derivatives are frequently employed to vola-tize alcohols, [14,203] carboxylic acids, [204,205] and other compounds [206,207] for mass spectrometry, and for GC-MS applications in particular. The El mass spectra of TMS derivatives exhibit weak molecular ion peaks, clearly visible [M-CHa]" signals and often [Si(Me)3], m/z 73, as the base peak. 

Chien, C.-J M. J. Charles, K. G. Sexton, and H. E. Jeffries, Analysis of Airborne Carboxylic Acids and Phenols as Their Pentafluo-robenzyl Derivatives Gas Chromatography/Ion Trap Mass Spectrometry with a Novel Chemical Ionization Reagent, PFBOH, Environ. Sci. Technol., 32, 299-309 (1998). 

Several enzymes catalyze stepwise removal of amino acids from one or the other end of a peptide chain. Carboxypeptidases232 remove amino acids from the carboxyl-terminal end, while aminopeptidases attack the opposite end. Using chromatographic methods, the amino acids released by these enzymes may be examined at various times and some idea of the sequence of amino acids at the chain ends may be obtained. A dipeptidyl aminopeptidase from bovine spleen cuts dipeptides one at a time from the amino terminus of a chain. These can be converted to volatile trimethylsilyl derivatives and identified by mass spectrometry.233 If the chain is shortened by one residue using the Edman degradation (Section 3) and the dipeptidyl aminopeptidase is again used, a different set of dipeptides that overlaps the first will be obtained and a sequence can be deduced. Carboxypeptidase Y can be used with MALDI mass spectrometry to deduce the C-terminal amino acid sequence for a peptide. However, He and Leu cannot be distinquished. 

Analytical pyrolysis with field ionization mass spectrometry (online Py-FIMS) or in combination with GC/MS (Curie point Py-GC/MS) led to a significant increased number of identified subunits (e.g., Bracewell et al., 1989 Schulten et al., 2002). In addition, the application of tetramethylammonium hydroxide (TMAH) methylation, followed by GC/MS, was successfully applied. The most abundant pyrolysis products identified are benzene, phenol and furan derivatives, aliphatic and carboxylic compounds, and indene derivatives (Schulten et al.,2002). New approaches have been used for the quantification of n-alkyl fatty acids of DOM and isolated fractions in the form of individual compounds after solvent extraction followed by derivatiza-tion with TMAH. 

Solutions of triethylamine (Et3N) 14 (1.0M), premixed carboxylic acid/alkyl chloroformate (1.0 M respectively), and 4-dimethylaminopyri-dine 15 (0.5 M) in MeCN were introduced into the reactor from separate inlets and the reaction products collected at the outlet in MeCN, prior to analysis by gas chromatography-mass spectrometry (GC-MS). Under optimized reaction conditions, the authors were able to synthesize the methyl 16, ethyl 17, and benzyl 18 esters in quantitative conversion, with no anhydride or deprotection by-products detected (as observed in conventional batch reactions). In addition to the Boc-glycine derivatives illustrated in Scheme 4, the authors also esterified a series of aromatic carboxylic acids with yields ranging from 91 to 100%, depending on the additional functional groups present. 

The special type of carbonyl-group derivatization is aimed for gas chromatography-mass spectrometry (GC-MS) determination of double-bond C = C positions in the unsaturated long-chain acids. The analytical derivatives for the solution of this problem are nitrogen-containing heterocycles. These compounds can be synthesized by condensation of carboxylic acids with 2-amino-2-methyl-l-propanol (2-substituted 4,4-dimethyloxazolines), 2-aminophenol (2-substituted benzoxazoles), and so forth. 

A serious limitation of this powerful analytical technique is the inability to detect and quantify Gin and Asn m an amino acid mixture In the acid-catalyzed esterification reaction, the amides (Gin and Asn) are rapidly deaminated and converted to the same derivatives as those formed by Glu and Asp, respectively. Consequently, summations of both Glu and Gin concentrations and Asp and Asn concentrations are determined by this method. It has been shown that when Gin is heated to a temperature of about 100°C in an acidified alcoholic medium, rapid conversion to glutamic acid diester takes place. Pyrrolidone carboxylic acid ester has been shown by mass spectrometry to be a cyclized intermediate (Fig 2). This intermediate reaches its maximum concentrahon after heating at 100°C for 5-10 mm (Collins and Summer, 1978). 

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