Pyrolysis of nucleic acids

Earlier studies related to the pyrolysis of nucleic acids were done on whole microorganisms when it was shown that pyrolysis of DNA generates a significant proportion of furfuryl alcohol [2], This compound was even used in a Py-GC/MS technique for the quantitation of DNA content in cultured mammalian cells [4]. 

The spectrum is dominated by the peaks corresponding to nicotinamide (MW 124) and adenine (MW 135) showing that the nature of the base is also important for the spectrum features. 

Several Py-MS fragmentations were reported for DNA when using DPI instrumentation, and they can be described by the following scheme [7]  

Based on Py-MS studies, it was possible to calculate the abundance of several bases in the nucleic acids and to obtain deviations between the composition of the nucleic acids of healthy and sick organs [7]. The abundance of the base expressed as deoxynucleoside dB % has been calculated from the ratio (BH ) peak height / S [(BH ) peak heights] defined as Deflection %. The sum is taken over the peak for adenine (m/z 135), cytosine (m/z 111), guanine (m/z 151), and thymine (m/z 126). The 

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Pyrolysis of nucleic acids compared to ion fragments formation from adenosine-5 -phosphate and 2-deoxyadenosine-5 -phosphate. 

The pyrolysis of nucleic acids generally occurs between 200 and 250°C, using a normal direct inlet probe. In order to decrease the possible contamination of the mass spectrometer, different pyroprobes - or even flash... 

Oxetan-2-one (j3-propanolactoiic) unci also oxetani-3-one are known, although most interest lies in the former compound. It is a carcinogen, possibly because it can alkylate guanidine, one of the constituent bases of nucleic acids. On standing, oxetan-2-one slowly polymerizes, and on pyrolysis it forms propenoic acid (Scheme 8.13). Alkaline hydrolysis gives a salt of 3-hydroxypropanoic acid. 

Phosphate esters show important thermal susceptibility (Fig. 1). Dialkyl phosphates, such as those found in nucleic acids (Fig. 2), decompose with the initial loss of one alkyl group, the concomitant transfer of protons, followed by the elimination of the second alkyl group and the subsequent loss of water. This thermal instability of phosphoesters has been used in the analysis of nucleic acids. Thus the pyrolysis that usually precedes the recording of a mass spectrum permits cleavage of the polymeric phosphoesters (nucleic acids), followed by phosphate extrusions, producing nucleotides or simple nucleosides as fragment ions. 

The main pyrolysis mechanism of nucleic acids (occurring at temperatures as low as 180°C) is the expulsion of the polysaccharide moiety with the simultaneous formation of base-phosphate condensates. The base phosphate complex is further pyrolysed, yielding the base fragments. Compared with polysaccharides and lignin, the application of Py-GC/MS techniques to the analysis of nucleic acids is still in its infancy. 

Understanding the product formation from the pyrolysis of amino acids is the first step in comprehending pyrolysis behavior for more complex systems, such as proteins and nucleic acids, which comprise a large quantity of the material in biological systems. Later in this chapter, in Section 10.5, proteins are discussed in more detail. 

HPLC in Nucleic Acid Research Methods and Applications, edited by Phyllis R. Brown 29. Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman andE. J. Levy 30. Modem Chromatographic Analysis of the Vitamins, edited by Andre P. De Leenheer, Willy E. 

One possibility for understanding the Py-MS results for nucleic acids is to use a parallel between the mass spectral fragmentation and the pyrolysis fragmentation. This can be exemplified for the mass spectra of the silylated 2 -deoxyadenosine-5 -phosphate (deoxy-AMP) and adenosine-5 -phosphate (AMP), which are shown in Figure 13.1.1 and 13.1.2 respectively (El spectra at 70 eV). The interpretation of several fragments in these spectra is given in Table 13.1.1. 

More valuable information on nucleic acids has been obtained from pyrolysis data when it was possible to evaluate the nature and abundance of the purine/pyrimidine bases. The information on these bases is important for monitoring in vitro DNA synthesis [5,6], for the evaluation of chromosome modifications [7], and for the study of complex formation of DNA with cisplatin [11,12]. As indicated previously, the DIP technique was reported to be more useful for detecting the base component of the nucleic acid. However, some information on the bases can be obtained also by Curie point Py-MS, as it can be seen from the spectrum of NADPH (nicotinamide adenine dinucleotide phosphate) shown in Figure 13.2.3. The spectrum was obtained in similar conditions as spectra for DNA and RNA shown previously [8]. 

Myers and Smith [133] showed that in identifying biological substances by Py-GC use should be made of a rubidium thermionic detector selective towards nitrogen-containing pyrolysis products of proteins and nucleic acids, because when a conventional flame-ionization detector is employed most characteristic structures are masked by the pyrolysis products of carbohydrates and lipids. 

An improved understanding of the quantity and quality of more complex pyrolysis products of organic matter can be obtained by first examining the relatively simpler pyrolysis characteristics of the major types of biomacromolecules polysaccharides, lignin, nucleic acids, proteins, and lipids (Figure 8.2). 

CDS Analytical was used with (jC/ion trap MS by Smith and Snyder to analyze lipid and fatty acid components in a variety of bacteria with a total analysis time under 10 min. Snyder et ah also used 13 strains of 8 bacterial species to demonstrate the use of Curie-point wire and quartz tube pyrolysis coupled to short-colunm GC/MS for the detection of biological warfare agents. Lipid and nucleic acid components were the primary target for discrimination purposes. Two articles by DeLuca et al. studied Curie-point pyrolysis-tandem MS for direct analysis of bacterial fatty acids. Py-MS results correlated well with previous Py-GC/MS data on the same bacteria. Interestingly, better classification results were obtained when just Py-MS fatty acid profiles were employed rather than total ion spectra. 

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