Synthesis of isotopically labelled DNA degradation products for use in mass spectrometric studies of cellular DNA damage

Abstract

SUMMARYThirteen substituted purines and pyrimidines bearing from three to five carbon, nitrogen and/or deuterium isotopic labels have been synthesized in yields ranging from . l to 70%. Most of the products originate from the same small number of commercially available labelled starting materials, and in several cases one intermediate leads to two products, thus minimizing the expense and time required. The parent compounds are found in tissue as the result of DNA damage often linked with carcinogenesis and mutagenesis. The synthesized compounds serve as internal standards for the study of DNA damage using mass spectrometry.

Salt shield: intracellular salts provide cellular protection against ionizing radiation in the halophilic archaeon, Halobacterium salinarum NRC‐1

“…FapyAde‐ 13 C 2 , 15 N 2 and FapyGua‐ 13 C 2 , 15 N 2 were purchased from Cambridge Isotope Laboratories (Cambridge, MA). 8‐OH‐Gua‐ 13 C 2 , 15 N 2 was synthesized as previously described (Nelson, 1996). DNA aliquots (50 μg) were supplemented with aliquots of the internal standards, and hydrolysed with 2 μg of Fpg for 1 h (Reddy et al ., 2004).…”

GC/MS Measurement of Nanomaterial-Induced DNA Modifications in Isolated DNA

“…Usage of stable isotope-labeled internal standards (ISTDs) from SRM 2396 enables absolute identification and quantification of the following DNA lesions: 4,6-diamino-5-formamidopyrimidine (FapyAde), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 8-hydroxyadenine (8-OH-Ade), 5-hydroxycytosine (5-OH-Cyt), 5-hydroxyuracil (5-OH-Ura), 5-(hydroxymethyl)uracil (5-(OHMe)Ura), thymine glycol (ThyGly) and 5-hydroxy-5-methylhydantoin (5-OH-5-MeHyd). Other important DNA lesions such as 8-hydroxyguanine (8-OH-Gua) can also be measured with this approach by hydrolyzing 8-hydroxy-2-deoxyguanosine-15 N 5 ISTD in SRM 2396 to 8-hydroxyguanine-15 N 5 (8-OH-guanine-15 N 5 ) or stable isotopelabeled versions of 8-OH-Gua may be synthesized directly [14]. In this procedure, 8-OHguanine-15 N 5 is obtained by hydrolysis of 8-hydroxy-2-deoxyguanosine-15 N 5 with 60% formic acid at 140 °C for 30 min followed by lyophilization.…”

GC-MS/MS Measurement of Nanomaterial- Induced DNA Modifications in Isolated DNA

“…Usage of stable isotope-labeled internal standards (ISTDs) from SRM 2396 enables absolute identification and quantification of the following DNA lesions: 4,6diamino-5-formamidopyrimidine (FapyAde), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 8-hydroxyadenine (8-OH-Ade), 5-hydroxycytosine (5-OH-Cyt), 5-hydroxyuracil (5-OH-Ura), 5-(hydroxymethyl)uracil (5-(OHMe)Ura), thymine glycol (ThyGly) and 5-hydroxy-5methylhydantoin (5-OH-5-MeHyd). Other important DNA lesions such as 8-hydroxyguanine (8-OH-Gua) can also be measured with this approach by hydrolyzing 8-hydroxy-2-deoxyguanosine- 15 [14]. In this procedure, 8-OHguanine-15 N 5 is obtained by hydrolysis of 8-hydroxy-2-deoxyguanosine-15 N 5 with 60% formic acid at 140 °C for 30 min followed by lyophilization.…”