Structural Categorization of Adenine, Guanine, and Xanthine Derivatives Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with 5-Nitrosalicylic Acid and 1,5-Diaminonaphtalene

Abstract

In this study, we analyzed purine derivatives using multimatrix variation matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) with α-cyano-4-hydroxycinnamic acid (CHCA), 1,5-diaminonaphtalene (DAN), 5-formylsalicylic acid (FSA), and 5-nitrosalicylic acid (NSA) as matrices. Further, we focused on the abstraction/attachment of hydrogen from/to analytes and detected [M - H]⁺, [M + 2H]^+• and/or [M + 3H]⁺ in MALDI MS spectra of compounds containing nitrogen and/or carbonyl oxygen. Although [M - H]⁺ generation of purine compounds in MALDI MS with conventional matrices was challenging, NSA-MALDI MS effectively yielded the [M - H]⁺species of purine derivatives compared with CHCA, FSA, and DAN, and the [M - H]⁺/[M + H]⁺ ratios reflected their structures, such as the substituting groups and positions. We speculated that the molecular ion [M]^+• generated and the subsequent hydrogen radical abstraction proceeded by NSA matrix from the α-carbon of the amine group. The nitro group (-NO₂) of NSA can withdraw hydrogen radicals in photochemical reactions. The [M - H]⁺ of adenosine, guanosine, and inosine suggested that hydrogen abstraction occurred in the ribose unit. The xanthine isomer of paraxanthine was distinguished from those of theophylline and theobromine using their [M - H]⁺/[M + H]⁺ ratios obtained with NSA-MALDI MS. Additionally, [M + 2H]^+• generated in DAN-MALDI MS of xanthine derivatives due to their carbonyl groups. The relative abundances of [M + 2H]^+• of xanthine derivatives were much higher than those of the other purine derivatives such as adenine derivatives which generated [M + 3H]⁺ in their DAN-MALDI MS. DAN induced the hydrogen attachment of purine compounds because the amine group (-NH₂) of DAN can give hydrogen radicals in photochemical reactions. NSA- and DAN-MALDI MS characterized purine derivatives and were useful for their structure categorization.

Figure 1

Structures of purine derivatives.

Figure 2

MALDI MS spectra of adenine 1 (A) and kinetin 2 (B). (a) CHCA-MALDI MS, (b) DAN-MALDI MS, (c) FSA-MALDI MS, and (d) NSA-MALDI MS of compounds 1 and 2. The peaks labeled by asterisks were from matrix.

Figure 3

Proposed hydrogen abstraction scheme for compound 2. (1) Ionization and molecular ion generation by UV laser irradiation; (2) hydrogen radical abstraction with NSA; (3) showing a possible electron conjugated structure; (4) tautomerism.

Figure 4

PSD MS/MS product ion spectra of [M + H]⁺ at m/z 216 (A) and [M – H]⁺ at m/z 214 as the precursor ion of kinetin 2 (B), and the corresponding PSD ion species (C). The proposed ion species in detail are summarized in Figure S3.

Figure 5

Relative abundances of [M–H]⁺ and [M]⁺ in NSA-MALDI MS of N⁶-benzyladenine (compound 3) (blue bars) and N⁶-benzoyladenine (compound 4) (orange bars), which were normalized from that of [M + H]⁺ ion abundance (100%). The abbreviation "Pwr" means the experimental laser powers, which were different from each matrix concentration. The NSA concentrations were changed (A)–(D). No NSA matrix was used in (A), 0.5 mM NSA was used and the molar ratio of NSA/analyte was 1/10 in (B), 5 mM NSA was used and the molar ratio of NSA/analyte was 5/5 in (C), and 50 mM NSA was used and the molar ratio of NSA/analyte was 10/1 in (D).

Figure 6

MALDI MS spectra of 3-methyl-7-propylxanthine 16 with (a) CHCA, (b) DAN, (c) FSA, and (d) NSA. The peak labeled with an asterisk (*) was from the matrix.

Figure 7

Proposed hydrogen attachment scheme of compound 16. (1) Ionization and molecular ion generation, (2) hydrogen radical attachment to the carboxy group by UV laser irradiation with DAN, and (3) tautomerism.