Formation of 13C-, 15N-, and 18O-labeled guanidinohydantoin from guanosine oxidation with singlet oxygen. Implications for structure and mechanism.

Full text of DOI:10.1021/ja0378660

Published on Web 10/28/2003
Formation of 13C-, 15N-, and 18O-Labeled Guanidinohydantoin from Guanosine
Oxidation with Singlet Oxygen. Implications for Structure and Mechanism
Yu Ye,† James G. Muller,† Wenchen Luo,† Charles L. Mayne,† Anthony J. Shallop,‡
Roger A. Jones,‡ and Cynthia J. Burrows*,†
Department of Chemistry, UniVersity of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, and
Department of Chemistry and Chemical Biology, Rutgers UniVersity, 610 Taylor Road,
Piscataway, New Jersey 08854
Received August 11, 2003; E-mail: burrows@chem.utah.edu
Scheme 1. Oxidation Pathways of Guanosine
The action of reactive oxygen species upon DNA bases damages
genomic and mitochondrial DNA as well as the cellular nucleotide
pool and is therefore implicated in cancer, aging, and neurological
disorders.1-3 Oxidation of guanosine by singlet oxygen has been
the focus of considerable interest because of its importance in both
cancer etiology and its cure via photodynamic therapy.4,5 Addition-
ally, 1O2 reaction with the guanine heterocycle presents a plethora
of pathways and potential products depending upon the reaction
conditions and the structural context - nucleoside versus double-
stranded DNA.6,7 It is now well established that the principal product
1
of G nucleoside oxidation by O2 above pH 7 is spiroiminodi-
hydantoin (Sp),8-10 although 8-oxo-7,8-dihydroguanosine (OG)
predominates in cellular DNA.11 We characterized Sp as the main
product of OG oxidation by one-electron oxidants in nucleoside
studies, while oxidation in ds-DNA led to a cationic base,
guanidinohydantoin (Gh).12 We report here that Gh is also the major
Table 1. Product Distribution (%) as a Function of pH
pH 4.5
pH 6
pH 7
pH 8.6
Gh
38
1
7
34
16
2
3
52
1
1
70
1
Sp
Ghox a
Izb
5
4
4
15
1
product of O2 reaction with G at pH < 7. Furthermore, isotopic
labeling resolves controversial issues concerning its structure and
points to a mechanistic explanation for products observed in our
laboratory and others.
a Ghox ) 5,6-dehydroGh. b Iz ) 2,5-diaminoimidazol-4-one.
160) led to a major fragmentation of the C5-N6 bond, forming
guanidinium ion at m/z ) 60. The second most intense ion at m/z
) 98 (shifted to 100 in H218O) is thought to be due to a two-bond
fragmentation whose product ion would include C4 and C5 of Gh
along with an 18O as a carbonyl group at C4 (originally C5 in G).
To gain further support for this assignment, guanosine isotopically
labeled at C8, N1, amino, and N716 was oxidized with Rose Bengal
photosensitization again in H216O versus H218O. As expected, the
guanidinium ion shifted to m/z ) 62 due to the presence of two
15N atoms (Figure 1). Significantly, the m/z ) 100 fragment, now
with two 15N atoms in the guanidinium group, was shifted to m/z
) 102 in the H218O experiment. The only pattern consistent with
such a labeling scheme involves introduction of an oxygen atom
from water at C5 of guanine (C4 of Gh) along with an oxygen
atom from O2 at C8 of G (C2 of Gh).17
The use of isotopes also permitted resolution of a controversy
surrounding the structure of Gh, a species formed as a mixture of
two slowly equilibrating isomers. Subject to debate was whether
the two HPLC peaks observed represent equilibrating diastereomers
via enolization of the C4 carbonyl18,19 or an additional set of
iminoallantoin isomers formed by ring closure of a guanidine
nitrogen attacking C4 followed by opposite ring opening.12 We
previously proposed the iminoallantoin structure on the basis of
an analogous isomerization observed in the urate pathway to
allantoin,20 as well as a related proposal from products of arylamine
adducts to C8 of G.21 To resolve this issue, Gh was formed by 1O2
oxidation of a G labeled at C2, N1, amino, and N722 at pH 4.5,
and the relevant NMR spectra in 10% D2O buffered to pH 7 are
shown in Figure 2.
Previously, we proposed that 5-hydroxy-OG is an unstable com-
mon intermediate in the pH and structure-dependent formation of
both Sp and Gh (Scheme 1),12 by analogy to uric acid oxidation to
allantoin via hydration and decarboxylation of 5-hydroxyurate.13
While an acyl shift to generate Sp is preferred at pH 7 and above,
formation of the tetrahedral carbon of Sp is inhibited in ds-DNA.
In addition, lower pH facilitates hydration of C6. In the present
study, oxidation of guanosine using Rose Bengal photosensitization
in buffered aqueous solution led to the pH-dependent product dis-
tribution shown in Table 1, as determined by quantitative LC-MS
analysis (see ref 14 and Supporting Information). The product distri-
bution alters dramatically in favor of Gh below pH 7, and overall
Sp and Gh account for the bulk of the products. Evidence for further
oxidation to Ghox 12 is also seen because of the high level of
conversion. 2,5-Diaminoimidazol-4-one15 accounts for a small
amount of the total products but is more pronounced at higher pH.
When the oxidation was conducted in 95% enriched H218O at
pH 4.5, MS analysis indicated that 84% of Gh and 83% of Sp
contained one atom of 18O. Because both products each gain two
oxygen atoms upon singlet oxygen oxidation, one oxygen atom
must be derived from O2 while the other is derived from H2O. That
both Sp and Gh show identical isotopic incorporation lends further
support to 5-OH-OG as a common intermediate.
The location of the oxygen atom derived from water was
determined by positive ion ESI-MS/MS studies of Gh formed with
H216O versus H218O. In-source fragmentation of the glycosidic bond
followed by mass selection of the Gh heterocycle (m/z ) 158 or
† University of Utah.
‡ Rutgers University.
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J. AM. CHEM. SOC. 2003, 125, 13926-13927
10.1021/ja0378660 CCC: $25.00 © 2003 American Chemical Society

C O MMU N I C A T I O N S
Scheme 2. Proposed Mechanism of the Formation of Gh and Sp
in H2O as Compared to Products Formed in Organic Solvents23
Figure 1. Positive ion ESI-MS/MS studies of labeled Gh in H216O (black)
versus H218O (red).
precursor to Sp and Gh. The mechanism in Scheme 2 explains all
of the major G nucleoside products observed from O2 in both
1
aqueous and nonaqueous solvents. That OG is the most common
product of singlet oxygen oxidation of cellular DNA is likely due
to interception of the hydroperoxide intermediate by cellular
reductants. While Gh may be less prevalent than OG in cellular
DNA, its biological effects are more dramatic. An in vivo
mutagenesis assay showed this lesion to cause 99% transversion
mutations (G to C) as compared to <5% (G to T) for OG.26
Acknowledgment. This work was supported by a grant from
the NIH (CA90689).
Note Added after ASAP: Scheme 2 contained errors in the
version published on the Web 10/28/2003. The version published
11/3/2003 and the print version are correct.
Supporting Information Available: Experimental procedures, 13
C
Figure 2. 15N and 13C NMR spectra of isotopically labeled Gh in 10%
D2O at pH 7. See Supporting Information for complete spectra and spectra
in other solvents.
and 15N NMR spectra in D2O (pH 4.5 and 7.0) and d6-DMSO, and
ESI-MS/MS data for labeled and unlabeled Gh (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
The 15N NMR spectrum of labeled guanosine starting material
displayed three resonances assigned as N7 (235.3 ppm), N1 (147.4
ppm, d, J ) 14.7 Hz), and amino (72.5, d, J ) 23.4 Hz). After
oxidation, the signal corresponding to the original N7 (now N3 of
Gh) was not seen at pH 7, although a weak resonance at 149.4
ppm was seen in the pH 4.5 spectrum.17 The only peaks observed
in the pH 7 15N spectrum were two overlapping doublets near 76
ppm, which became clearly resolved at pH 4.5.17 These data are
consistent with the formation of two diastereomers of Gh in which
the two 15N-labeled nitrogen atoms of the guanidinium group are
equivalent and coupled to the adjacent 13C. Even more convincing
is the observation of two triplets (one for each diastereomer) in
the 13C NMR spectrum of the same material, showing the
equivalency of the two nitrogen atoms attached to the 13C. While
it is still possible that iminoallantoin isomers exist, we could not
detect them by NMR, and the upper limit of the equilibrium constant
must therefore be about 0.03.
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