5-Cyanoamino-4-imidazolecarboxamide and Nitrosative Guanine Deamination: Experimental Evidence for Pyrimidine Ring-Opening during Deamination

Article abstract of DOI:10.1021/ja0389523

5-Cyanoamino-4-imidazolecarboxamide 4a (R = CH₂-O-CH₂-CH₂-OH) has been synthesized, purified, and fully characterized by MS, MS/MS, HRMS, IR spectroscopy, and by ¹H and ¹³C NMR spectroscopy. It is shown that cyclization of 4a yields the guanine 6a and the isoguanine 12a. Our findings provide experimental evidence in support of our hypothesis that the formation of oxanine and xanthine in nitrosative guanine deamination may proceed via pyrimidine ring-opened intermediates. The observed formation of 6a from the amide 4a (XH₂ = NH₂) shows that, in analogy, oxanine can be formed from 3 (XH = OH). The formation of 12a from 4a reveals for the first time the possibility that oxanine might be formed by a second pathway that involves electrocyclic reaction of 3. Finally, the new chemistry suggests the possibility for a new dG-to-dG cross-link. Copyright

Full text of DOI:10.1021/ja0389523

Published on Web 02/07/2004
5-Cyanoamino-4-imidazolecarboxamide and Nitrosative Guanine
Deamination: Experimental Evidence for Pyrimidine Ring-Opening during
Deamination
Ming Qian and Rainer Glaser*
UniVersity of Missouri-Columbia, Department of Chemistry, 605 South College AVenue, Columbia, Missouri 65211
Received October 8, 2003; E-mail: glaserr@missouri.edu
Nitrosative guanine deamination via guanine diazonium ion 1
Scheme 1
1
to xanthine was discovered by Strecker in 1861 before Kossel
2
identified guanine as a DNA base in 1884. A century later this
3
chemistry became a central topic in toxicology, and it is now well
4
understood that endogenous nitrosation occurs and that dG-to-dG
5
and dG-to-dA cross-links also are formed. Guanine deamination
chemistry became interesting once again with the 1996 discovery
by Suzuki, et al. that deamination may form oxanine.6 We
proposed a reaction mechanism that involves dediazoniation of 1
-8
9
10
with concomitant pyrimidine ring-opening and deprotonation
leading to intermediate 211 (Scheme 1). It is our hypothesis that
the highly reactive dihydroimidazole 2 adds water to form the
cyanoamine 3 which then cyclizes to oxanine 5. The cyanoimine 2
also may add water to cyanoimine instead of the ketene moiety,
and the resulting ketene might then ring-close to xanthine. Our
published theoretical predictions are now being tested experiment-
ally, and as a first step, we targeted the amide 412 for one practical
and one conceptual reason (vide infra).
Here, we report the synthesis of 4a and its recyclization to
guanine 6a and isoguanine 12a. Importantly, we were able to purify
4
a so that we can firmly establish that 4a is the precursor for the
formations of 6a and 12a. We employed the R-group CH -O-
CH -CH -OH as a sugar model. This (2-hydroxyethoxy)methyl
group occurs in acyclovir, and we indicate this R-group by adding
2
2
2
Scheme 2
“
a” to the compound numbers.
The synthesis of 4a is outlined in Scheme 2. Compound 16 was
synthesized with the efficient method by Clausen13 via alkylation
of 13, condensation of 14 with benzoyl isothiocyanate, and
hydrolysis of 15. The methylation of 16 in basic aqueous solution
14
gave low yields even though this procedure works well for the
related ester.15 We found the methylation of 16 in the mixed solvent
5/1 acetone/methanol to give 17 in good yield.
Cyanoamine 4a was obtained by methylthioether elimination
16
from 17a. Yamazaki, Okutsu, and Yamada studied the base-
catalyzed methylthioether elimination from 17 (R ) H, 17h) leading
to guanine 6 and isoguanine 12 via cyanoamine 4 (R ) H, 4h).
The unstable cyanoamine 4h was not isolated and was characterized
-
1
only by its IR spectrum (ν(CtN) ) 2190 cm ). We have now
succeeded in the preparation of the cyanoamine 4a and the isolation
of this reactive intermediate. Reactions to form 4a were quenched
in liquid nitrogen after 3 min, and after HPLC separation and
lyophilization, 4a was stored at -18 °C to prevent cyclization. The
cyanoamine 4a was characterized by mass spectrometry (MS, MS/
reason for the choice of 4 is now clear. While 3 cannot be
synthesized starting with the acid analogue of 13, with the
experience of the synthesis of 4 it appears possible to prepare 3
starting with an ester of 13.
-
1
MS, HRMS), by IR spectroscopy (ν(CtN) ) 2138.6 cm ), and
1
13
1
by H and C NMR spectroscopy. The H NMR spectrum (Figure
The cyclization of 4a was studied at room temperature in 0.2M
1) demonstrates the purity of the synthetic 4a. The cyanoamine 4a
2 4 2 4
K HPO /KH PO buffer solution (pH ) 6.0, 7.0, 8.0, 9.0). Figure
may exist in equilibrium with the carbodiimide. Ab initio studies
of the parent cyanamide and carbodiimide show a preference of
about 7 kcal/mol for the cyanamide,17 and our IR data also show
that the observed tautomer is the cyanoamine 4a. The practical
2 shows the HPLC chromatograms recorded at pH ) 7.0. There
are two products, the guanine 6a and the isoguanine 12a, and they
were identified by comparison to authentic samples. 6a is com-
mercially available, and 12a was synthesized with an adaptation
2274
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J. AM. CHEM. SOC. 2004, 126, 2274-2275
10.1021/ja0389523 CCC: $27.50 © 2004 American Chemical Society

C O MMU N I C A T I O N S
Scheme 3
formation of oxanine 5 from 3. (2) Interestingly, the formation of
2a and the explanation of its formation suggests that there is a
1
second pathway to oxanine via an electrocyclic reaction (3 to 7,
carbonyl-O as nucleophile), and for X ) O, oxanine is the final
product of that path because it is known that xanthine 11 does not
Figure 1. 1H NMR spectrum (250 MHz, D2O, 0.05 N NaOD at 4 °C) of
4
a shows signals at δ 7.0 (s, 1H, C2-H), 5.0 (s, 2H, N1-CH2), 3.1-3.3 (m,
4
H, -CH2-CH2-) and establishes the purity of 4a.
6
form from oxanine 7 via rearrangement. (3) Finally, the conceptual
reason for the choice of 4 is as follows: Any potentially formed 2
in nitrosative DNA deamination might add to the amino group of
a proximate DNA base, and this addition would lead to N-
substituted derivatives of 4, as exemplified in Scheme 3. We have
synthesized the classical G-to-G cross-link and its structure-isomer
1
9 by addition of 4a to guanosine.20 The reaction of 4a to 6a
strongly suggests that 18 would cyclize to 19 and provide a second
path to this cross-link.
Acknowledgment. We are grateful for support by the National
Institutes of Health (GM61027).
Supporting Information Available: Details for the syntheses of
1
13
1
7, 4a, and 12a; H and C NMR spectra of 17, 4a, and 12a; the IR
spectrum of 4a; and HPLC chromatograms of the pH-dependent
cyclization of 4a (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
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Figure 2. HPLC chromatograms of the cyclization of cyanoamine 4a in
buffer solution at pH 7 show formation of 6a (aGua) and 12a (aIGua).
Final yields are 74.2% 12a and 5.8% 6a with 4.3% 4a remaining.
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18
(12)
O-Labeling studies confirm the dominance of the formation of 5 via 2
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(
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(
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(
Scheme 1). This mechanism involves electrocyclic cyclization,
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tautomerization, and electrocyclic ring-opening to achieve the
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(
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We have achieved the synthesis of the pure cyanoamine 4a. With
the availability of pure 4a, we were able to study its cyclization
chemistry. The results of this study have several important
implications for nitrosative guanine deamination. (1) The demon-
strated formation of 6a from 4a is the model reaction for the
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(
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(
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