Trapping a labile adduct formed between an ortho -quinone methide and 2′-deoxycytidine

Article abstract of DOI:10.1021/ol200071p

Selective oxidation by bis[(trifluoroacetoxy)iodo]benzene (BTI) provides an effective trap for quenching adducts formed reversibly between dC and an ortho-quinone methide (QM) under physiological conditions. A model adduct generated by 4-methyl-o-QM and 2

Full text of DOI:10.1021/ol200071p

ORGANIC
LETTERS
2011
Vol. 13, No. 5
1186–1189
Trapping a Labile Adduct Formed between
an ortho-Quinone Methide and 20-
Deoxycytidine
Michael P. McCrane,† Emily E. Weinert,† Ying Lin,† Eugene P. Mazzola,§ Yiu-Fai Lam,†
Peter F. Scholl,‡ and Steven E. Rokita*,†
Department of Chemistry and Biochemistry and the Joint Institute for Food Safety and
Applied Nutrition (JIFSAN), University of Maryland, College Park, Maryland 20742,
United States, and Division of Analytical Chemistry, Spectroscopy and Mass
Spectroscopy Branch in the Center for Food Safety and Nutrition of the Food and Drug
Administration, College Park, Maryland 20740, United States
Rokita@umd.edu
Received January 10, 2011
ABSTRACT
Selective oxidation by bis[(trifluoroacetoxy)iodo]benzene (BTI) provides an effective trap for quenching adducts formed reversibly between dC
and an ortho-quinone methide (QM) under physiological conditions. A model adduct generated by 4-methyl-o-QM and 20-deoxycytidine is rapidly
converted by intramolecular cyclization and loss of aromaticity to a characteristic product for quantifying QM alkylation. However, BTI induces a
surprising rearrangement driven by overoxidation of a derivative lacking an alkyl substituent at the 4-position of the QM.
The reversibility of DNA alkylation by certain quinone
methide intermediates (QM) is advantageous for the design
of cross-linking reagents,1 target-promoted reactions,2,3
covalent combinatorial systems,4,5 and protocols for chiral
resolution.6 Conversely, such reversibility is detrimental
to the detection of QM adducts formed in DNA since only
the most stable derivatives may persist through standard
enzymatic digestion and chromatographic separation.7-9
Identifying the full profile and yield of QM capture by
DNA is a prerequisite for understanding the genotoxicity of
QMs generated by metabolism of food additives, natural
products, and synthetic drugs.10-14 Satisfactory analysis
awaits development of a method to suppress QM release
(7) Lewis, M. A.; Yoerg, D. G.; Bolton, J. L.; Thompson, J. A. Chem.
Res. Toxicol. 1996, 9, 1368.
(8) Pande, P.; Shearer, J.; Yang, J.; Greenberg, W. A.; Rokita, S. E.
J. Am. Chem. Soc. 1999, 121, 6773.
(9) Weinert, E. E.; Frankenfield, K. N.; Rokita, S. E. Chem. Res.
Toxicol. 2005, 18, 1364.
(10) Egholm, M.; Koch, T. J. Am. Chem. Soc. 1989, 111, 8291.
(11) Tomasz, M. Chem. Biol. 1995, 2, 575.
(12) Hulsman, N.; Medema, J. P.; Bos, C.; Jongejan, A.; Luers, R.;
Smit, M. J.; de Esch, I. J. P.; Richel, D.; Witjmans, M. J. Med. Chem.
2007, 50, 2424.
† Department of Chemistry and Biochemistry, University of Maryland.
§ Joint Institute for Food Safety and Applied Nutrition (JIFSAN), Uni-
versity of Maryland.
‡ Division of Analytical Chemistry, Spectroscopy and Mass Spectroscopy
Branch in the Center for Food Safety and Nutrition of the Food and Drug
Administration.
(1) Veldhuyzen, W. F.; Pande, P.; Rokita, S. E. J. Am. Chem. Soc.
2003, 125, 14005.
(2) Zhou, Q.; Rokita, S. E. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
15452.
(3) Rossiter, C. S.; Kumar, D.; Modica, E.; Rokita, S. E. Chem.
Commun. 2011, 47, 1476.
(13) Kashfi, K.; Rigas, B. Biochem. Biophys. Res. Commun. 2007,
358, 1096.
(4) Wang, H.; Wahi, M. S.; Rokita, S. E. Angew. Chem., Int. Ed. 2008,
47, 1291.
(5) Wang, H.; Rokita, S. E. Angew. Chem., Int. Ed. 2010, 49, 5957.
(6) Colloredo-Mels, S.; Dorr, R. T.; Verga, D.; Freccero, M. J. Org.
Chem. 2006, 71, 3889.
(14) Bolton, J. L., Thompson, J. A. Reactive Intermediates in Chem-
istry and Biology: Quinone Methides; Rokita, S. E., Ed.; Wiley: Hoboken,
NJ, 2009; Vol. 1, p 329.
(15) Veldhuyzen, W. F.; Shallop, A. J.; Jones, R. A.; Rokita, S. E.
J. Am. Chem. Soc. 2001, 123, 11126.
r
10.1021/ol200071p
Published on Web 02/09/2011
2011 American Chemical Society

from its most labile adducts. Model studies based on deoxy-
nucleosides have previously indicated that strong nitrogen
nucleophiles of DNA react with the parent ortho-QM most
efficiently, but reversibly.9,15 Lifetimes of these adducts
(>2 h) are sufficient to elicit a variety of biological
responses although not adequate for easy detection. Reac-
tion with weak nitrogen nucleophiles of DNA is signifi-
cantly less efficient but irreversible and amenable to routine
detection.7,8
(BTI) in particular effect equivalent dearomatization
and function well under aqueous conditions.25-27 Most
importantly, a control study indicated that BTI did not
react with deoxynucleotides under the conditions des-
cribed below.28
Scheme 1. Quinone Methide Generation, Reversible Alkyla-
tion, and Trapping
Most techniques for quantifying alkylation are predi-
cated on the formation of stable adducts. For example,
polymerase stop assays have detected reaction of tamoxifen
metabolites with the weakly nucleophilic exo-amino group
of dG.16 However, no comparable information is available
on possible adduct formation with the more nucleophilic
N7 of dG. Labile adducts generated by other potential
carcinogens can often be stabilized for detection by an acid
or alkaline quench prior to enzymatic analysis,17 but neither
treatment is appropriate for QM adducts.15,18,19 Alterna-
tively, mass spectrometry offers an excellent method for
simultaneously observing a range of DNA products,20 yet
certain QM adducts and benzyl substituted phenols in
general (e.g., 1, below) often lack sufficient stability for
detecting their parent ions. Still, adducts formed between a
QM-like intermediate derived from the natural product
lucidin and the weakly nucleophilic exo-amino groups of
dA and dG have been observed by electrospray mass
spectrometry, but again no equivalent data are available
on potential reaction with the strongly nucleophilic sites of
dG N7 and dA N1.21
Acylation, alkylation, silylation, or reduction of the
resulting phenolic product of QM alkylation was not con-
sidered for quenching the reversible reaction since these
approaches are not likely to exhibit sufficient selectivity for
this product when studies involve genomic DNA. In
contrast, oxidative dearomatization has the potential to
target the phenolic products uniquely. Singlet oxygen is
often used for such dearomatization but is not suitable for
this application based on its oxidation of guanine.22,23
Potassium nitrosodisulfonate (Fremy’s salt) was very se-
lective for oxidation of the QM adducts, but the reaction
could not be driven to completion, a necessary criterion for
quantifying the adducts formed.24b Hypervalent iodine re-
agents in general and bis[(trifluoroacetoxy)iodo]benzene
The goalof thisstudywas to testwhether oxidation of an
o-QM-deoxynucleoside (QM-dN) adduct forms a stable
and identifiable compound under physiological condi-
tions. 20-Deoxycytidine (dC) was chosen as the initial
deoxynucleoside of interest since it forms only a single
QM adduct (Scheme 1).29 Similarly, 6-methylene-cyclo-
hexa-2,4-dienone (2) was chosen as the first model QM
since its dC adduct exhibits only modest reversibility for
challenging the oxidative trapping by BTI. In the absence
of such a trap, the dC adduct decomposes over days by
releasing the QM for irreversible addition by water.9
o-(tert-Butyldimethylsilyl)-2-(bromomethyl)phenol (1)
was prepared as the source of QM 2 according to a
literatureprocedure,9,29 and thedC adduct6was generated
in situ under standard conditions (37 °C).29 After 20 min, a
4-fold excess of BTI in acetonitrile was added and the
mixture was cooled to room temperature. Reversed-phase
chromatography indicated complete consumption of the
dC adduct and formation of a new compound (8) within 20
min.28 This product was isolated under equivalent condi-
tions and remained stable (90%) over 6 days in aqueous
acetonitrile. Initial characterization by 1H and 13C NMR
spectroscopy indicated that the expected product (7,
(16) Lowes, D. A.; Brown, K.; Heydon, R. T.; Martin, E. A.; Gant,
T. W. Biochemistry 1999, 38, 10989.
(17) Marsch, G. A.; Mundkowski, R. G.; Morris, B. J.; Manier,
M. L.; Hartmann, M. K.; Guengerich, F. P. Chem. Res. Toxicol. 2001,
14, 600.
(18) Rodia, J. S.; Freeman, J. H. J. Org. Chem. 1959, 24, 21.
(19) Wakselman, M. New J. Chem. 1983, 7, 439.
1
Scheme 2) had not formed based on the lack of H-1H
coupling anticipated for the benzoquinone protons and the
absence of signals for two quinone carbons.
(20) Banoub, J. H., Limbach, P. A., Eds. Mass Spectrometry of
Nucleosides and Nucleic Acids; CRC Press: Boca Raton, FL, 2010.
(21) Ishii, Y.; Okamura, T.; Inoue, T.; Fukuhara, K.; Umemura, T.;
Nishikawa, A. Chem. Res. Toxicol. 2010, 23, 134.
(25) Tamura, Y.; Yakura, T.; Tohma, H.; Kikuchi, K.; Kita, Y.
Synthesis 1989, 126.
(26) McKillop, A.; McLaren, L.; Taylor, R. J. K. J. Chem. Soc.,
~
ꢀ
(22) Carreno, M. C.; Gonzalez-Lopez, M.; Urbano, A. Angew.
ꢀ
Chem., Int. Ed. 2006, 45, 2737.
(23) Cadet, J.; Ravanat, J.-L.; Martinez, G. R.; Medeiros, M. H. G.;
Di Mascio, P. Photochem. Photobiol. 2006, 82, 1219.
Perkin Trans. 1 1994, 2047.
(27) Moriarty, R. M.; Prakash, O. Org. React. 2001, 57, 327.
(28) See Supporting Information.
(29) Rokita, S. E.; Yang, J.; Pande, P.; Greenberg, W. A. J. Org.
Chem. 1997, 62, 3010.
(24) (a) Weinert, E. E.; Dondi, R.; Colloredo-Mels, S.; Frankenfield,
K. N.; Mitchell, C. H.; Freccero, M.; Rokita, S. E. J. Am. Chem. Soc.
2006, 128, 11940. (b) Weinert, E. E. Ph.D. Dissertation. University of
Maryland, College Park, MD, 2006.
Org. Lett., Vol. 13, No. 5, 2011
1187

this product forms and incorporates the bromide released
from the QM precursor 1 is far from obvious. Hence, its
use for quantifying QM alkylation is dubious.
Scheme 2. Extensive Oxidation of an Unsubstituted Quinone
Methide Adduct by BTI
Overoxidation and rearrangement of the dC adduct was
subsequently prevented by use of an alternative QM that
generated products containing an alkyl group para to the
phenolic -OH.25,26 The necessary precursor 2-bromo-
methyl-4-methyl-O-(tert-butyldimethylsilyl)phenol (9) was
prepared from 5-methylsalicylaldehyde using standard
procedures.28 The presence of the methyl substituent was
not expectedtoalter the profileofDNA adducts formedby
4-methyl-6-methylene-cyclohexa-2,4-dienone (MeQM)
after deprotection of 9 since a related set of substituents
had not significantly altered the profiles of equivalent QM
adducts.24 However, the electron-donating properties of
the methyl group stabilize the electron-deficient QM inter-
mediate, leading to faster generation and increased lability
of the resulting adducts formed by MeQM vs those of QM
(2).24 Thus, the methyl-substituted model may block over-
oxidation but poses a greater challenge for the trapping
system due to the increased reversibility of the MeQM
adduct.
Loss of the two carbons was confirmed by electrospray
mass spectrometry (ESI-MS), and surprisingly the parent
ion revealed the presence of one bromine by its distinct
isotope ratio.28 MS/MS experiments generated the char-
acteristic deglycosylation and debromination products to
support the structural assignment based on extensive
NMR data. Signals (1H and 13C) based on literature
values30 for both the pyrimidine and ribose groups were
observed, and their assignments were confirmed by
1H-13C HSQC and 1H-13C HMBC analysis.28
Scheme 3. Oxidative Trapping of a Methyl-Substituted Quinone
Methide Adduct
Figure 1. 1H-13C HMBC of 8 in DMSO-d6 at 600 MHz.
The unsaturated nature of the o-QM remnant was
apparent from the remaining 13C signals that all ranged
between 116.6 and 187.6 ppm (Figure 1). The large 2-bond
coupling (2JCH = 43 Hz) observed for the cross peak
between C11 and H12 in the HMBC spectrum is unique to
aldehydes (Figure 1).31,32 Connectivities between C7
through C12 were established by a combination of
1H-13C HSQC, H-13C HMBC, and H-15N HMBC
analysis.28 Finally, the bromine was placed on C11 to
satisfy its valence and lack of attached proton. The entire
conjugated system is illustrated in a favorable thermody-
namic configuration although the stereochemistry has not
yet been confirmedexperimentally. A mechanism bywhich
The MeQM-dC adduct (10) was prepared in situ by
deprotection of 9 with aqueous KF in the presence of dC
under conditions similar to those used to prepare the dC
adduct 6 (Scheme 3). The structure of 10 was confirmed by
1H NMR and UV-vis data based on previous analysis of
the parent adduct 6 (Scheme 2).29 Most diagnostic are the
benzylic protons that vary by less than 0.1 ppm (4.96 ppm for
6,29 4.89 ppm for 10) and the λmax values that vary by only
1 nm (278 nm for 6,29 279 nm for 10). The absorption data
1
1
(30) Becker, E. D.; Miles, H. T.; Bradley, R. B. J. Am. Chem. Soc.
1965, 87, 5575.
(31) Pople, J. A.; Bothner-By, A. A. J. Chem. Phys. 1965, 42, 1339.
(32) Hansen, P. E. Prog. Nucl. Magn. Reson. Spectrosc. 1981, 14, 175.
(33) Sun, L.; Singer, B. Biochemistry 1974, 13, 1905.
Org. Lett., Vol. 13, No. 5, 2011
1188

are also most similar to those of N3-ethyl dC (λmax = 280
nm) rather than those of N4-ethyl dC (λmax = 270 nm).33
Treatment of the alkylation mixture above with 4 equiv
of BTI transformed the dC adduct 10 into a new com-
pound within 20 min as detected by reversed phase chro-
matography.28 This compound also persisted unchanged
(>85%, 24 h) in solution (9 mM ammonium formate pH
6.8, 12% CH3CN) for convenient analysis and future
application with DNA. Initial characterization by 1H
NMR immediately confirmed the ability of the para-
methyl substituent to block the overoxidation that had
plagued the reaction of its unsubstituted parent 6. Cou-
pling between adjacent vinyl protons was now evident for
the oxidized product of 10, and none of its 1H signals were
observed downfield of 8 ppm.28 ESI-MS confirmed that
oxidative trapping of adduct 10 did not cause loss of
carbon atoms. Instead, the (M þ H)þ of m/z 346.18
provided the first suggestion that the oxidized product 12
(calculated m/z 346.14 (M þ H)þ) had formed rather than
the alternative product 11 (calculated m/z 364.15 (M þ
H)þ) (Scheme 3).
hybridization of the spiro carbon (Figure 2). This chemical
shift is quite distinct from that predicted for the correspond-
ing sp2 carbon in 11 (ca. 133 ppm) based on the model
compound 4-hydroxy-2,4-dimethyl-2,5-cyclohexadien-1-
one (13).22 Additionally, a correlation between the para-
methyl protons (H14) and the adjacent vinyl proton (H13)
was observed in a 2D-COSY experiment as expected for 12.
Such a correlation would not be likely for 1128 and was not
previously detected for the model 13.22 Finally, restricted
rotation of the former benzylic carbon (C7) is apparent
from the diastereotopic relationship of the attached protons
(H7A and H7B) and their proximity to the carbonyl oxygen
of C9 that alternatively extends in front or back of C8. This
1
configuration creates a pair of doublets in the H NMR
spectrum, but these signals are further complicated by the
diastereomeric mixture of 12 (12a and 12b) formed by
oxidation of the dC adduct by BTI (Figure 2).
The general ability of BTI to promote intramolecular
cyclization and dearomatization was reported earlier and
has since been integrated into a number of synthetic
strategies.27,34,35 This process obviously dominates the oxi-
dation of 10 and may even begin to explain the origins of the
imidazole ring in the overoxidized product 8. In both cases,
intramolecular addition of the exo-imine to a position ortho
to the phenolic oxygen is favored over the intermolecular
addition of water at the para-position. At least for 4-alkyl
QMs such as that formed by the precursor 9, BTI is an
effective quench for preventing the reversible reactions of
QM. Oxidation proceeds rapidly under physiological con-
ditions and is selective for the phenolic product formed by
QM alkylation of dC. Use of this reagent should provide an
opportunity to study the intrinsic susceptibility of dC
toward alkylation in DNA without worry of spontaneous
QM release from its adducts or adventitious oxidation of
the parent nucleosides. This approach of oxidative quench-
ing should also be applicable to adducts of the other
deoxynucleosides in DNA and ultimately should allow for
quantifying the kinetic products of reaction between QMs
and genomic DNA for the first time.
Acknowledgment. We thank Michael Novak (Miami
University) for suggesting use of the methyl-substituted
quinone methide and the NSF (CHE-0517498) for finan-
cial support.
Figure 2. 1H-13C HMBC of 12 in DMSO-d6 at 400 MHz.
NMR signals (1H and 13C) corresponding to the pyrimi-
dine and ribose moieties of 12 were again assigned from
Supporting Information Available. Synthesis and char-
acterization of all compounds including 2D NMR and MS
spectra of 8, 10, and 12; HPLC chromatograms of dC
adducts and their oxidized derivatives. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
1
literature values30 and confirmed by H-13C HSQC and
1H-13C HMBC spectra.28 These spectra also provided the
necessary data to establish the connectivities for the oxi-
dized QM adduct. For example, C8 was identified by its
proximity to the protons of C7, and the 13C chemical shift
of C8 (73.1 ppm) was most consistent with the sp3
(34) Tohma, H.; Kita, Y. Top. Curr. Chem. 2003, 224, 209.
(35) Uyanik, M.; Yasui, T.; Ishihara, K. Angew. Chem., Int. Ed. 2010,
49, 2175.
Org. Lett., Vol. 13, No. 5, 2011
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