Transimination of quinone imines: A mechanism for embedding exogenous redox activity into the nucleosome

Article abstract of DOI:10.1021/tx3004517

Aminophenols can redox cycle through the corresponding quinone imines to generate ROS. The electrophilic quinone imine intermediate can react with protein thiols as a mechanism of immobilization in vivo. Here, we describe the previously unkown transimination of a quinone imine by lysine as an alternative anchoring mechanism. The redox properties of the condensation product remain largely unchanged because the only structural change to the redox nucleus is the addition of an alkyl substituent to the imine nitrogen. Transimination enables targeting of histone proteins since histones are lysine-rich but nearly devoid of cysteines. Consequently, quinone imines can be embedded in the nucleosome and may be expected to produce ROS in maximal proximity to the genome.

Full text of DOI:10.1021/tx3004517

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Transimination of Quinone Imines: A Mechanism for Embedding
Exogenous Redox Activity into the Nucleosome
Wenjie Ye, Uthpala I. Seneviratne, Ming-Wei Chao, Kodihalli C. Ravindra, Gerald N. Wogan,†
†
†
†,§
†
Steven R. Tannenbaum,†,‡ and Paul L. Skipper*,†
†
‡
Department of Biological Engineering and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts
Avenue, Cambridge, Massachusetts 02139, United States
*
S Supporting Information
ABSTRACT: Aminophenols can redox cycle through the
corresponding quinone imines to generate ROS. The electro-
philic quinone imine intermediate can react with protein thiols
as a mechanism of immobilization in vivo. Here, we describe
the previously unkown transimination of a quinone imine by
lysine as an alternative anchoring mechanism. The redox
properties of the condensation product remain largely
unchanged because the only structural change to the redox
nucleus is the addition of an alkyl substituent to the imine
nitrogen. Transimination enables targeting of histone proteins
since histones are lysine-rich but nearly devoid of cysteines. Consequently, quinone imines can be embedded in the nucleosome
and may be expected to produce ROS in maximal proximity to the genome.
1
2,13
niline and its various ring-alkyl congeners are transformed
in vivo principally to aminophenols through oxidative
described previously,
whereby a primary amine displaces
A
ammonia from the quinone imine. We demonstrate here that
this occurs in cells with lysine residues. Histones, which are
virtually devoid of cysteine residues, were of particular interest
because lysine transimination of histones places the redox-
active structure in the closest possible location to oxidizable
DNA nucleobases and creates a potentially potent mechanism
for genetic damage by aminophenols that is distinct from
covalent modification of the bases.
Synthesis of the lysyl transimination product of 3,5-
dimethylquinone imine (3,5-DMQI) is shown in Scheme 1.
,5-DMAP was synthesized from 2,6-dimethylphenol as
described for the isomeric 2,6-DMAP. To make [ring- C]-
1
,2
metabolism. This pathway is generally considered, at least
with respect to carcinogenesis, as one of detoxification, but that
assessment may represent a bias arising out of the under-
standing that N-, rather than ring-, hydroxylation of multicyclic
aromatic amines is the key metabolic step in their activation to
3
−5
an ultimate carcinogenic form.
Aminophenol formation is
not necessarily the end point of oxidative metabolism if the
substitution is ortho or para: it has recently been shown that
these structures may be further transformed in vivo to quinone
6
,7
imines. Quinone imines are electrophiles and can undergo
Michael addition with thiols and amines. They might, therefore,
be capable of reacting with DNA nucleobases as a means of
bringing about the mutagenic and carcinogenic effects observed
3
14
14
14
3
,5-DMAP, [ring- C]-2,6-dimethylphenol was obtained by
decomposition of [ring- C]-2,6-dimethylbenzenediazonium
14
8
with some alkylanilines such as 3,5-dimethylaniline, the focus
of the present work. There is as yet no evidence for this
mechanism of action.
ε
Scheme 1. Synthesis of N -(4-Hydroxy-3,5-dimethylphenyl)-
lysine
In addition to their electrophilic character, 1,2- and 1,4-
quinone imines are redox active structures that can cycle
through the corresponding aminophenols to generate reactive
oxygen species (ROS). As we have recently demonstrated with
9
p-hydroxy-3,5-dimethylaniline (3,5-DMAP) and p-hydroxy-
2
,6-dimethylaniline (2,6-DMAP), aminophenols do generate
10
ROS intracellularly. ROS generation in these experiments
persisted for at least 24 h, indicating that the source was
effectively immobilized within the cells. Immobilization could
be achieved through Michael reaction with cellular thiols, as
such products have been shown to retain their redox activity,
but this is not the only mechanism available to quinone imines.
They are also capable of undergoing transimination, as
11
Received: November 9, 2012
Published: November 29, 2012
©
2012 American Chemical Society
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Figure 1. (A) HPLC-MS-MS analysis of synthetic 2b (top), isolate from histones in cells treated with 3,5-DMAP (bottom), and isolate from
histones in untreated cells (middle). (B) CID spectra of synthetic 2b (top) and 2b isolated from 3,5-DMAP-treated cells (bottom).
ion in H O. Compound 1a was produced by reaction of 3,5-
2
α
DMQI, formed transiently by PbO oxidation, with N -(t-
2
Boc)lysine. Following the transimination reaction, product 1a
was reduced with ascorbic acid to the more stable aminophenol
α
ε
form N -(t-Boc)-N -(4-hydroxy-3,5-dimethylphenyl)lysine
2a). With care to avoid oxidation, 2a could be isolated from
(
the reaction mixture by preparative HPLC, and its structure was
confirmed by 1D and 2D NMR and mass spectrometry. After
deprotection, 2b could not similarly be isolated because its
increased polarity did not permit separation from other
reactants. Nevertheless, it could readily be detected and
characterized by HPLC-MS (Figure 1A, upper trace, and
Figure 1B, upper spectrum). Reaction of unprotected lysine
with 3,5-DMQI apparently produced 1b since, after treatment
with ascorbate, the product was indistinguishable from 2b by
HPLC-MS.
1
4
Figure 2. HPLC analysis of histones isolated from [ C]3,5-DMAP-
ε
Formation of N -(4-hydroxy-3,5-dimethylphenyl)-lysine (2b)
treated cells showing UV detector response (continuous trace) and
14
C concentration in collected fractions.
by histones in vivo was investigated using Chinese hamster
ovary AA8 cells grown in culture. Cells were treated with 25
μM 3,5-DMAP for 1 h, washed with fresh medium, and isolated
by centrifugation. Histones were isolated and digested with
protease in sodium acetate buffer containing 1 mM ascorbate to
inhibit oxidation. After initial purification by semipreparative
HPLC, 2b was analyzed by HPLC-MS using a triple-quad mass
spectrometer. Data acquired in multiple reaction monitoring
mode are shown in Figure 1A, which shows traces produced by
synthetic 2b as well as isolates from control histones (not
treated with 3,5-DMAP) and histones from treated cells. Figure
counting to achieve the requisite sensitivity. Figure 2 reveals the
correspondence of 14C concentration with histone fractions
generally as well as two notable features that may be indicative
of site-specificity in the labeling reaction. The arrow marked
with an asterisk at 60.5 min indicates a region of the
chromatogram where histone H2A.Z elutes. With the exception
of one other fraction, this region of the chromatogram exhibits
the highest levels of C. Histone H2A.Z appears to be the
target of more extensive acetylation than other variants and,
by implication, might be more highly targeted by 3,5-DMAP,
thereby accounting for the higher degree of labeling relative to
the more abundant histones. The second notable feature of
14
15
1
A demonstrates cochromatography of histone isolate with
synthetic 2b; Figure 1B demonstrates that the CID spectra are
identical, confirming that the product isolated from histones is
indeed the lysine transimination product.
14
Figure 2 is the C peak at 63.2 min that corresponds to a
As evidence that 2b originated from histone protein and not
minor peak observed in the UV trace. The identity of this peak
is unknown, so we can only speculate that it represents a
hydroxydimethylphenylated histone variant. Alternatively, it
might correspond to phenylated H4 whose retention on the
HPLC column was increased by the modification.
The transimination reaction described here, between a
quinone imine and a primary amine, is not unknown but has
received little attention. The analogous reaction between
quinone imides, specifically, N-acetylated quinone imines, has
been investigated with divergent results. 1,2-Fluorenoquinone-
2-acetimide reacted with lysine in serum albumin via 1,4-
from any other protein that might have contaminated the
1
4
histone preparation, cells were treated with [ C]-3,5-DMAP
and the isolated histone fraction was subjected to HPLC for
analysis of isotopic labeling. The chromatograpic separation
described by Boyne et al. for top down MS characterization of
1
5
histones was replicated for the present study, and the results
are given in Figure 2 with identification of the histone peaks as
given in the cited reference. In developing this separation, we
confirmed that the indicated peaks had masses appropriate for
histones but could not confirm the assignments made by Boyne
because of the complexity associated with post-translational
16
addition. In contrast, N-acetyl-3,5-dimethyl-p-benzoquinone
imine reacted with aniline to give the N-phenyl quinone imine,
whereas the 2,6-isomer gave a tetrahedral adduct in which the
1
4
modifications. Quantitation of C was performed on collected
fractions by accelerator mass spectrometry rather than decay
2
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Chemical Research in Toxicology
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1
7
acetamido group was retained. In the present work, we
(3) Delclos, K. B., and Kadlubar, F. F. (1997) Carcinogenic Aromatic
Amines and Amides, in Comprehensive Toxicology (Bowden, G. T., and
Fischer, S. M., Eds.) Vol. 12, Chemical Carcinogens and
Anticarcinogens, pp 141−170, Elsevier Science, New York.
looked for and found no evidence by mass spectrometry for
,4--addition of N -(t-Boc)lysine to 3,5-DMQI when forming
a. 1,4-Addition is clearly not blocked by the methyl
substituents, but it is directed toward the unsubstituted
positions on the ring: we found that 3,5-DMQI reacts with
N-acetylcysteine at the 2-position at much greater rates than
the rate of transimination with N -(t-Boc)lysine, and similarly,
N-acetyl-3,5-dimethyl-p-benzoquinone imine readily forms a
Michael product with ethanethiol. In both instances, Michael
addition takes place relative to the ketone, not the imine.
The biological consequences of quinone imine transimina-
tion may be considerable, especially insofar as they relate to
hydroxyphenylation of the lysine side chains of the histones. As
noted above, this reaction embeds a redox-active center in the
nucleosome where it is positioned for maximum efficacy in
creating genetic damage via ROS. Additionally, modification
α
1
2
(4) Beland, F. A., and Kadlubar, F. F. (1990) Metabolic Activation
and DNA Adducts of Aromatic Amines and Nitroaromatic Hydro-
carbons, in Handbook of Experimental Pharmacology: Chemical
Carcinogenesis and Mutagenesis (Cooper, C. S., and Grover, P. L.,
Eds.) Vol. 94/I, pp 267−325, Springer-Verlag, Berlin, Germany.
(5) Kadlubar, F. F., and Beland, F. A. (1985) Chemical Properties of
Ultimate Carcinogenic Metabolites of Arylamines and Arylamides, in
Polycyclic Hydrocarbons and Carcinogenesis (Harvey, P. G., Ed.) ACS
Symposium Series No. 283, pp 341−370, American Chemical Society,
Washington, D.C.
α
1
7
(6) Jefferies, P. R., Quistad, G. B., and Casida, J. E. (1998)
Dialkylquinonimines validated as in vivo metabolites of alachlor,
acetochlor, and metolachlor herbicides in rats. Chem. Res. Toxicol. 11,
3
(
53−359.
7) Martínez-Cabot, A., Morato, A., and Messeguer, A. (2005)
́
(
acetylation and methylation) of certain lysine side chains is
Synthesis and stability studies of the glutathione and N-acetylcysteine
adducts of an iminoquinone reactive intermediate generated in the
biotransformation of 3-(N-phenylamino)propane-1,2-diol: Implica-
tions for Toxic Oil Syndrome. Chem. Res. Toxicol. 18, 1721−1728.
involved in epigenetic change. Their nonbiological modification
through hydroxyphenylation may inhibit or replicate these
changes inappropriately. The combination of two comple-
mentary modes of action may serve to endow certain
monocyclic aminophenols with considerably more biological
activity than has historically been attributed to them.
(8) Gan, J.-P., Skipper, P. L., Gago-Dominguez, M., Arakawa, K.,
Ross, R. K., Yu, M. C., and Tannenbaum, S. R. (2004) Alkylaniline−
hemoglobin adducts and risk of non−smoking-related bladder cancer.
J. Natl. Cancer Inst. 96, 1425−1431.
(9) This nomenclature differs from the IUPAC system for the
ASSOCIATED CONTENT
■
purpose of preserving the numbering used for the amines from which
the aminophenols and quinone imines are derived.
(10) Chao, M.-W., Kim, M. Y., Ye, W., Ge, J., Trudel, L. J., Belanger,
C., Skipper, P., Engelward, B., Tannenbaum, S., and Wogan, G. N.
*
S
Supporting Information
1
Experimental details and H NMR and mass spectral data
identifying structure 2b. This material is available free of charge
via the Internet at http://pubs.acs.org.
(
2012) Genotoxicity of 2,6- and 3,5-dimethylaniline in cultured
mammalian cells: the role of reactive oxygen species. Toxicol. Sci. 130,
8−59.
11) Eyer, P. (1994) Reactions of oxidatively activated arylamines
4
AUTHOR INFORMATION
■
(
Corresponding Author
with thiols: reaction mechanisms and biologic implications. An
overview. Environ. Health Perspect. 102 (6), 123−132.
*E-mail: skipper@mit.edu.
Present Address
Department of Bioscience and Technology, Chung Yuan
Christian University, 200 Chung-Pei Road, Chungli, Taiwan
(12) Yang, Q.-Z., Siri, O., and Braunstein, P. (2005) Tunable N-
substitution in zwitterionic benzoquinonemonoimine derivatives:
Metal coordination, tandemlike synthesis of zwitterionic metal
complexes, and supramolecular structures. Chem.Eur. J. 11, 7237−
§
3
20.
7
246.
Funding
(13) Lee, Y., and Sayre, L. M. (1995) Model reactions for the
This work was supported by NIEHS (PO1-ES006052 and P30-
ES002109).
Notes
quinone-containing copper amine oxidases. Anaerobic reaction
pathways and catalytic aerobic deamination of activated amines in
buffered aqueous acetonitrile. J. Am. Chem. Soc. 117, 3096−3105.
(
14) Gan, J., Skipper, P. L., and Tannenbaum, S. R. (2001) Oxidation
The authors declare no competing financial interest.
of 2,6-dimethylaniline by recombinant human cytochrome P450s and
human liver microsomes. Chem. Res. Toxicol. 14, 672−677.
ACKNOWLEDGMENTS
■
(15) Boyne, M. T., II, Pesavento, J. J., Mizzen, C. A., and Kelleher, N.
L. (2006) Precise characterization of human histones in the H2A gene
family by top down mass spectrometry. J. Proteome Res. 5, 248−253.
Radiocarbon measurements were made by Rosa G. Liberman in
the MIT BEAMS laboratory. We thank Agilent Technologies
for graciously providing access to the 6430 triple quadrupole
mass spectrometer and the 1290 HPLC system.
(
16) Irving, C. C., and Gutmann, H. R. (1961) Protein binding of
e
model quinone imides. III. Preparation of N -(1-hydroxy-2-acetamido-
-fluorenyl)-DL-lysine. J. Org. Chem. 26, 1859−1861.
17) Fernando, C. R., Calder, I. C., and Ham, K. N. (1980) Studies
4
(
ABBREVIATIONS
■
on the mechanism of toxicity of acetaminophen. Synthesis and
reactions of N-acetyl-2,6-dimethyl- and N-acetyl-3,5-dimethyl-p-
benzoquinone imines. J. Med. Chem. 23, 1153−1158.
CID, collision-induced dissociation; DMAP, dimethylamino-
phenol; DMQI, dimethyl quinone imine; MRM, multiple
reaction monitoring; ROS, reactive oxygen species; t-Boc, tert-
butyloxycarbonyl
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