Stereocontrolled syntheses of all four stereoisomeric 1,N2-deoxyguanosine adducts of the lipid peroxidation product trans-4-hydroxynonenal

Article abstract of DOI:10.1021/ol016810c

(formula presented) trans-4-Hydroxynonenal (4-HNE) is a unique product from the peroxidation of Α-6 polyunsaturated fatty acids. The major reaction of racemic 4-HNE with DNA is with deoxyguanosine to give four stereoisomeric exocyclic propano adducts. The

Full text of DOI:10.1021/ol016810c

ORGANIC
LETTERS
2001
Vol. 3, No. 22
3603-3605
Stereocontrolled Syntheses of All Four
Stereoisomeric 1,N -Deoxyguanosine
2
Adducts of the Lipid Peroxidation
Product trans-4-Hydroxynonenal
Hao Wang and Carmelo J. Rizzo*
Department of Chemistry and Center in Molecular Toxicology, Vanderbilt UniVersity,
VU Station B 351822, NashVille, Tennessee 37235-1822
c.j.rizzo@Vanderbilt.edu
Received September 24, 2001
ABSTRACT
trans-4-Hydroxynonenal (4-HNE) is a unique product from the peroxidation of ω-6 polyunsaturated fatty acids. The major reaction of racemic
4-HNE with DNA is with deoxyguanosine to give four stereoisomeric exocyclic propano adducts. The stereospecific syntheses of these four
adducts has been achieved at the nucleoside level. The synthetic approach is amenable to the synthesis of structurally defined oligonucleotides
containing these endogenous genotoxins.
Reactive oxygen species such as hydroxyl radical are
produced by cellular respiration. These species abstract
hydrogen atoms from polyunsaturated fatty acids to generate
carbon-centered radicals, which in turn react with molecular
oxygen to generate the corresponding hydroperoxy radicals.1
Subsequent fragmentation of the lipid hydroperoxides gener-
ates complex arrays of lipid peroxidation products. A number
of these products have been identified as relatively simple
aliphatic R,â-unsaturated aldehydes (enals) such as acrolein,
crotonaldehyde, 4-hydroxynonenal, and 4-oxononenal.2 Ac-
rolein and crotonaldehyde are also ubiquitous environmental
pollutants and are found in cigarette smoke. These substances
react with proteins and are toxic. The simpler enals acrolein
and crotonaldeyde induce revertants in the Ames Salmonella
tester strains TA100 and TA104;3 thus the lipid peroxidation
products represent a class of endogenous mutagens.4 4-Hy-
droxynonenal (4-HNE), the major lipid peroxidation product
derived from ω-6 polyunsaturated fatty acids, was reported
to be inactive in these Salmonella tester strains, while its
lower homologue 4-hydroxypentenal was shown to be
mutagenic. It is likely that the toxicity of 4-HNE masked its
genotoxicity in these assays. Consistent with this hypothesis
is that 4-HNE induced mutations in V79 Chinese hamster
ovary cells and induced an SOS response in Salmonella
typhimurium.5,6
The reaction of enals with nucleobases produces exocyclic
propano adducts. For deoxyguanosine, this involves initial
Michael addition of the exocyclic amino group followed by
ring closure of N1 onto the aldehyde group. The reaction of
deoxyguanosine with 4-HNE gives three new stereogenic
centers. The relative stereochemistry of the C8-hydroxy
group and the C6-hydroxyalkyl side chain has been deter-
mined to be trans.7 Thus, four diastereomeric adducts, 1-4
(Figure 1), are possible, which may have different properties
when incorporated into oligonucleotides.8 We report here the
stereocontrolled syntheses of all four stereoisomers of the
(1) Porter, N. A.; Caldwell, S. E.; Mills, K. A. Lipids 1995, 30, 277.
(2) Esterbauer, H.; Schaur, R. J.; Zollner, H. Free Radical Biol. Med.
1991, 11, 81.
(3) Marnett, L. J.; Hurd, H. K.; Hollstein, M. C.; Levin, D. E.; Esterbauer,
H.; Ames, B. N. Mutat. Res. 1985, 148, 25-34.
(4) Chung, F.-L.; Nath, R. G.; Nagao, M.; Nishikawa, A.; Zhou, G.-D.;
Randerath, K. Mutat. Res. 1999, 424, 71.
(5) Cajelli, E.; Ferraris, A.; Brambilla, G. Mutat. Res. 1987, 190, 169.
(6) Benamira, M.; Marnett, L. J. Mutat. Res. 1992, 293, 1.
(7) (a) Eder, E.; Hoffman, C. Chem. Res. Toxicol. 1992, 5, 802. (b)
Chung, F.-L.; Young, R.; Hecht, S. S. Cancer Res. 1984, 44, 990.
10.1021/ol016810c CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/12/2001

Figure 2. Amino alcohols for the synthesis of 1-4.
Figure 1. 1,N2-Deoxyguanosine adducts of 4-HNE.
Dihydroxylation gave a 1:1 mixture of vicinal diols; since
the vicinal diol is eventually oxidatively cleaved to the
corresponding aldehyde by periodate, the mixture of stereo-
isomers at this center is of no consequence. Deprotection
gave the desired anti amino alcohol (9) in enantiomerically
pure form. The enantiomeric amino alcohol 8 was prepared
using (+)-diethyl tartrate as the ligand for the Sharpless
epoxidation.
Using a similar synthetic strategy, we have synthesized
the syn amino alcohol 11 (Scheme 3). The stereochemistry
is established through a Sharpless kinetic resolution of
racemic 3-hydroxy-1-octene using (+)-diisopropyl tartrate
as the chiral ligand for the titanium catalyst. We estimate
that the epoxy alcohol (18) was produced in 92% ee on the
basis of high field NMR analysis of the corresponding
Mosher’s ester.11b Treatment of 18 with benzyl isocyanate
gave a single cyclic N-benzyl urethane 19. The remainder
of the synthesis proceeded by an identical sequence of
reactions as for the anti amino alcohol discussed above to
give 11. Once again, the enantiomeric syn amino alcohol
10 was synthesized by simply changing to the antipodal
tartrate ligand during the Sharpless kinetic resolution.
Amino alcohols 8-11 were individually condensed with
2′-deoxy-O6-(2-trimethylsilylethyl)-2-fluoroinosine (5a) to
1,N2-deoxyguanosine adduct of 4-HNE. The synthesis is
amenable to site-specific syntheses of oligonucleotides
containing stereochemically defined adducts of 4-HNE.
The site-specific syntheses of oligonucleotides containing
1,N2-deoxyguanosine adducts of acrolein (7) have been
reported by Johnson and by Harris.9,10 In both cases, a vicinal
diol unit was used as a surrogate for the aldehyde; the vicinal
diol was cleaved with sodium periodate after assembly of
the oligonucleotides. The three-carbon acrolein unit was
introduced via a nucleophilic aromatic substitution reaction
of amino alcohol 6 with an O6-protected 2-fluoroinosine
derivative 5 (Scheme 1).
We envisioned a similar strategy for the synthesis of 1,N2-
deoxyguanosine adducts of 4-HNE. Enantiomeric amino
alcohols 8 and 9, possessing the anti stereochemistry, are
required for the synthesis of adducted nucleosides 1 and 2,
respectively (Figure 2), whereas syn amino alcohols 10 and
11 would give 3 and 4. Enantioselective syntheses of amino
alcohols 9 and 11 are shown in Schemes 2 and 3.
The synthesis of 9 began with a Sharpless asymmetric
epoxidation of (E)-oct-2-en-1-ol using (-)-diethyl tartrate
as the ligand, to give the epoxy alcohol 13; a minor
stereoisomer could not be detected by high field NMR
analysis of the corresponding Mosher’s ester of 13.11
Reaction of 13 with benzyl isocyanate afforded a separable
mixture of two cyclic N-benzyl urethanes 14 and 15; the
minor isomer 14 could be equilibrated by treatment with
sodium hydride.12 At this point, a two-carbon extension of
the aliphatic chain was accomplished by converting the
primary alcohol of 15 to the corresponding iodide followed
by displacement with vinylmagnesium bromide to give 16.
Scheme 2a
Scheme 1
a Reagents: (a) Ti(OiPr)4, tBuOOH, (-)-DET, 4 Å MS, CH2Cl2,
-20 °C, 85%; (b) BnNCO, NaH, THF, reflux, 50%; (c) Ph3P, I2,
imidazole, 84%; (d) H2CdCHMgBr, CuI, HMPA, THF, 67%; (e)
OsO4, NMO, THF, tBuOH, H2O, 72%; (f) KOH, EtOH, reflux,
68%; (g) H2, Pd(OH)2, MeOH, 100%
3604
Org. Lett., Vol. 3, No. 22, 2001

or the corresponding 3′,5′-bisphosphate can be partially
separated by reverse phase HPLC. It was observed that two
stereoisomers coeluted while the two others were cleanly
resolved; no definitive stereochemical assignments were
made. With stereospecific syntheses of 1-4, we are now in
a position to assign the absolute stereochemistry of the
4-HNE adducts.
Scheme 3a
Using the published HPLC conditions we were able to
reproduce the reported elution profile. On the basis of the
HPLC retention times and published 1H NMR data, we have
established that the two resolved isomers are 3 and 4, derived
from the syn amino alcohols. This is consistent with the
previous observation that acid deglycosylation of these
isomers gave a single, presumably racemic guanine deriv-
ative.13a Interestingly, the reaction of racemic 4-HNE with
calf thymus DNA followed by digestion and analysis by
HPLC with 32P postlabeling detection has been reported to
give only two of the stereoisomers.13c On the basis of a
comparison of the reported HPLC retention time with our
own analysis, we tentatively assign the major adduct as 4
and the minor adduct as 3. It is worth noting that all four
stereoisomers have been detected from animal tissue using
the 32P postlabeling method, although not always in equal
concentrations.13c
Harris has shown that oligonucleotides containing an O6-
protected 2-fluoroinosine residue can undergo nucleophilic
aromatic substitution with aliphatic and aromatic amines to
give site-specifically adducted oligonucleotides.10 The O6-
protected 2-fluoroinosine was incorporated into oligonucleo-
tides using standard phosphoramidite chemistry. Reaction of
amino alcohols 8-11 with oligonucleotides containing
2-fluoroinosine followed by deprotection and periodate
cleavage should be a viable route to structurally and
stereochemically defined oligonucleotides containing 4-HNE
adducts. These modified oligonucleotides will be essential
for mutagenesis, repair, and structural studies on this
endogenous genotoxin.
a Reagents: (a) Ti(OiPr)4, tBuOOH, (+)-DIPT, CH2Cl2, -20 °C,
40%, 91% ee; (b) BnNCO, NaH, THF, reflux, 58%; (c) Ph3P, I2,
imidazole, 82%; (d) H2CdCHMgBr, CuI, HMPA, THF, 69%; (e)
OsO4, NMO, THF, tBuOH, H2O, 69%; (f) KOH, EtOH, reflux,
70%; (g) H2, Pd(OH)2, MeOH, 100%
give the corresponding N2-dG derivative (i.e., 21 from 9) in
57-64% yield after deprotection of the O6-(2-trimethylsilyl-
ethyl) group (Scheme 4). In the case of the syn amino
Scheme 4a
a Reagents: (a) iPr2NEt, DMSO, 70 °C; (b) 5% AcOH, 64%
from 5a; (c) NaIO4, H2O, 87%
alcohols, the minor stereoisomer from the Sharpless kinetic
resolution could be separated at this stage by HPLC.
Oxidative cleavage of the vicinal diol with sodium periodate
gave the 1,N2-propano adducts 1-4 in 80-87% yield.
Acknowledgment. We are grateful to Dr. Lubomir
Nechev, Dr. Ivan Kozekov, and Professors Tom and Connie
Harris for helpful suggestions. This work was supported by
the American Cancer Society (research grant RPG-96-061-
04-CDD) and the National Institutes of Health (research grant
ES05355 and center grant ES00267).
Methods have been developed for the detection of DNA
adducts of 4-HNE and related species.13 This involves
digestion of the oligonucleotide and analysis by HPLC. The
four stereoisomers of the 4-HNE-dG adduct at the nucleoside
1
Supporting Information Available: Copies of the H
and 13C NMR spectra of all new compounds and CD spectra
of 1-4. This material is available free of charge via the
Internet at http://pubs.acs.org.
(8) Winter, C. K.; Segall, H. J.; Haddon, W. F. Cancer Res. 1986, 46, 6,
5682.
(9) Khullar, S.; Varaprasad, C. V.; Johnson, F. J. Med. Chem. 1999, 42,
947.
(10) Nechev, L. V.; Harris, C. M.; Harris, T. M. Chem. Res. Toxicol.
2000, 13, 421.
(11) (a) Katsuki, T.; Martin, V. S. Org. React. 1996, 48, 1. (b) Gao, Y.;
Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune, H.; Sharpless K. B.
J. Am. Chem. Soc. 1987, 109, 5765.
OL016810C
(13) (a) Douki, T,; Ames, B. N. Chem. Res. Toxicol. 1994, 7, 511. (b)
Yi, P.; Zhan, D.; Samokyszyn, V. M.; Doerge, D. R.; Fu, P. P. Chem. Res.
Toxicol. 1997, 10, 1259. (c) Chung, F.-L.; Nath, R. G.; Ocando, J.;
Nishikawa, A.; Zhang, L. Cancer Res. 2000, 60, 1507. (d) Wacker, M.;
Schuler, D.; Wanek, P.; Eder, E. Chem. Res. Toxicol. 2000, 13. 1165.
(12) (a) Ager, D. J.; Prakash, I.; Schaad, D. R. Chem. ReV. 1996, 96,
835. (b) Nagamitsu, T.; Sunazuka, T.; Tanaka, H.; Omura, S.; Sprengeler,
P. A.; Smith, A. B. J. Am. Chem. Soc. 1996, 118, 3584.
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