Site-specific synthesis and reactivity of oligonucleotides containing stereochemically defined 1,N2-deoxyguanosine adducts of the lipid peroxidation product trans-4-Hydroxynonenal

Article abstract of DOI:10.1021/ja0288800

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of ω-6 polyunsaturated fatty acids. The reaction of HNE with DNA gives four diastereomeric 1,N²-γ-hydroxypropano adducts of deoxyguanosine; background levels of these adducts have been detected in animal tissue. Stereospecific syntheses of these four adducts at the nucleoside level have been accomplished. In addition, a versatile strategy for their site-specific incorporation into oligonucleotides has been developed. These adducts are destabilizing as measured by melting temperature when compared to an unadducted strand. The thermal destablization of the adducted 12-mers ranged from 5 to 16 °C and is dependent on the absolute stereochemistry of the adduct. The HNE adducts were also examined for their ability to form interstrand DNA-DNA cross-links when incorporated into a CpG sequence. We find that only one of the HNE stereoisomers formed interstrand DNA-DNA cross-links.

Full text of DOI:10.1021/ja0288800

Published on Web 04/19/2003
Site-Specific Synthesis and Reactivity of Oligonucleotides
Containing Stereochemically Defined 1,N2-Deoxyguanosine
Adducts of the Lipid Peroxidation Product
trans-4-Hydroxynonenal
Hao Wang, Ivan D. Kozekov, Thomas M. Harris, and Carmelo J. Rizzo*
Contribution from the Department of Chemistry and Center in Molecular Toxicology,
Vanderbilt UniVersity, NashVille, Tennessee 37235-1822
Received October 9, 2002; E-mail: c.j.rizzo@vanderbilt.edu
Abstract: trans-4-Hydroxynonenal (HNE) is a major peroxidation product of ω-6 polyunsaturated fatty acids.
The reaction of HNE with DNA gives four diastereomeric 1,N2-γ-hydroxypropano adducts of deoxyguanosine;
background levels of these adducts have been detected in animal tissue. Stereospecific syntheses of these
four adducts at the nucleoside level have been accomplished. In addition, a versatile strategy for their
site-specific incorporation into oligonucleotides has been developed. These adducts are destabilizing as
measured by melting temperature when compared to an unadducted strand. The thermal destablization of
the adducted 12-mers ranged from 5 to 16 °C and is dependent on the absolute stereochemistry of the
adduct. The HNE adducts were also examined for their ability to form interstrand DNA-DNA cross-links
when incorporated into a CpG sequence. We find that only one of the HNE stereoisomers formed interstrand
DNA-DNA cross-links.
Introduction
A major product of the peroxidation of ω-6 polyunsaturated
fatty acids is (()-4-hydroxynonenal (HNE).9 HNE is highly
cytotoxic and induces apoptosis and necrosis in tumor cells.10
HNE is inactive in Ames assays; however, the lower homologue
4-hydroxypentenal was found to be mutagenic.8 It is likely that
the toxicity of HNE masks its genotoxicity in these assays.
Consistent with this hypothesis is the fact that HNE induces
mutations in V79 Chinese hamster ovary cells and induces a
dose-dependent SOS response in S. typhimurium.11,12 Recently,
livers from humans who had suffered from Wilson’s disease
and hemochromatosis were examined and found to contain
mutations at C:G sites in the coding regions of the p53 tumor
suppressor gene.13 Evidence was presented that these mutations
may be caused by HNE modification of a deoxyguanosine.
The autoxidation of polyunsaturated fatty acids is a free
radical process initiated by reactive oxygen species such as
hydroxyl radical. The initial product is a lipid hydroperoxide
which can undergo metal ion mediated fragmentation to produce
a complex array of products.1 Among the peroxidation products
are R,â-unsaturated aldehydes (enals).2 Enals react with sulf-
hydryl and amino groups of proteins and are toxic.2a They also
react with DNA bases and have mutagenic potential.3 Thus, the
peroxidation of lipids represents an endogenous source of
cytotoxins and genotoxins. The simplest enals, acrolein and
crotonaldehyde, have been shown to be products of lipid
peroxidation although the mechanism of their formation is not
obvious.4,5 Acrolein can also arise by the oxidative degradation
of threonine.6 In addition, acrolein, and crotonaldehyde are
widely dispersed environmental pollutants and are constituents
of cigarette smoke.7 Acrolein and crotonaldehyde induce
revertants in the Ames Salmonella typhimurium tester strains
TA100 and TA104.8
Enals are bis-electrophiles and can potentially undergo tandem
reactions with pairs of nucleophilic sites on the DNA bases.
Reaction with deoxyguanosine gives the cyclic 1,N2-hydroxy-
propano adducts (Figure 1). Two regioisomers are possible. The
one having the hydroxyl group at C8, i.e., proximal to the purine
ring system, arises by conjugate addition of the exocyclic N2-
(1) (a) Porter, N. A.; Caldwell, S. E.; Mills, K. A. Lipids 1995, 30, 277. (b)
Porter, N. A. Acc. Chem. Res. 1986, 19, 262. (c) Dix, T. A.; Aikens, J.
Chem. Res. Toxicol. 1993, 6, 2.
(2) (a) Esterbauer, H.; Schaur, R. J.; Zollner, H. Free Radical Biol. Med. 1991,
11, 81. (b) Marnett, L. J. Mutat. Res. 1999, 424, 83.
(3) Chung, F.-L.; Nath, R. G.; Nagao, M.; Nishikawa, A.; Zhou, G.-D.;
Randerath, K. Mutat. Res. 1999, 424, 71.
(4) Uchida, K.; Kamematsu, M.; Morimitsu, Y.; Osawa, T.; Noguchi, N.; Niki,
E. J. Biol. Chem. 1998, 273, 16058.
(5) Pan, J.; Chung, F.-L. Chem. Res. Toxicol. 2002, 15, 367.
(6) Hazen, S. L.; Hsu, F. F.; d’Avignon, A.; Heinecke, J. W. Biochemistry,
1998, 37, 6864.
(7) Nath, R. G.; Ocando, J. E.; Guttenplan, J. B.; Chung, F. L. Cancer Res.
1998, 58, 581.
(8) Marnett, L. J.; Hurd, H. K.; Hollstein, M. C.; Levin, D. E.; Esterbauer, H.;
Ames, B. N. Mutat. Res. 1985, 148, 25.
(9) Schneider, C.; Tallman, K. A.; Porter, N. A.; Brash, A. R. J. Biol. Chem.
2001, 276, 20831.
(10) (a) Ji, C.; Amarnath, V.; Pietenpol, J. A.; Marnett, L. J. Chem. Res. Toxicol.
2001, 14, 1090. (b) Haynes, P. L.; Brune, B.; Townsend, A. J. Free Radical
Biol. Med. 2001, 30, 884.
(11) Cajelli, E.; Ferraris, A.; Brambilla, G. Mutat. Res. 1987, 190, 169.
(12) Benamira, M.; Marnett, L. J. Mutat. Res. 1992, 293, 1.
(13) Hussain, S. P.; Raja, K.; Amstad, P. A.; Sawyer, M.; Trudel, L. J.; Wogan,
G. N.; Hofseth, L. J.; Shields, P. G.; Billiar, T. R.; Trautwein, C.; Ho¨hler,
T.; Galle, P. R.; Phillips, D. H.; Markin, R.; Marrogi, A. J.; Harris, C. C.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12770.
9
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J. AM. CHEM. SOC. 2003, 125, 5687-5700
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A R T I C L E S
Wang et al.
Figure 1. 1,N2-hydroxypropano adducts of deoxyguanosine.
amino group to the enal followed by ring closure of N1 with
the aldehyde. The acrolein adduct has a new stereogenic center
at C8. For crotonaldehyde and higher enals, two new stereogenic
centers are formed; however, only the stereoisomers with the
C8-hydroxyl group trans to the C6-substituent are observed.14
The second type of adduct, i.e., the 6-hydroxy or distal adduct,
results from initial conjugate addition of N1 followed by ring
closure of the aldehyde with the N2-amino group or, alterna-
tively, by formation of the Schiff base with the N2-amino group
followed by ring closure onto N1 and rehydration of the Schiff
base. The distal adduct has only been observed for the reaction
Figure 2. 1,N2-deoxyguanosine adducts of HNE.
form both interstrand and intrastrand DNA-DNA cross-
links.24-26 Hecht and co-workers have also isolated a formal
crotonaldehyde cross-link between two deoxyguanosines from
the reaction of calf thymus DNA with acetaldehyde.27 The
mechanism by which that cross-link is formed probably does
not involve the reaction of DNA with crotonaldehyde, which
is the aldol product of acetaldehyde, but rather the reaction of
acetaldehyde with the acetaldehyde-deoxyguanosine Schiff
base.17c,27 The ability of enals to form DNA-DNA cross-links
could play a significant role in their biological processing.
The reaction of HNE with deoxyguanosine creates three new
stereogenic centers in the nucleoside. One center arises from
the HNE itself; the other two are generated by the adduction
reaction. As in the proximal crotonaldehyde adducts, the relative
stereochemistry between C8 and C6 is trans. Thus, there are
four possible stereoisomers of the HNE adducts of deoxygua-
nosine, 1-4 (Figure 2).28 Oligonucleotides containing the
diastereomeric HNE adducts may have different local confor-
mations. Consequently, it is possible that the various stereo-
isomers of the HNE adduct could elicit different DNA-DNA
and DNA-protein cross-linking reactions and different biological
responses.
of deoxyguanosine with acrolein and methyl vinyl ketone.15
A
number of studies have demonstrated the formation of proximal
deoxyguanosine adducts in DNA incubated with acrolein and
other enals and in DNA samples isolated from enal-exposed
and unexposed animal tissue, but distal adducts have not been
identified in any of these DNA samples.16
Recently, 1,N2-deoxyguanosine adducts of acrolein and
crotonaldehyde have been site-specifically incorporated into
oligonucleotides.17 Site-specific mutagenesis studies of the
acrolein adduct revealed this lesion to be essentially nonmu-
tagenic in point mutation assays in E. coli, but mutagenic in
mammalian cells.18,19 The synthesis of the distal acrolein
deoxyguanosine adduct has recently been reported by Johnson
and by our laboratory, and we have also reported the preparation
of oligonucleotides containing this lesion.20,21 Mutagenesis
studies for the distal adducts have not yet been reported.
At the nucleoside level, the 1,N2-deoxyguanosine adducts of
enals exist exclusively in the ring-closed form. However, recent
NMR structures of an oligonucleotide duplex containing the
proximal acrolein and closely related malondialdehyde adducts
of deoxyguanosine showed that the ring-opened form predomi-
nated.22,23 The ring-opened structure leaves an electrophilic
aldehyde group in the minor groove of DNA, which can react
with nucleophilic sites of other DNA bases to form intra- or
interstrand DNA-DNA cross-links or with proteins to give
DNA-protein cross-links. Recent chemical and spectroscopic
studies have demonstrated the ability of the acrolein adduct to
We report here the stereocontrolled syntheses of all four
stereoisomers of the proximal 1,N2-deoxyguanosine adduct of
HNE.29 In addition, oligonucleotides containing the four stereo-
isomers of the HNE adduct have been prepared in a structurally
specific manner. Finally, we have examined the ability of these
adducts to form interchain cross-links and found the cross-
linking reaction is strongly dependent on adduct stereochemistry
and gives a remarkably stable cross-link.
Results
Stereospecific Synthesis of the Deoxyguanosine Adducts
of HNE. Syntheses of the proximal 1,N2-deoxyguanosine adduct
of acrolein (7a) and of oligonucleotides containing 7a have been
independently reported by Johnson and Harris.17a,b Similar
syntheses of the two diastereomers of the crotonaldehyde adduct
(14) (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.
(15) Chung, F.-L.; Roy, K. R.; Hecht, S. S. J. Org. Chem. 1988, 53, 14.
(16) (a) Nath, R. G.; Chung, F.-L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7491.
(b) Nath, R. G.; Ocando, J. E.; Chung, F.-L. Cancer Res. 1996, 56, 452.
(17) (a) Khullar, S.; Varaprasad, C. V.; Johnson, F. J. Med. Chem. 1999, 42,
947. (b) Nechev, L. V.; Harris, C. M.; Harris, T. M. Chem. Res. Toxicol.
2000, 13, 421. (c) Nechev, L. V.; Kozekov, I.; Harris, C. M.; Harris, T. M.
Chem. Res. Toxicol. 2001, 14, 1506.
(18) (a) Yang, I.-Y.; Hossian, M.; Miller, H.; Khullar, S.; Johnson, F.; Grollman,
A.; Moriya, M. J. Biol. Chem. 2001, 276, 9071. (b) VanderVeen, L. A.;
Hashim, M. F.; Nechev, L. V.; Harris, T. M.; Harris, C. M.; Marnett, L. J.
J. Biol. Chem. 2001, 276, 9066.
(19) Kanuri, M.; Minko, I. G.; Nechev, L. V.; Harris, T. M.; Harris, C. M.;
Lloyd, R. S. J. Biol. Chem. 2002, 277, 18257.
(20) Huang, Y., Johnson, F. Chem. Res. Toxicol. 2002, 15, 236.
(21) Nechev, L. V.; Kozekov, I. D.; Brock, A. K.; Rizzo, C. J.; Harris, T. M.
Chem. Res. Toxicol. 2002, 15, 607.
(22) de los Santos, C.; Zaliznyak, T.; Johnson, F. J. Biol. Chem. 2001, 276,
9077.
(23) Mao, H.; Schnetz-Boutaud, N. C.; Weisenseel, J. P.; Marnett, L. J.; Stone,
M. P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6615.
(24) Kozekov, I. D.; Nechev, L. V.; Sanchez, A.; Harris, C. M.; Lloyd, R. S.;
Harris, T. M. Chem. Res. Toxicol. 2001, 14, 1482.
(25) Kim, H.-Y.; Voehler, M.; Harris, T. M.; Stone, M. P. J. Am. Chem. Soc.
2002, 124, 9324.
(26) Kawanishi, M.; Matsuda, T.; Nakayama, A.; Takebe, H.; Matsui S.; Yagi,
T. Mutat. Res. 1998, 417, 65.
(27) (a) Wang, M.; McIntee, E. J.; Cheng, G.; Shi, Y.; Villalta, P. W.; Hecht,
S. S. Chem. Res. Toxicol. 2000, 13, 1149. (b) Wang, M.; McIntee, E. J.;
Cheng, G.; Shi, Y.; Villalta, P. W.; Hecht, S. S. Chem. Res. Toxicol. 2001,
14, 423.
(28) Winter, C. K.; Segall, H. J.; Haddon, W. F. Cancer Res. 1986, 46, 5682.
(29) Wang, H.; Rizzo, C. J. Org. Lett. 2001, 3, 3603. In our initial report,
compounds 2 and 3 in Figure 1 were interchanged and should be as shown
in Figure 2 of this manuscript. Corrrection: Wang, H.; Rizzo, C. J. Org.
Lett. 2002, 4, 155.
9
5688 J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003

Adducts of trans-4-Hydroxynonenal
A R T I C L E S
Scheme 1
Figure 3. Amino alcohols for the synthesis of 1-4.
7b were reported as well as site-specific syntheses of oligo-
nucleotides containing the individual diastereomers.17c In each
case, a vicinal diol unit was used as a surrogate for the aldehyde
group and the vicinal diol was cleaved with sodium periodate
to yield an aldehyde which then cyclized (Scheme 1). The three-
and four-carbon units for acrolein and crotonaldehyde, respec-
tively, were introduced via a nucleophilic aromatic substitution
reaction of amino alcohol 6 with an O6-protected 2-fluoroinosine
derivative 5. In the oligonucleotide syntheses by Harris et al.,
these displacements were accomplished on an oligonucleotide
containing O6-protected 2-fluoroinosine nucleotide 5a.17b,c
A similar strategy can be envisioned for the synthesis of 1,N2-
deoxyguanosine adducts of HNE and of oligonucleotides
containing these adducts. The stereocontrolled synthesis of the
four diastereomeric HNE adducts would require enantioselective
syntheses of the amino alcohols shown in Figure 3. The
antipodal amino alcohols 8 and 9 possessing the anti relative
stereochemistry between the critical amino and hydroxyl groups
are required for the synthesis of adducted nucleosides 1 and 2,
respectively, 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.
Scheme 2 a
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%.
The enantioselective synthesis of 9 began with a Sharpless
asymmetric epoxidation of E-oct-2-en-1-ol (12) using (-)-
diethyl tartrate as the ligand, to give the epoxy alcohol 13.30
Reaction of 13 with benzyl isocyanate afforded a separable
mixture of two cyclic N-benzyl urethanes 14 and 15; the
undesired isomer 14 could be equilibrated to form additional
amounts of 15 by treatment with sodium hydride.31 A two-
carbon extension of the aliphatic chain was accomplished by
converting the primary hydroxyl group of 15 to the correspond-
ing iodide (16) followed by displacement with vinylmagnesium
bromide in the presence of copper iodide to give 17. Dihy-
droxylation gave a 1:1 mixture of vicinal diols (18). Because
the vicinal diol is eventually oxidatively cleaved to the corre-
sponding aldehyde by periodate, the mixture of stereoisomers
at this center is of no consequence. Deprotection gave the desired
anti amino alcohol 9 in high enantiomeric purity at carbons 4
and 5. The enantiomeric amino alcohol 8 was prepared in an
identical fashion using (+)-diethyl tartrate as the ligand for the
Sharpless epoxidation.
Scheme 3 a
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%.
Using a related synthetic strategy, we have synthesized the
syn amino alcohol 11 (Scheme 3). The absolute configuration
was established through a Sharpless kinetic resolution of racemic
3-hydroxy-1-octene (20) using (+)-diisopropyl tartrate as the
chiral ligand for the titanium catalyst.30,32 Treatment of 21 with
benzyl isocyanate gave a single cyclic N-benzyl urethane 22.
The remainder of the synthesis to give 11 proceeded by a
sequence of reactions identical to those for anti amino alcohols
8 and 9 discussed above. Once again, the enantiomeric syn
amino alcohol 10 was synthesized by simply changing to the
antipodal tartrate ligand during the Sharpless kinetic resolution.
The optical purity of epoxy alcohols 13 and 21 was
established by analysis of the corresponding (-)-Mosher’s esters
by HPLC and high-field NMR. Richardson and Rychnovsky
previously reported that the Sharpless asymmetric epoxidation
(30) (a) Katsuki, T.; Martin, V. S. Org. React. 1996, 48, 1. (b) Gao, Y.; Klunder,
J. M.; Hanson, R. M.; Masamune, H.; Ko, S. Y.; Sharpless K. B. J. Am.
Chem. Soc. 1987, 109, 5765.
(31) (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.
(32) Mori, K.; Otsuka, T. Tetrahedron 1985, 41, 553.
9
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A R T I C L E S
Wang et al.
Scheme 4 a
Figure 5. HMBC spectra of 4.
Table 1. 1H NMR Spectra of Nucleosides 1-4 in Methanol-d4
assignment
1
2
3
4
2
6
7.91
7.91
7.91
3.61
2.18
1.60c
6.40
3.47
NDd
0.93
6.23
2.68
2.33
4.51
3.98
3.78
3.70
7.91
3.61
2.19
1.60c
6.40
3.47
NDd
0.93
6.23
2.68
2.33
4.51
3.99
3.78
3.71
3.75-3.70a
2.10
3.75-3.70a
2.11
i
a Reagents: (a) Pr2NEt, DMSO, 70 °C, (b) 5% AcOH, 57-64% from
7a
7b
8
11
12-15
16
1′
2′
2′′
3′
5a, (c) NaIO4, H2O, 80-87%.
1.74
1.74
6.42
6.42
3.82-3.75b
3.81-3.75b
NDd
NDd
0.92
6.23
2.67
2.33
4.51
3.98
3.78
3.72
0.93
6.24
2.69
2.33
4.51
3.98
3.78
3.73
Figure 4. HMBC correlation establishing the structures of 1-4.
4′
5′
5′′
of 12 proceeded in greater than 99% ee as determined by chiral
GC analysis.33 We could not detect a minor stereoisomer of
the corresponding (-)-Mosher’s ester of 13 by HPLC with UV
detection or by NMR. From a similar analysis of the corre-
sponding (-)-Mosher’s ester of 21, we estimate that the
Sharpless kinetic resolution of 20 proceeded in 92-94% ee.30b,32
Amino alcohols 8-11 were individually condensed with
2-fluoro-O6-(2- trimethylsilylethyl)-2′-deoxyinosine (5a) to give
the corresponding N2-deoxyguanosine derivatives 27-30 in 57-
64% yield after deprotection of the O6-(2-trimethylsilylethyl)
group (Scheme 4). In the case of the syn amino 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,N2-8-hydroxypro-
pano deoxyguanosine derivatives 1-4 in 80-87% yield.
The structures of HNE adducts 1-4 were firmly established
a The peak is overlapped with H-5′ and its chemical shift was estimated
from COSY. b The peak is overlapped with H-5′′ and its chemical shift
was estimated from COSY. c The peak is hidden under the bigger peaks of
protons from H12-H15, and its chemical shift was estimated from COSY.
d Not determined.
virtually superimposable. Likewise, the spectra of 3 and 4 are
essentially identical. This relationship is not unexpected because
each pair is enantiomeric with respect to the three stereogenic
sites in the base. The deoxyribosyl moiety is too distant from
the stereogenic centers of the adducted base to produce more
than miniscule perturbations in the chemical shifts.
We have examined the circular dichroism spectra of nucleo-
sides 1-4. In contrast to NMR, each diastereomer gives a unique
CD spectrum (Figure 6). Nevertheless, the 280 nm band arises
primarily from the nucleobase. Thus, 1 and 3, which have the
(6R,8S) configuration show negative ellipticity at 280 nm,
whereas 2 and 4, which have mirror image stereochemistry at
these sites, show positive ellipticity. A similar result had been
observed for the crotonaldehyde adducts which have only a
methyl group at C6 (Figure 7).17c,34 A negative ellipticity was
observed for crotonaldehyde adduct 31 having the (6S,8S)
configuration whereas diastereomer 32 gave a positive signal.
It should be noted that the designation of absolute configuration
at C6 for the crotonaldehyde adducts is opposite that for the
1
by H-13C long-range heteronuclear correlation NMR spectra
(HMBC). For each sample we observed correlation between
H8 and the C10 carbonyl carbon as well as C4a (Figure 4). For
4 these resonances were at 6.40 and 157.8 and 152.3 ppm
(Figure 5). No such correlations would have been observed in
the alternative hemi-acetal structure shown in Figure 4; thus,
this structure is ruled out for all four diastereomers.
The 1H NMR spectra of the four nucleosides 1-4 are listed
in Table 1. It should be noted that the spectra of 1 and 2 are
(33) Richardson, T. I.; Rychnovsky, S. D. Tetrahedron 1999, 55, 8977.
(34) Chung, F.-L.; Hecht, S. S. Cancer Res. 1983, 43, 1230.
9
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Adducts of trans-4-Hydroxynonenal
A R T I C L E S
Scheme 5
Figure 6. CD Spectra of 1-4. Sample concentration is 1 mg/mL in
methanol.
Figure 7. 1,N2-deoxyguanosine adducts of crotonaldehyde.
have included the preparation of oligonucleotides bearing 1,N2-
deoxyguanosine adducts of acrolein and crotonaldehyde.17
Stereo- and site-specific syntheses of 12-mer oligodeoxy-
nucleotides containing the four 1,N2-deoxyguanosine adducts
of HNE was accomplished according to Scheme 5. Oligonucleo-
tide 33 was prepared using standard solid-phase methods with
phenoxyacetyl-protected phosphoramidite reagents for the un-
modified bases. Deprotection of the oligonucleotide without
hydrolysis of the O6-(2-(trimethylsilyl)ethyl)-2-fluoroinosine
base was accomplished with 0.1 M NaOH at room temperature
as previously described.17b,c,36 Aminotriols 8-11 were individu-
ally reacted with oligonucleotide 33 in DMSO at 70 °C in the
presence of Hu¨nig’s base to give the corresponding site-
specifically adducted oligonucleotides 34-37 after deprotection
of O6 with dilute acetic acid. Confirmation that the adduction
reaction had proceeded as planned was provided by MALDI-
TOF mass spectrometric analysis of 34-37. In addition, the
oligonucleotides were subjected to enzymatic digestion followed
by HPLC analysis to confirm the presence of modified nucleo-
sides 27-30 (Figure 7). Periodate cleavage of the vicinal diol
unit provided the corresponding stereochemically defined HNE-
adducted oligonucleotides 38-41. Aminotriols 10 and 11 had
been prepared in 92-94% ee. However, HPLC purification of
the oligonucleotides both before and after periodate cleavage
HNE adducts because of a change of priority for the side-chain
(a methyl group in 31 and 32 versus a hydroxyhexyl group for
1-4). The trans relative stereochemistry between H6 and H8
is observed for both types of adducted nucleosides. For the six
example, we have examined thus far (1-4, 31-32), the sign
of the CD signal at 280 nm correlates with the absolute
configuration at C6 and C8.
Site-Specific Synthesis of Oligonucleotides Containing
Stereochemically Defined Adducts of HNE. A post-oligo-
merization strategy was the method of choice for the synthesis
of oligonucleotides containing stereochemically defined HNE
adducts. The alternative use of phosphoramidite derivatives of
HNE-adducted nucleosides 1-4 or of aminotriol-adducted
nucleosides 27-30 would require cumbersome strategies for
orthogonal protection of the side-chain functionality. The
phosphoramidite reagent of 5a has previously been incorporated
into oligonucleotides.35 Many examples have been reported of
oligonucleotides containing 5a undergoing substitution by amine
analogues of mutagens to give sequence specific mutagen-linked
oligonucleotides at N2 of deoxyguanosine.17,36 These syntheses
(35) Harris, C. M.; Harris, T. M. Curr. Prot. Nucleic Acid Chem. Boyle, A. L.,
Ed.; J. Wiley & Sons: New York: 1999; p 1.3.1-1.3.15.
(36) DeCorte, B. L.; Tsarouhtsis, D.; Kuchimanchi, S.; Cooper, M. D.; Horton,
P.; Harris, C. M.; Harris, T. M. Chem. Res. Toxicol. 1996, 9, 630.
9
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A R T I C L E S
Wang et al.
Figure 8. HPLC analysis of the enzymatic digestion of oligonucleotides
34 (top) and 38 (bottom).
Scheme 6
Figure 9. Cross-linking of (6S,8R,11S)-HNE adducted oligonucleotide 41
with a complementary 12-mer in 50 mM phosphate buffer, pH 7.0 containing
1 M KCl at 37 °C.
can undergo reversible dehydration to the corresponding imine
cross-link (44). The imine cross-link has not been detected
spectroscopically but its presence, at least in small quantities,
has been established by chemical probes.
Interchain cross-linking of oligonucleotides containing the
(6S,8S)- and (6R,8R)-crotonaldehyde adducts (31 and 32,
respectively, in Figure 7) has also been studied.37 Interestingly,
a strong stereochemical preference for the interstrand cross-
linking reaction was observed with the (6R,8R)-stereoisomer
(32) cross-linking efficiently (38%), whereas the (6S,8S)-
stereoisomer cross-linked only to the extent of about 5%.
Duplexes were formed by hybridization of 38-41 with the
complementary strand and their ability to form interstrand cross-
links examined. As with the crotonaldehyde adducts, we
observed a strong stereochemical preference in the cross-linking
reaction. Oligonucleotides 38-40 showed no evidence of cross-
linking with a complementary strand after incubation for 60
days in 50 mM, pH 7.0 phosphate buffer with 1M KCl.
Oligonucleotide 41 having the (6S,8R,11S) configuration,
however, formed a new species over time which proved to be
the interstrand cross-link. With the oligonucleotide containing
the (6R,8R)-crotonaldehyde adduct, the rate of cross-linking had
been significantly slower than for the acrolein adduct. The cross-
linking reaction with oligonucleotide 41 was slower yet. Figure
9 shows the electrophoretograms obtained when the reaction
was monitored for more than eight weeks by CZE.
gave purified oligonucleotides that were totally free of diaster-
eomeric contamination. Again, the structures of 38-41 were
confirmed by MALDI-TOF mass spectrometry and enzymatic
digestion, which established the presence of HNE-adducted
nucleosides 1-4 (Figure 8).
DNA-DNA Cross-Linking Studies of HNE-Adducted
Oligonucleotides. We have previously shown that 1,N2-deoxy-
guanosine adducts of acrolein are capable of forming interstrand
DNA-DNA cross-links in a CpG sequence. In DNA duplexes
the hydroxypropano adduct opens to give the free aldehyde (42,
Scheme 6). Nucleophilic addition by the exocylic amino group
gives a carbinolamine interstrand cross-link (43). Recent NMR
studies of a cross-linked DNA duplex have provided compelling
evidence for the carbinolamine cross-link.25 The carbinolamine
(37) Kozekov, I. D.; Nechev, L. V.; Moseley, M. S.; Harris, C. M.; Rizzo, C.
J.; Stone, M. P.; Harris, T. M. J. Am. Chem. Soc. 2003, 125, 50.
9
5692 J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003

Adducts of trans-4-Hydroxynonenal
A R T I C L E S
Figure 10. Enzyme digest of the cross-linking reaction of 41 with its
complement after 61 days at 37 °C.
The cross-linking reaction with 41, although slow, gave a
remarkably high yield. The duplex containing the acrolein-
adducted oligonucleotide had come to equilibrium within 7 days
and gave approximately 50% of the cross-link, whereas the
(6R,8R)-crotonaldehyde-adducted oligonucleotide had come to
equilibrium within 24 days and gave 38% of the cross-link. For
41, the cross-linking reaction did not appear to reach equilib-
rium, even after 61 days. When the reaction was stopped after
this time, we estimated that the yield of cross-link was 85%
based on integration of the UV absorbance in the electrophore-
togram.
Multiple lines of evidence had been obtained to establish that
acrolein could mediate interstrand cross-links in a CpG se-
quence. MALDI-TOF mass spectrometry of the cross-linked
oligonucleotide had supported the presence of carbinolamine
and imine species. Enzymatic digestion of the cross-linked
oligonucleotides with nucleases gave pyrimidopurinone bis-
nucleoside 45a. When the cross-linked oligonucleotide was
treated with sodium cyanoborohydride prior to enzymatic
digestion, the N2-N2 trimethylene-linked bis-nucleoside was
obtained. Authentic samples of the pyrimidopurinone and
trimethylene bis-nucleoside standards were prepared by inde-
pendent routes. The presence of the carbinolamine has been
confirmed by NMR spectroscopy. Similar lines of evidence have
established the interstrand cross-link in a duplex containing the
(6R,8R)-adduct of crotonaldehyde.
The presence of an interstrand cross-link in the duplex formed
from oligonucleotide 41 was established by enzymatic digestion
of the cross-linking reaction with snake venom phospho-
diesterase, DNase I, and alkaline phosphatase which gave
pyrimidopurinone bis-nucleoside 45c along with the four
unmodified nucleosides in the proper relative amounts for a
duplex of 41 with its complement (Figure 10). In addition, some
of nucleoside 4 was observed; it arose from un-cross-linked
oligonucleotide 41 and also by hydrolysis of the cross-link
during the course of the enzymatic digestion. The assignments
of nucleosides 4 and 45c in the digestion reaction were
confirmed by HPLC co-injection with authentic samples.
Nucleoside 45c was prepared according to Scheme 7 in low
yield. The pyrimidopurine structure 45c was established by
Figure 11. 1H NMR spectrum of 45c in DMSO-d6.
Table 2. Tm Studies of Adducted Oligonucleotide Duplexes
5′-d(G-G-A-C-T-C-G-C-T-A-G-C)-3′
3′-d(C-C-T-G-A-X-C-G-A-T-C-G)-5′
Unadducted (X ) dG)
Acroleina (46)
65° C
55 °C (55 + 91°C)d
Crotonaldehydeb
47: X ) 31 (6S,8S)
48: X ) 32 (6R,8R)
HNEc
51°C
51 °C (51 + 88 °C)e
38: X ) 1 (6R,8S,11S)
39: X ) 2 (6S,8R,11R)
40: X ) 3 (6R,8S,11R)
41: X ) 4 (6S,8R,11S)
60 °C
60 °C
53 °C
49 °C (49 + >90 °C)f
Temperature was raised 1 °C per minute. Conditions: 1M NaCl, 10 mM
pH 7 Phosphate, 0.05 mM EDTA, 0.5 AU/mL of each Oligonucleotide).
a Ref. b Ref. c This work. d After 7 days at 37 °C. e After 24 days at 37 °C.
f After 61 days at 37 °C.
observation of the 1H-1H coupling of the hydroxyl protons in
the NMR spectrum taken in DMSO. The proton assignments
were made by a COSY spectrum. The C11 hydroxyl proton
was observed as a doublet, which is consistent with structure
45c (Figure 11). The structure was further confirmed by HMBC.
Melting Studies. Modified oligonucleotides 38-41 were
hybridized with the complementary 12-mer and the influence
of the HNE adducts on the thermal stability of the duplexes
was assessed via melting studies monitored by the increase in
molar absorptivity at 260 nm which occurs when a DNA duplex
dissociates. The results of this study are summarized in Table
2. A melting experiment carried out with the unadducted 12-
mer duplex gave a Tm value of 65 °C (Figure 12, Panel A). For
the modified oligonucleotides, the Tm values were measured
immediately after annealing with the complementary strands.
The samples were then incubated at 37 °C to allow cross-linking
to occur.
The initial Tm values for the duplexes formed from HNE-
adducted oligonucleotides 38-41 varied with the stereochem-
istry of the adduct. In general, DNA adducts destabilize duplex
structure and lower the Tm of the duplex. Oligonucleotides 38
and 39 containing the (6R,8S,11S) and (6S,8R,11R) stereochem-
istry in the HNE adduct, respectively, had Tm values of 60 °C,
only 5° lower than the unmodified duplex. Oligonucleotides 40
and 41 were significantly more destabilized and had Tm values
of 53 and 49 °C, respectively. With all four adducted duplexes,
there was only a small hysteresis effect between the melting
and annealing processes, comparable to that observed with the
unadducted duplex.
Scheme 7
9
J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003 5693

A R T I C L E S
Wang et al.
27-30 are significantly slower than the corresponding reactions
of the aminodiols used to produce the acrolein and crotonal-
dehyde adducts. The basis for this reduced reactivity may, in
part, be steric because of the greater bulk of the R-hydroxyhexyl
moiety attached to the aminodiol. However, electronic factors
stemming from the vicinal amino alcohol structure are believed
to play a greater role. The strong dipole of the hydroxyl group
reduces electron density on the amine, thus decreasing its
nucleophilicity. Furthermore, the hydroxyl group is able to
hydrogen bond with the amine, further decreasing its reactivity
with electrophiles. Attenuated reactivity has also been observed
in similar reactions of aminotriols derived from PAH diol
epoxides, even in cases where steric factors do not play a major
role.38 Despite this problem, the reactivity of aminotriols 27-
30 was sufficient for the syntheses of adducted nucleosides 1-4
and oligonucleotides containing them to be achieved in a
reasonably efficient fashion.
Figure 12. Melting curves for (A) the unadducted 12-mer duplex, (B) the
duplex containing the (6S,8R,11S) adduct of HNE (41) immediately after
duplex preparation, (C) the same sample after incubation for 61 days at 37
°C, (D) a second melting-annealing cycle carried out immediately after the
measurements shown in Panel C.
The HPLC analysis of 1,N2-deoxyguanosine adducts of HNE,
as well as the corresponding 5′-phosphates, 3′-phosphates, and
3′,5′-bis-phosphates, has been reported in connection with the
development of methods to detect these adducts in animal tissue
using 32P-postlabeling or electrochemical detection.39 In all of
these reports, the elution profile of the four stereoisomers shows
that the first two diastereomers that eluted from reverse phase
columns are only partially resolved, whereas the subsequent pair
are cleanly resolved from the first pair and from each other.
Importantly, Fu showed that the four diastereomers of the
nucleosides eluted in the same relative order as the correspond-
ing phosphate derivatives.39b It was previously demonstrated
that acid-catalyzed deglycosylation of the partially resolved pair
of nucleoside adducts gave a single, presumably racemic,
guanine derivative.39b Deglycosylation of the second pair also
gave a single guanine derivative, which had a diastereomeric
relationship to that derived from the partially resolved pair of
HNE adducts.39a This result indicates that the partially resolved
pair of adducts have the same relative stereochemistry, but
opposite absolute configurations at C6, C8, and C11. Likewise,
the second pair have the same relative stereochemistry, but
opposite configurations, at C6, C8, and C11. The two pairs are
diastereomeric at C11 relative to C8 and C6.
Using our nucleosides 1-4, we are now able to assign the
absolute configurations of the HNE adducts. Using the published
HPLC conditions, we have reproduced the chromatogram with
mixtures of the synthetic HNE-adducted nucleosides (Figure
13). In the order of elution, the unresolved pair of diastereomers
are assigned as diastereomers 1 (6R,8S,11S) and 2 (6S,8R,11R)
and the resolved pair as 3 (6R,8S,11R) and 4 (6S,8R,11S). (Our
numbering of 1-4 in this paper has been chosen so that the
numbers would correspond to the order of HPLC elution.) A
comparison of the NMR data (Table 1) for nucleosides
characterized by Fu with those of the synthetic samples prepared
in the present investigation, particularly protons 7a and 7b, fully
supports these assignments.28,39b
Figure 13. HPLC chromatogram of 1-4.
The oligonucleotides were then incubated at 37 °C for
extended periods of time to allow an opportunity for interstrand
cross-linking to occur. There was little or no change in the
melting curves for the duplexes formed from oligonucleotides
38-40, which had also shown no evidence of cross-link
formation by the CZE assay. However, for the duplex containing
adducted oligonucleotide 41 with the (6S,8R,11S)-adduct con-
figuration, a second melting transition was observed. Panel C
of Figure 12 shows a biphasic melting curve observed after the
sample had incubated for 61 days. The higher transition occurred
at >90 °C. It should be noted that the cooling curve is
substantially displaced from the heating curve although clearly
not identical with the curve in Panel B. Immediately after the
melting-annealing cycle had been completed the cycle was
repeated. Two melting transitions can still be seen (Panel D),
although the high melting transition is less pronounced than in
Panel C.
Discussion
Syntheses of the HNE-adducted nucleosides 1-4 and oligo-
nucleotides containing them have been accomplished in a
structurally specific manner by taking advantage of the displace-
ment of fluoride from 2-fluorohypoxanthine derivatives by
aminotriols. The present syntheses are modeled on previous
work from our laboratory for the syntheses of acrolein- and
crotonaldehyde-adducted nucleosides and oligonucleotides.17b,c,37
The present reactions differ in one key aspect from the earlier
reports, namely, the condensation reactions using aminotriols
The reaction of racemic HNE with calf thymus DNA followed
by enzymatic digestion and detection by 32P-postlabeling has
(38) McNees, A. G.; O’Donnell, M.; Horton, P. H.; Kim, H.-Y.; Kim, S. J.;
Harris, C. M.; Harris, T. M.; Lloyd, R. S. J. Biol. Chem. 1997, 272, 33 211.
(39) (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.
9
5694 J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003

Adducts of trans-4-Hydroxynonenal
A R T I C L E S
Scheme 8
been reported to give only two of the four possible stereoisomers
of the deoxyguanosine adducts.39b The major adduct can now
be assigned as 4 possessing the 6S,8R,11S stereochemistry and
the minor adduct is 3, possessing the 6R,8S,11R stereochemistry.
However, it should be noted that all four adducts have been
detected in animal tissue using the 32P-postlabeling method.39c
The Tm measurements summarized in Table 2 provide some
insight into the structure of DNA duplexes containing the enal
adducts. Previous studies have confirmed that the acrolein
adducted oligonucleotide (46) is present entirely in the ring-
opened N2-(3-oxopropyl) form (42, Scheme 6) in the DNA
duplexes rather than in the pyrimidopurinone form.22-25 The
ring opening allows for the stabilizing effects of Watson-Crick
hybridization to be maintained. We believe that the (6R,8R)-
crotonaldehyde (32) and (6S,8R,11S)-HNE (41) adducts also
exist, at least in part, in the ring-opened form because they are
able to form cross-links and ring-opening is a prerequisite for
the cross-linking reaction. Adducted oligonucleotides 47 and
38-40 form interstrand cross-links inefficiently or not at all
and may exist predominantly in the ring-closed pyrimidopu-
rinone form. The adducted duplexes are all destabilized relative
to the unmodified duplex.
hypothesis, the resulting cross-link has much higher thermal
stability than those derived from the acrolein and crotonaldehyde
adducts. A possible explanation for the high yield and thermal
stability of the HNE interstrand cross-link, as well as the
dependence on the C11 stereochemistry for the HNE adducts
is that the side-chain hydroxyl group of 41 can trap the imine
form of the cross-link to form tetrahydrofuran 49 (Scheme 8),
which we believe would be more stable than either the
carbinolamine or imine cross-links. The disposition of the pentyl
group in cross-link 49 would be dependent upon the C11
stereochemistry of 44c and one could easily imagine that the
configuration of the carbon bearing the hydroxyl group could
greatly influence the stability of tetrahydrofuran 49.
An alternative explanation for the stereochemical dependence
for the cross-linking reaction is that only the (6R,8R)-crotonal-
dehyde and (6S,8R,11S)-HNE adducts open to the N2-3-
oxopropyl structure (42, Scheme 6) when hybridized to a
complementary strand of DNA and the other diastereomers
remain in the ring-closed pyrimidopurinone form. It is likely
that the modified base would exist in the syn conformation about
the glycosidic bond for the ring closed structure as was shown
for oligonucleotides containing 1,N2-propano-2′-deoxyguanosine
(PdG).40 UV melting studies showed that a PdG-containing 13-
mer was destabilized by 14 °C when hybridized to a comple-
mentary strand under low salt (1 mM NaCl) conditions relative
to an unadducted oligonucleotide. This Tm difference is the same
as that observed for the crotonaldehyde adducted oligonucle-
otides. The thermal destabilization of the HNE-adducted oli-
gonucleotides due to a possible syn conformation of the
nucleobase could be partially offset by formation of a hydrogen
bond by the side chain hydroxyl group. We plan to establish
the structure and conformation of both the cross-linked and un-
cross-linked crotonaldehyde and HNE-adducts by NMR spec-
troscopy.
The reported Tm of the acrolein-modified oligonucleotide was
depressed by 10 °C, whereas the slightly bulkier crotonaldehyde
adducts caused a 14 °C depression. Duplexes containing HNE-
adducted oligonucleotides 40 and 41 have Tm values comparable
to those for crotonaldehyde whereas those containing 38 and
39 are less destabilizing than the acrolein adduct. We hypoth-
esize that HNE adducts, in particular those in oligonucleotides
38 and 39 are able to adopt conformations in which the side-
chain hydroxyl groups can hydrogen bond with basic sites in
the duplex, thus offsetting the inherent destabilization by the
hydrophobic hexyl chain.
For the acrolein-, (6R,8R)-crotonaldehyde-, and (6S,8R,11S)-
HNE-adducted oligonucleotides, an additional, higher melting
transition had been observed at approximately 90 °C after the
duplexes have been allowed to incubate for an extended period
of time to permit cross-linking (Table 2). The higher melting
transition is assigned to the interstrand cross-link. When the
cross-link was present, a large hysteresis effect was seen during
the cooling phase of the experiment, indicating that the cross-
link was undergoing cleavage after the second melting transition
took place. In the melting studies, a second melting-annealing
cycle was routinely carried out immediately after completion
of the first one. For the oligonucleotides containing the acrolein
and (6R,8R)-crotonaldehyde adducts, the higher melting species
was no longer observed in the second melting cycle. However,
the cross-link derived from the (6S,8R,11S)-HNE adduct was
much more robust; only partial loss of the cross-linked species
occurred during the first melting cycle.
Summary
We have developed stereospecific syntheses of nucleosides
and oligonucleotides containing 1,N2-deoxyguanosine adducts
of HNE. We have also demonstrated that one of these adducts,
i.e., that possessing the (6S,8R,11S) stereochemistry, is capable
of efficiently forming a very stable interstrand cross-link in a
CpG context. The cross-linking adduct shares a common C6
stereochemistry with the crotonaldehyde adduct that also forms
interstrand cross-links.
Oligonucleotides containing the (6R,8R)-crotonaldehyde ad-
duct (48) and the (6S,8R,11S)-HNE adduct (41) have the same
relative stereochemistry at C6 and cross-link effectively.
Interestingly, the oligonucleotide containing the (6S,8R,11R)
adduct of HNE (40) failed to cross-link even though it has the
same relative stereochemistry at C6. In addition, the (6S,8R,11S)
adduct cross-linked to a greater extent than the (6R,8R) adduct
of crotonaldehyde or even the acrolein adduct. This suggests
that the C11 side-chain hydroxyl group in the (6S,8R,11S) adduct
of HNE stabilizes the cross-linked product. In support of this
DNA-DNA cross-links represent a severe form of DNA
damage that will interfere with DNA replication. We have now
shown that simple enals that arise endogenously from lipid
peroxidation are able to form interstrand DNA-DNA cross-
links. In these in vitro experiments, the rate of cross-link was
on the order of days to weeks. However, this rate could be
(40) (a) Weisenseel, J. P.; Reddy, R.; Marnett, L. J.; Stone, M. P. Chem. Res.
Toxicol. 2002, 15, 127. (b) Singh, U. S.; Moe, J. G.; Reddy, G. R.;
Weisenseel, J. P.; Marnett, L. J.; Stone, M. P. Chem. Res. Toxicol. 1993,
6, 825.
9
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A R T I C L E S
Wang et al.
(2S,3S)-3-Pentyloxirane-2-methanol (ent-13). The Sharpless epox-
idation of 12 was performed in the same manner as described above
dramatically influenced by the local structure of DNA such as
those imposed by supercoiling. In addition, the persistence of
the DNA lesion is an important issue. In preliminary structural
work, the ring-opened form of the acrolein adduct did not
perturb the duplex structure. The synthesis of authentic standards
of the cross-linked nucleosides will greatly aid in the identifica-
tion of these cross-links in the DNA of exposed cells and tissue
samples. Work along these lines is currently in progress.
for 13 using diethyl L-(+)-tartrate to give ent-13 in 82% yield. [R]20
D
-40.4° (c 1.08, CHCl3).
(4S,5R)-3-Benzyl-4-hydroxymethyl-5-pentyl-2-oxazolidinone (15)
and 3-Benzyl-4-(1-hydroxyhexyl)-2-oxazolidinone (14). A suspension
of 60% NaH (390 mg, 1.51 mmol) in dry THF (12 mL) was cooled to
0 °C, and a solution of 13 (360 mg, 2.50 mmol) in dry THF (2 mL)
was added dropwise over 5 min. The reaction was stirred for an
additional 10 min, then freshly distilled benzyl isocyanate (460 µL,
53.8 mmol) was added dropwise over 5 min. The reaction was heated
at reflux for 2 h. The mixture was cooled to 0 °C and 60% NaH (85
mg, 2.13 mmol) was added. The reaction was heated at reflux for an
additional 1 h after which time the solution was cooled to 0 °C and
quenched with saturated NH4Cl (10 mL). The layers were separated
and the aqueous phase was extracted with methylene chloride (3 × 10
mL). The combined organic extracts were washed with brine, dried
over Na2SO4, filtered and concentrated. Purification by flash chroma-
tography on silica, eluting with 5-20% ethyl acetate in hexane gave a
mixture of 14 and 15. The mixture was separated by flash chromatog-
raphy on silica, eluting with 5-50% methylene chloride in 2:1 ether
in hexane to give 15 (258 mg, 37%). The remaining 14 was recycled
by the treatment with NaH in THF and the resulting mixture purified
as above to afford additional 15 (91 mg, 13%). 14: 1H NMR (CDCl3)
δ 7.60-7.26 (m, 5H), 4.69 (d, J ) 15.2 Hz, 1H), 4.34 (dd,
J ) 8.5, 6.9 Hz, 1H), 4.30 (d, J ) 15.2 Hz, 1H), 4.20 (t, J ) 8.8 Hz,
1H), 3.81-3.78 (m, 1H), 3.60 (m, 1H), 2.21 (br s, 1H), 1.45-1.05 (m,
8H), 0.87 (t, J ) 6.8 Hz, 3H); 13C NMR δ 159.2, 136.2, 129.1, 128.2,
Experimental Section
All commercially obtained chemicals were used as received.
Methylene chloride was freshly distilled from calcium hydride.
Anhydrous THF was freshly distilled from a sodium/benzophenone
ketyl. Anhydrous tert-butyl hydroperoxide in isooctane was prepared
as previously described.30b All reactions were performed under an argon
atmosphere. Glassware was oven-dried and cooled under argon. Proton
NMR data were recorded at 300 and 400 MHz; carbon-13 data was
recorded at 75 and 100 MHz. High-resolution FAB mass spectra were
obtained from the University of Notre Dame Mass Spectrometry Center
using nitrobenzyl alcohol (NBA) as the matrix. MALDI-TOF mass
spectra (negative ion) of modified oligonucleotides were obtained on
a Voyager Elite DE instrument (Perseptive Biosystems) at the Vander-
bilt Mass Spectrometry Facility using a 3-hydroxypicolinic acid (HPA)
matrix containing ammonium hydrogen citrate (7 mg/mL) to suppress
sodium and potassium adducts. The cross-linked oligonucleotides were
unstable in the MALDI matrix, rapidly reverting to the individual
strands. Flash column chromatography was performed using silica gel
(70-230 mesh). Nucleosides were purified by C-18 reverse phase
HPLC in water/acetonitrile with a diode array detector monitoring at
260 nm. Gradient A and B were used for HPLC purification and
analysis: gradient A, 99% water initially, 15 min linear gradient to
90% water, 5 min linear gradient to 80% water, and 10 min linear
gradient to 0% water followed by 5 min linear gradient to initial
conditions; and gradient B, 85% water initially, 40 min linear gradient
to 70% water, 5 min linear gradient to 0% water, followed by 5 min
linear gradient to initial conditions. Electrophoretic analyses were carried
out using a Beckman P/ACE Instrument System 5500 Series monitored
at 260 nm on a 27 cm × 100 µm column packed with the manufac-
turer’s 100-R gel (for ss DNA) using a Tris-borate buffer system
containing 7 M urea.
128.0, 67.5, 62.0, 59.2, 46.6, 31.7, 31.5, 25.5, 22.4, 13.9. 15: [R]20
D
+13.1° (c 0.61, CHCl3); 1H NMR (CDCl3) δ 7.39-7.26 (m, 5H), 4.79
(d, J ) 15.2 Hz, 1H), 4.47 (ddd, J ) 9.9, 7.8, 3.6 Hz, 1H), 2.43 (br s,
1H), 4.25 (d, J ) 15.2 Hz, 1H), 3.80-3.67 (m, 2H), 3.55 (ddd, J )
7.8, 3.8, 3.2 Hz, 1H), 1.93-1.83 (m, 1H), 1.69-1.56 (m, 2H),1.42-
1.26 (m, 5H), 0.88 (t, J ) 6.5 Hz, 3H); 13C NMR δ 158.8, 136.5, 128.8,
128.0, 127.8, 77.5, 58.6, 58.3, 46.5, 31.4, 29.0, 25.8, 22.4, 13.9; HRMS
(FAB, NBA) m/z calcd for C16H24NO3 (M+H) 278.1756, found
278.1757. Anal. Calcd for C16H23NO3: C, 69.29; H, 8.36; N, 5.05.
Found: C, 69.18; H, 8.47; N, 4.91.
(4R,5S)-3-Benzyl-4-hydroxymethyl-5-pentyl-2-oxazolidinone (ent-
15). Following the procedure described above for 15, ent-13 was
converted to ent-15 in 49% yield: [R]20 -13.3° (c 0.55, CHCl3).
D
(2R,3R)-3-Pentyloxirane-2-methanol (13).30,33 To a 200 mL flame-
dried flask were added activated, powdered 4 Å molecular sieves (0.70
g) and anhydrous methylene chloride (80 mL). A solution of diethyl
D-(-)-tartrate (0.69 g, 3.37 mmol) in methylene chloride (5 mL) was
stirred over activated 4 Å molecular sieves (pellets) for 15 min, then
transferred to the reaction flask via cannula. The reaction mixture was
cooled to -25 °C then titanium tetraisopropoxide (5.42 mL, 18.2 mmol)
and anhydrous tert-butyl hydroperoxide (6.0 M in isooctane, 6.30 mL,
37.86 mmol) were successively added dropwise and the mixture stirred
at -25 °C for 40 min. A solution of E-2-octenol (12, 1.80 g, 14.1
mmol) in methylene chloride (10 mL) was stirred over activated 4 Å
molecular sieves (pellets) for 15 min, then transferred to the reaction
flask dropwise via cannula. The mixture was stirred at -25 °C over 4
h, then quenched by the addition of a solution of ferrous sulfate (15 g)
in 10% aqueous tartaric acid (50 mL) to the cooled reaction mixture.
The mixture was stirred for 1 h and the organic phase was separated,
washed with water, dried over Na2SO4, filtered and concentrated. The
residue was diluted with ether (100 mL) and stirred with 1 N NaOH
(50 mL) at 0 °C for 40 min. The ether solution was separated, washed
with brine, dried over MgSO4, filtered and evaporated. Purification by
(4S,5R)-3-Benzyl-4-iodomethyl-5-pentyl-2-oxazolidinone (16). Tri-
phenylphosphine (706 mg, 2.68 mmol) and imidazole (183 mg, 2.68
mmol) were dissolved in ether (4 mL) and acetonitrile (1 mL). The
mixture was cooled to 0 °C, and iodine (680 mg, 2.68 mmol) was added
with vigorous stirring. The resulting slurry was warmed to 20 °C, then
cooled to 0 °C again, and a solution of 15 (248 mg, 0.89 mmol) in
ether/acetonitrile/benzene (3 mL, 1:1:1) was added dropwise over 5
min. The mixture was warmed to 20 °C and stirred for 1 h, then cooled
to 0 °C and quenched with 5% sodium bicarbonate (10 mL). The layers
were separated and the aqueous phase was extracted with ether (3 ×
10 mL). The combined organic extracts were dried over MgSO4, filtered
and concentrated. Purification by flash chromatography on silica, eluting
with 5-7% ethyl acetate in hexanes afforded 16 (291 mg, 84.0%):
1
[R]20 + 40.6° (c 1.30, CHCl3); H NMR (CDCl3) δ 7.41-7.26 (m,
D
5H), 4.85 (d, J ) 15.4 Hz, 1H), 4.50 (ddd, J ) 10.4, 7.5, 3.3 Hz, 1H),
4.13 (d, J ) 15.4 Hz, 1H), 3.71 (ddd, J ) 7.5, 7.2, 3.1 Hz, 1H), 3.21-
3.08 (m, 2H), 1.71-1.92 (m, 2H), 1.60-1.63 (m, 1H), 1.26-1.42 (m,
5H), 0.90 (t, J ) 6.6 Hz, 3H); 13C NMR δ 157.8, 135.7, 129.0, 128.1,
128.0, 77.5, 56.9, 46.4, 31.4, 27.8, 25.6, 22.4, 13.9, -0.9.
(4R,5S)-3-Benzyl-4-iodomethyl-5-pentyl-2-oxazolidinone (ent-16).
Following the procedure described above for 16, ent-15 was converted
flash chromatography on silica, eluting with 10% ethyl acetate in
to ent-16 in 83% yield. [R]20 - 40.6 ° (c 0.725, CHCl3).
1
hexanes gave 13 (1.73 g, 85.0%): [R]20 +40.0° (c 0.56, CHCl3); H
D
D
NMR (CDCl3) δ 3.94-3.88 (m, 1H), 3.65-3.60 (m, 1H), 2.97-2.90
(m, 2H), 1.70 (br s, 1H),1.61-1.24 (m, 8H), 0.90 (t, J ) 6.7 Hz, 3H);
13C NMR δ61.7, 58.5, 56.0, 31.5, 31.4, 25.6, 22.6,13.9.
(4S,5R)-4-Allyl-3-benzyl-5-pentyl-2-oxazolidinone (17). To a solu-
tion of 16 (294 mg, 0.759 mmol), cuprous iodide (15 mg, 0.08 mmol),
and HMPA (0.56 mL, 3.04 mmol) in THF (3 mL) was added
9
5696 J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003

Adducts of trans-4-Hydroxynonenal
A R T I C L E S
vinylmagnesium bromide (1.52 mL, 1.0 M solution in THF) dropwise
at -23 °C over 5 min. The resulting mixture was stirred for 40 min at
-23 °C, then quenched with saturated NH4OH (10 mL). The layers
were separated and the aqueous phase was extracted with ether (3 ×
10 mL). The combined organic extracts were dried with MgSO4,
filtered, and concentrated. Purification by flash chromatography on
silica, eluting with 5-7% ethyl acetate in hexanes afforded 17 (146
mg, 67%). [R]20D +23.2 (c 1.34 in CHCl3); 1H NMR (CDCl3) δ 7.37-
7.26 (m, 5H), 5.74-5.67 (m, 1H), 5.17-5.12 (m, 2H), 4.85 (d, J )
15.3 Hz, 1H), 4.44-4.40 (m, 1H), 4.08 (d, J ) 15.3 Hz, 1H), 3.63-
3.58 (m, 1H), 2.38-2.34 (m, 2H), 1.80-1.72 (m, 1H), 1.67-1.30 (m,
7H), 0.89 (t, J ) 6.1 Hz, 3H); 13C NMR δ 158.2, 136.2, 133.0, 128.8,
127.9, 127.8, 118.9, 78.1, 56.5, 46.1, 31.9, 31.4, 29.1, 25.6, 22.4, 13.9;
HRMS (FAB, NBA) m/z calcd for C18H26NO2 (M+H) 288.1964, found
288.1964. Anal. Calcd for C18H25NO2: C, 75.22; H, 8.77; N, 4.87.
Found: C, 74.97; H, 8.62; N, 4.87.
34.8 and 34.6, 33.0 and 32.9, 31.5, 27.2 and 27.1, 23.7, 14.4; HRMS
(FAB, NBA) m/z calcd for C17H30NO3 (M+H) 296.2226, found
296.2221.
(4R,5S)-4-(Benzylamino)-1,2,5-decanetriol (ent-19). Following the
procedure described above for 19, ent-18 was converted to ent-19 in
69% yield.
(4R,5S)-4-Amino-1,2,5-decanetriol (8). Following the procedure
described below for 9, ent-19 was converted to 8 as an inseparable
mixture of diastereomers in 98% yield. The spectra properties were
identical to 9.
(4S,5R)-4-Amino-1,2,5-decanetriol (9). A suspension of 20% Pd-
(OH)2 on carbon (5 mg) in methanol (1 mL) was stirred at room
temperature under a hydrogen atmosphere for 30 min. A solution of
19 (18 mg, 0.061 mmol) in methanol (1 mL) was added via cannula,
and the reaction mixture was stirred under a hydrogen atmosphere for
5 h. The catalyst was removed by filtration, and the filtrate was
concentrated to give 9 (12.5 mg, 100%) as an inseparable mixture of
diastereomers: 1H NMR (CD3OD) 3.82-3.73 (m, 1H), 3.62-3.40 (m,
3H), 2.98-2.88 (m, 1H), 1.88-1.16 (m, 10 H), 0.93-0.89 (m, 3H);
13C NMR (assignment of diastereomeric pair based on relative intensity
and DEPT) δ 75.9 and 75.6, 73.0 and 70.4, 67.8 and 67.5, 56.4 and
53.5, 35.7 and 34.6, 33.6, 33.0, 26.9, 23.7, 14.4; HRMS (FAB, NBA)
m/z calcd for C10H24NO3 (M+H) 206.1756, found 206.1759.
(2R,3S)-1,2-Epoxy-3-octanol (21).30b,32 Dry methylene chloride (120
mL) was added to a 500 mL flame-dried flask under argon. A solution
of (()-octen-3-ol (20, 2.37 g, 18.2 mmol) and diisopropyl L-(+)-tartrate
(5.13 g, 21.8 mmol) in 10 mL of CH2Cl2 was stirred with 4 Å molecular
sieves (pellets) for 15 min, then transferred to the reaction flask via
cannula, and the mixture was cooled to -20 °C. Titanium tetraisopro-
poxide (5.42 mL, 18.2 mmol) was added dropwise, and the mixture
was stirred for 15 min at -20 °C. Anhydrous tert-butyl hydroperoxide
(6.0 M in isooctane, 6.30 mL, 37.86 mmol) was added dropwise. The
mixture was stirred at -20 °C for 5 h, after which time GC-MS
analysis showed 45% conversion. To the stirred and cooled solution
was added a solution of ferrous sulfate (15 g) in 10% aqueous tartartic
acid (50 mL). The mixture was stirred for 1 h and then the organic
phase was separated, washed with water, dried over Na2SO4, filtered,
and evaporated. The residue was diluted with ether (100 mL) and stirred
with 1N NaOH (50 mL) at 0 °C for 40 min. The ether phase was
separated, washed with brine, dried over MgSO4, filtered and evapo-
rated. Purification by flash chromatography on silica, eluting with 8%
ethyl acetate in hexanes gave 21 (1.06 g, 40%): [R]20D +21.4° (c 1.17,
CHCl3); 1H NMR (CDCl3) δ 3.81-3.80 (m, 1H), 3.02-2.99 (m, 1H),
2.81 (dd, J ) 5.0, 2.9 Hz, 1H), 2.73 (dd, J ) 5.0, 4.1 Hz, 1H), 2.10 (br
s, 1H), 1.31-1.55 (m, 8H), 0.90 (t, J ) 6.7 Hz, 3H); 13C NMR δ 68.4,
54.5, 43.4, 33.4, 31.8, 24.9, 22.5, 13.9.
(4R,5S)-4-Allyl-3-benzyl-5-pentyl-2-oxazolidinone (ent-17). Fol-
lowing the procedure described above for 17, ent-16 was converted to
ent-17 in 70% yield. [R]20 -23.8° (c 1.40, CHCl3).
D
(4S,5R)-3-Benzyl-4-(2′,3′-dihydroxypropyl)-5-pentyl-2-oxazoli-
dinone (18). To a stirred solution of 17 (66 mg, 0.23 mmol),
N-methylmorpholine N-oxide (31 mg, 0.253 mmol), THF (0.8 mL),
t-BuOH (0.32 mL), and water (0.16 mL) was added osmium tetroxide
(14 µL, 0.1 M in benzene, 1.4 µmol). The mixture was stirred for 12
h, then a second portion of osmium tetroxide (9 µL, 0.1 M in benzene
0.9 µmol) was added and stirring continued for 8 h. The reaction was
quenched with 5% aqueous NaHSO3 (5 mL) with vigorous stirring for
15 min then poured into water (5 mL), transferred to a separatory funnel
and extracted with methylene chloride (10 × 3 mL). The combined
organic extracts were dried over Na2SO4, filtered and concentrated.
Purification by flash chromatography on silica, eluting with 1-2%
methanol in chloroform gave 18 (53 mg, 72%) as an inseparable mixture
of diastereomers: 1H NMR (CDCl3, assignment of diastereomeric pairs
based on coupling pattern and COSY) δ 7.35-7.22 (m, 5H), 4.79 and
4.67 (diastereomers, d, d, J ) 15.4 and 15.4 Hz, 1H), 4.50-4.40 (m,
1H), 4.21 and 4.13 (diastereomers, d, d, J ) 15.4 and 15.4 Hz, 1H),
3.87 and 3.76 (diastereomers, m, m, 1H), 3.75 and 3.64 (diastereomers,
m, m, 1H), 3.59-3.53 (m, 1H), 3.38-3.32 (m, 1H), 2.99 (d, J ) 21.3
Hz, 1H), 2.89 (br s, 1H), 1.74-1.46 (m, 5H), 1.37-1.29 (m, 5H), 0.90-
0.86 (m, 3H); 13C NMR (CDCl3, assignment of diastereomeric pairs
based on relative intensity and DEPT) δ 159.9 and 158.6, 136.5 and
136.2, 128.8 and 128.7, 127.9, 127.8 and 127.8, 79.0 and 78.2, 69.1
and 68.7, 67.8 and 66.7, 55.2 and 54.7, 46.8 and 45.9, 31.5, 30.6 and
30.1, 29.4 and 29.4, 25.5, 22.5 and 22.4, 13.9; HRMS (FAB, NBA)
m/z calcd for C18H28NO4 (M+H) 322.2018, found 322.2010.
(4R,5S)-3-Benzyl-4-(2′,3′-dihydroxypropyl)-5-pentyl-2-oxazolidi-
none (ent-18). Following the procedure described above for 18, ent-
17 was converted to ent-18 in 75% yield.
(2S,3R)-1,2-Epoxy-3-octanol (ent-21). The Sharpless kinetic resolu-
tion of 20 was performed in the same manner as described above for
21 using diisopropyl D-(-)-tartrate to give ent-21 in 41% yield: [R]20
D
(4S,5R)-4-(Benzylamino)-1,2,5-decanetriol (19). A solution of 18
(32 mg, 0.108 mmol) and 50% aqueous NaOH (0.25 g) in absolute
ethanol (1 mL) was heated at reflux overnight. The reaction was cooled
to room temperature and poured into water (5 mL). The reaction was
transferred to a separatory funnel and extracted with ethyl acetate
(3 × 10 mL). The combined organic extracts were dried over Na2SO4,
filtered and concentrated. Purification by flash chromatography, eluting
with 4-6% methanol in hexanes afforded 19 (20 mg, 68%) as an
inseparable mixture of diastereomers: 1H NMR (CD3OD, assignment
of diastereomeric pairs based on coupling pattern and COSY) δ 7.35-
7.26 (m, 5H), 3.98 and 3.90 (diastereomers, d, d, J ) 12.8 and 12.4
Hz, 1H), 3.89-3.76 (m, 2H), 3.77 and 3.74 (diastereomers, d, d, J )
12.7 and 12.7, 1H), 3.47-3.37 (m, 2H), 2.79-2.74 (m, 1H), 1.72-
1.31 (m, 10 H), 0.93-0.89 (m, 3H); 13C NMR (assignment of
diastereomeric pairs based on relative intensity and DEPT) δ 141.0
and 140.8, 129.6 and 129.5, 129.5 and 129.4, 128.3 and 128.2, 73.6
and 71.9, 71.5 and 71.1, 67.5 and 67.4, 62.1 and 59.4, 52.1 and 51.6,
-20.2° (c 1.29, CHCl3).
(4S,5S)-3-Benzyl-4-hydroxymethyl-5-pentyl-2-oxazolidinone (22).
A solution of 21 (450 mg, 3.12 mmol) in dry THF (10 mL) was treated
with 60% NaH (269 mg, 6.72 mmol) under argon and stirred for 10
min at room temperature. Freshly distilled benzyl isocyanate (0.50 mL,
4.06 mmol) was added dropwise over 10 min, and the resulting mixture
heated at reflux for 1.5 h. The mixture was then cooled to 0 °C and
quenched with saturated NH4Cl (10 mL) and the layers separated. The
aqueous layer was extracted with methylene chloride (3 × 10 mL).
The combined organic layers were washed with brine, dried over Na2-
SO4, filtered, and concentrated. Purification by flash chromatography
on silica, eluting with 20% ethyl acetate in hexane gave a partially
purified product. Further purification was accomplished by flash
chromatography on silica, eluting with 1-18% ethyl acetate in hexane
to afford 22 (0.502 g, 58%): [R]20 -72.0 (c 1.02, CHCl3); H NMR
1
D
(CDCl3) δ 7.36-7.26 (m, 5H), 4.72 (d, J ) 15.3 Hz, 1H), 4.39 (ddd,
J ) 7.0, 5.6, 5.6 Hz, 1H), 4.26 (d, J ) 15.3 Hz, 1H), 3.73-3.69 (m,
9
J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003 5697

A R T I C L E S
Wang et al.
1H), 3.54-3.48 (m, 1H), 3.25 (ddd, J ) 5.6, 3.3, 3.3 Hz, 1H), 2.67 (br
s, 1H), 1.67-1.46 (m, 2H), 1.44-1.19 (m, 6H), 0.860 (t, 6.7 Hz, 3H);
13C NMR δ 156.9, 144.9, 135.2, 128.4, 127.7, 127.3, 81.5, 78.4, 45.2,
35.3, 31.3, 23.5, 22.4, 13.9; HRMS (FAB, NBA) m/z calcd for C16H24-
NO3 (M+H) 278.1756, found 278.1767. Anal. Calcd for C16H23NO3:
C, 69.29; H, 8.36; N, 5.05. Found: C, 69.06; H, 8.29; N, 5.11.
(4R,5R)-3-Benzyl-4-hydroxymethyl-5-pentyl-2-oxazolidinone (ent-
22). Following the procedure described above for 22, ent-21 was
assignment of diastereomeric pairs based on coupling pattern and
COSY) δ 7.37-7.26 (m, 5H), 4.82 and 4.73 (diastereomers, d, d, J )
15.3 and 15.2 Hz, 1H), 4.40-4.20 (m, 1H), 4.12 and 4.10 (diastere-
omers, d, d, J ) 15.3 and 15.2 Hz, 1H), 3.72-3.71 (m, 1H), 3.58-
3.51 (m, 1H), 3.48-3.44 and 3.25-3.28 (diastereomers, m, m, 1H),
3.40-3.33 (m, 1H), 2.63 (m, 1H), 2.25 (br s, 1H), 1.81-1.50 (m, 4H),
1.39-1.15 (m, 6H), 0.86 (t, J ) 6.4 Hz, 3H); 13C NMR (CDCl3,
assignment of diastereomeric pairs based on relative intensity and
DEPT) δ 158.3, 135.8, 128.8, 128.0, 127.9 and 127.9, 80.1 and 79.2,
69.2 and 67.6, 66.9 and 66.8, 57.9 and 57.0, 46.0 and 45.7, 35.0 and
34.9, 34.6 and 34.2, 31.3 and 31.3, 24.0 and 24.0, 22.3, 13.8; HRMS
(FAB, NBA) m/z calcd for C18H28NO4 (M+H) 322.2018, found
322.2017.
converted to ent-22 in 56% yield: [R]20 +67.5 (c 0.830, CHCl3).
D
(4S,5S)-3-Benzyl-4-iodomethyl-5-pentyl-2-oxazolidinone (23). Tri-
phenylphosphine (1.11 g, 4.22 mmol) and imidazole (288 mg, 4.22
mmol) were dissolved in ether (4.8 mL) and acetonitrile (1.2 mL). The
mixture was cooled to 0 °C, and iodine (1.07 g, 4.22 mmol) was added
with vigorous stirring. The resulting slurry was warmed to 20 °C and
then cooled to 0 °C again, and a solution of 22 (390 mg, 1.41 mmol)
in ether/acetonitrile/benzene (3 mL, 1:1:1) was added dropwise over 5
min. The mixture was warmed to 20 °C and stirred 1 h, then cooled to
0 °C and quenched with 5% sodium bicarbonate (10 mL). The layers
were separated and the aqueous phase extracted with ether (3 × 10
mL). The combined organic extracts were dried with MgSO4, filtered
and concentrated. Purification by flash chromatography on silica, eluting
(4R,5R)-3-Benzyl-4-(2′,3′-dihydroxypropyl)-5-pentyl-2-oxazolidi-
none (ent-25). Following the procedure described above for 25, ent-
24 was converted to ent-25 as an inseparable mixture of diastereomers
in 71% yield.
(4S,5S)-4-(Benzylamino)-1,2,5-decanetriol (26). A solution of 25
(70 mg, 0.218 mmol) and 50% NaOH (0.5 mL) in 95% ethyl alcohol
(2 mL) was heated at reflux overnight, cooled to room temperature
and poured into water (10 mL). The reaction was transferred to a
separatory funnel and extracted with ethyl acetate (4 × 10 mL). The
combined organic extracts were dried over MgSO4, filtered and
concentrated. Purification by flash chromatography, eluting with 4-6%
ethanol in hexanes afforded 26 (45 mg, 70%): 1H NMR (CDCl3,
assignment of diastereomeric pairs based on coupling pattern and
COSY) δ 7.35-7.26 (m, 5H), 3.98 and 3.93 (diastereomers, d, d, J )
12.8 and 12.4 Hz, 1H), 3.96-3.94 and 3.91-3.87 (diastereomers, m,
m, 1H), 3.84 and 3.71) (diastereomers, d, d, J ) 12.4 and 12.8, 1H),
3.74-3.67 and 3.68-3.65 (diastereomers, m, m, 1H), 3.61-3.52 (m,
1H), 3.46-3.39 (m, 1H), 3.24 (br s, 4H), 2.90-2.86 and 2.82-2.78
(diastereomers, m, m, 1H), 1.95-1.26 (m, 10H), 0.92-0.87 (m, 3H);
13C NMR (CDCl3, assignment of diastereomeric pairs based on relative
intensity and DEPT) δ 139.3 and 139.2, 128.5, 128.3 and 128.2, 127.3
and 127.2, 72.9 and 71.9, 71.5 and 69.9, 67.0 and 66.9, 60.8 and 60.2,
51.8 and 51.5, 33.9 and 33.9, 33.3 and 30.1, 31.8, 25.8 and 25.2, 22.6,
14.0; HRMS (FAB, NBA) m/z calcd for C17H30NO3 (M+H) 296.2226,
found 296.2221.
with 4% ethyl acetate in hexanes afforded 23 (449 mg, 82%): [R]20
D
-10.5° (c 1.22, CHCl3); 1H NMR (CDCl3) δ 7.37-7.26 (m, 5H), 4.84
(d, J ) 15.3 Hz, 1H), 4.22-4.16 (m, 1H), 4.08 (d, J ) 15.3 Hz, 1H),
3.20-3.15 (m, 3H), 1.61-1.59 (m, 2H), 1.45-1.20 (m, 6H), 0.87 (m,
3H); 13C NMR δ 157.4, 135.4, 129.0, 128.2, 128.1, 79.1, 59.0, 46.0,
35.0, 31.3, 23.9, 22.4, 13.9, 6.9.
(4R,5R)-3-Benzyl-4-iodomethyl-5-pentyl-2-oxazolidinone (ent-23).
Following the procedure described above for 23, ent-22 was converted
to ent-23 in 83% yield: [R]20 + 10.9° (c 0.86, CHCl3).
D
(4S,5S)-4-Allyl-3-benzyl-5-pentyl-2-oxazolidinone (24). To a solu-
tion of 23 (530 mg, 1.37 mmol), cuprous iodide (27 mg, 0.14 mmol)
and HMPA (1.01 mL, 5.48 mmol) in THF (5 mL) was added
vinylmagnesium bromide (2.74 mL, 1.0 M solution in THF) dropwise
at -30 °C over 5 min. The resulting mixture was stirred for 1 h at
-30 °C then quenched with saturated NH4OH (10 mL). The layers
were separated and the aqueous phase extracted with ether (3 × 10
mL). The combined organic extracts were dried with MgSO4, filtered
and concentrated. Purification by flash chromatography on silica, eluting
(4R,5R)-4-(Benzylamino)-1,2,5-decanetriol (ent-26). Following the
procedure described above for 26, ent-25 was converted to ent-26 in
68% yield.
(4R,5R)-4-Amino-1,2,5-decanetriol (10). Following the procedure
described below for 11, ent-26 was converted to 10 as an inseparable
mixture of diastereomers in 100% yield.
with 3-7% ethyl acetate in hexanes afforded 24 (274 mg, 70%). [R]20
D
-52.4° (c 0.95, CHCl3); 1H NMR (CDCl3) δ 7.39-7.26 (m, 5H), 5.62
(m, 1H), 5.16 (m, 2H), 4.84 (d, J ) 15.2 Hz, 1H), 4.16 (ddd, J ) 7.2,
5.1, 5.1 Hz, 1H), 4.07 (d, J ) 15.2 Hz, 1H), 3.21 (ddd, J ) 7.2, 5.1,
4.1 Hz, 1H), 2.39-2.22 (m, 2H), 1.67-1.26 (m, 8H), 0.86 (t, J ) 6.6
Hz, 3H); 13C NMR δ 157.9, 135.8, 131.4, 128.7, 128.0, 127.8, 119.7,
78.1, 58.4, 45.8, 36.0, 34.8, 31.3, 24.1, 22.3, 13.8; HRMS (FAB, NBA)
m/z calcd for C18H26NO2 (M+H) 288.1964, found 288.1973. Anal.
Calcd for C18H25NO2: C, 75.22; H, 8.77; N, 4.87. Found: C, 75.12;
H, 8.80; N, 4.83.
(4S,5S)-4-Amino-1,2,5-decanetriol (11). A suspension of 20% Pd-
(OH)2 on carbon (10 mg) in methanol (1 mL) was stirred at room
temperature under a hydrogen atmosphere for 30 min. A solution of
26 (29 mg, 0.098 mmol) in methanol (1 mL) was added via cannula
and the reaction mixture stirred under a hydrogen atmosphere for 5 h.
The catalyst was removed by filtration and the filtrate concentrated to
give 11 (20 mg, 100%) as an inseparable mixture of diastereomers:
1H NMR (CD3OD, assignment of diastereomeric pairs based on cou-
pling pattern and COSY) δ 3.83-3.76 (m, 1H), 3.53-3.43 (m, 2H),
3.50-3.44 (m, 1H), 2.96-2.93 (m, 1H), 1.81-1.18 (m, 10 H), 0.93-
0.89 (m, 3H); 13C NMR (CD3OD, assignment of diastereomeric pairs
based on relative intensity and DEPT) δ 75.9 and 75.0, 72.6 and 70.6,
67.8 and 67.5, 55.4 and 53.4, 38.0 and 37.4, 34.5, 33.1, 26.8 and 26.7,
23.7, 14.4; HRMS (FAB, NBA) m/z calcd for C10H24NO3 (M+H)
206.1756, found 206.1764.
N2-[1-(R)-(2,3-Dihydroxypropyl)-2-(S)-hydroxyheptyl]deoxygua-
nosine (27). Following the procedure described below for 28, the
reaction of (4R,5S)-8 (11 mg, 0.053 mmol) with 5a (13.0 mg, 0.035
mmol) gave 27 (10 mg, 60%) after hydrolysis. 1H NMR (DMSO-d6) δ
10.21 (m, 1H, N1-H), 7.89 and 7.88 (diastereomers, s, s, 1H, H-8),
6.43-6.34 (m, 1H, N2-H), 6.13-6.08 (m, 1H, H-1′), 5.25 (d, J ) 3.7
Hz, 1H, 3′-OH), 4.86 (t, J ) 3.4 Hz, 1H, 5′-OH), 4.87-4.58 (m, 2H,
(4R,5R)-4-Allyl-3-benzyl-5-pentyl-2-oxazolidinone (ent-24). Fol-
lowing the procedure described above for 24, ent-23 was converted to
ent-24 in 72% yield. [R]20 + 51.8° (c 0.845, CHCl3).
D
(4S,5S)-3-Benzyl-4-(2′,3′-dihydroxypropyl)-5-pentyl-2-oxazoli-
dinone (25). To a stirred solution of 24 (150 mg, 0.522 mmol),
N-methylmorpholine N-oxide (70 mg, 0.574 mmol), THF (1 mL),
t-BuOH (0.4 mL), and water (0.2 mL) was added osmium tetroxide in
benzene (32 µL, 0.1 M in benzene, 3.2 µmol). The mixture was stirred
for 12 h, then a second portion of osmium tetroxide (20 µL, 0.1 M in
benzene, 2.0 µmol) was added and stirring continued for 8 h. The
reaction was quenched with 5% aqueous NaHSO3 (10 mL) with
vigorously stirring for 15 min. The mixture was poured into water,
transferred to a separatory funnel and extracted with methylene chloride
(3 × 20 mL). The combined organic extracts were dried over Na2SO4,
filtered, and concentrated. Purification by flash chromatography on
silica, eluting with 1-2% methanol in chloroform gave 25 (116 mg,
69%) as an inseparable mixture of diastereomers: 1H NMR (CDCl3,
9
5698 J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003

Adducts of trans-4-Hydroxynonenal
A R T I C L E S
5*-OH and 2*-OH), 4.58-4.40 (m,1H, 1*-OH), 4.32 (m, 1H, H-3′),
4.013 and 3.99 (diastereomers, m, m, 1H, H-4*), 3.79 (m, 1H, H-4′),
3.63-3.42 (m, 4H, H-5′, H-5′′, H-2*, H-5*), 3.30-3.19 (m, 2H, H-1*,
H-1**, hidden under DMSO peak but visible in CD3OD), 2.58 (m,
1H, H-2′), 2.18 (m, 1H, H-2′′), 1.78 (m, 1H, H-3*), 1.57-1.14 (m,
9H), 0.85 (t, J ) 6.6 Hz, 3H); HRMS (FAB, NBA) m/z calcd for
C20H34N5O7 (M+H) 456.2458, found 456.2440.
overlapped with H-5′), 3.72 (dd, J ) 12.0, 3.9 Hz, 1H, H-5′′), 3.75-
3.70 (m, 1H, H-6, overlapped with H-5′′), 2.67 (m, 1H, H-2′), 2.33
(m, 1H, H-2′′), 2.10 (m, 1H, H-7a), 1.74 (m, 1H, H-7b), 1.57-1.27
(m, 8H), 0.923 (t, J ) 6.5 Hz, 3H); HRMS (FAB, NBA) m/z calcd for
C19H30N5O6 (M+H) 424.2196, found 424.2190.
3-(2-Deoxy-â-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-6-(S)-
[1-(R)-hydroxyhexyl]pyrimido[1,2-a]purin-10(3H)-one (2). Following
the procedure described below for 4, 28 was converted to 2 in 84%
N2-[1-(S)-(2,3-Dihydroxypropyl)-2-(R)-hydroxyheptyl]deoxygua-
nosine (28). A solution of (4S,5R)-9 (10.0 mg, 0.049 mmol) in
anhydrous DMSO (200 µL) was added to a mixture of 5a (11.9 mg,
0.032 mmol), DMSO (100 µL), and diisopropylethylamine (100 µL).
The mixture was stirred at 65-70 °C for 2 days, then cooled, and
concentrated under high vacuum using a centrifugal evaporator. The
residue was dissolved in 5% acetic acid (1 mL) and stirred at room
temperature for 1 h. Purification by HPLC (gradient B) gave 28 (8.3
mg, 57%): 1H NMR (DMSO-d6) δ 10.41 (m, 1H, N1-H), 7.88 and
7.87 (diastereomers, s, s, 1H, H-8), 6.41-6.33 (m, 1H, N2-H), 6.12-
6.08 (m, 1H, H-1′), 5.25 (d, J ) 4.0 Hz, 1H, 3′-OH), 4.87-4.80 (m,
2H, 5′-OH and 5*-OH), 4.73-4.63 (m, 1H, 2*-OH), 4.51 and 4.45
(diastereomers, t, t, J ) 5.6 and 5.5 Hz, 1H, 1*-OH), 4.32 (m, 1H,
H-3′), 4.02 and 3.91 (diastereomers, m, m, 1H, H-4*), 3.79 (m, 1H,
H-4′), 3.57-3.44 (m, 4H, H-5′, H-5′′, H-2*, H-5*), 3.26 (m, 2H, H-1*,
H-1**, hidden under DMSO peak but visible in CD3OD), 2.57 (m,
1H, H-2′), 2.17 (m, 1H, H-2′′), 1.79 (m, 1H, H-3*), 1.57-1.23 (m,
9H), 0.85 (t, J ) 6.6 Hz, 3H); HRMS (FAB, NBA) m/z calcd for
C20H34N5O7 (M+H) 456.2458, found 456.2450.
1
yield. H NMR (CD3OD) δ 7.91 (s, 1H, H-2), 6.42 (t, J ) 2.5, 1H,
H-8), 6.24 (dd, J ) 7.6, 6.2 Hz, 1H, H-1′), 4.51 (m, 1H, H-3′), 3.98
(m, 1H, H-4′), 3.78 (dd, J ) 12.0, 3.4 Hz, 1H, H-5′), 3.81-3.75 (m,
1H, H-11, overlapped with H-5′), 3.73 (dd, 12.0, 3.9 Hz, 1H, H-5′′),
3.75-3.70 (m, 1H, H-6, overlapped with H-5′′), 2.69 (m, 1H, H-2′),
2.33 (m, 1H, H-2′′), 2.11 (m, 1H, H-7a), 1.74 (m, 1H, H-7b), 1.57-
1.28 (m, 8H), 0.93 (t, J ) 6.7 Hz, 3H); HRMS (FAB, NBA) m/z calcd
for C19H30N5O6 (M+H) 424.2196, found 424.2177.
3-(2-Deoxy-â-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-6-(R)-
[1-(R)-hydroxyhexyl]pyrimido[1,2-a]purin-10(3H)-one (3). Following
the procedure described below for 4, 29 was converted to 3 in 83%
1
yield. H NMR (CD3OD) δ 7.91 (s, 1H, H-2), 6.40 (t, J ) 2.5, 1H,
H-8), 6.23 (t, J ) 6.9 Hz, 1H, H-1′), 4.51 (m, 1H, H-3′), 3.98 (dt, J )
3.3, 3.0 Hz, 1H, H-4′), 3.78 (dd, J ) 12.1, 3.4 Hz,1H, H-5′), 3.70 (dd,
J ) 12.1, 3.8 Hz, 1H, H-5′′), 3.61 (m, 1H, H-6), 3.47 (m, 1H, H-11),
2.68 (m, 1H, H-2′), 2.33 (m, 1H, H-2′′), 2.18 (m, 1H, H-7a), 1.64-
1.28 (m, 9H), 0.93 (t, J ) 6.6 Hz, 3H); HRMS (FAB, NBA) m/z calcd
for C19H30N5O6 (M+H) 424.2196, found 424.2192.
N2-[1-(R)-(2,3-Dihydroxypropyl)-2-(R)-hydroxyheptyl]deoxygua-
nosine (29). Following the procedure described below for 30, the
reaction of (4R,5R)-28 (15.2 mg, 0.074 mmol) with 5a (13.7 mg, 0.037
mmol) gave 29 (10.7 mg, 63.5%) after hydrolysis of the O6-trimeth-
ylsilylethyl group. 1H NMR (DMSO-d6) δ 10.35 (m, 1H, N1-H), 7.89
and 7.86 (diastereomers, s, s, 1H, H-8), 6.34 (m, 1H, N2-H), 6.08 (m,
1H, H-1′), 5.24 (m, 1H, 3′-OH), 4.99 and 4.90 (diastereomers, m, 1H,
5*-OH), 4.88 (m, 1H, 5′-OH), 4.60 and 4.49 (diastereomers, s, s, 1H,
2*-OH), 4.47 (m, 1H, 1*-OH), 4.33 (m, 1H, H-3′), 4.11-4.05 (m, 1H,
H-4*), 3.79 (m, 1H, H-4′), 3.60-3.40 (m, 4H, H-5′, H-5′′, H-2*, H-5*),
3.30-3.15 (m, 2H, H-1*, H-1**, hidden under water peak but visible
in CD3OD), 2.58 (m, 1H, H-2′), 2.18 (m, 1H, H-2′′), 1.72 (m, 1H,
H-3*), 1.50-1.10 (m, 9H), 0.81 (t, J ) 6.8 Hz, 3H); HRMS (FAB,
NBA) m/z calcd for C20H34N5O7 (M+H) 456.2458, found 456.2439.
N2-[1-(S)-(2,3-Dihydroxypropyl)-2-(S)-hydroxyheptyl]deoxygua-
nosine (30). A solution of (4S,5S)-11 (20.1 mg, 0.098 mmol) in
anhydrous DMSO (200 µL) was added to a solution of 5a (18.2 mg,
0.049 mmol), DMSO (100 µL), and diisopropylethylamine (100 µL).
The mixture was stirred at 70-75 °C for 26 h, cooled, and concentrated
under high vacuum using a centrifugal evaporator. The residue was
dissolved in 5% acetic acid (1 mL) and stirred at room temperature for
1 h. Purification by HPLC (gradient A) gave 30 (13.5 mg, 60.5%) as
3-(2-Deoxy-â-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-6-(S)-
[1-(S)-hydroxyhexyl]pyrimido[1,2-a]purine-10(3H)-one (4). A solu-
tion of sodium periodate (505 µL, 0.0101 mmol, 20 mM in water) was
added to a solution of 30 (4.6 mg, 0.010 mmol) in 50 mM, pH 7
phosphate buffer (500 µL). The reaction mixture was stirred at room
temperature for 10 min and immediately purified by HPLC (gradient
1
A) to give 4 (3.7 mg, 87%). H NMR (CD3OD) δ 7.91 (s, 1H, H-2),
6.40 (t, J ) 2.5 Hz, 1H, H-8), 6.23 (t, J ) 6.9 Hz, 1H, H-1′), 4.51 (m,
1H, H-3′), 3.99 (m, H-4′), 3.78 (dd, J ) 12.1, 3.4 Hz, H-5′), 3.71 (dd,
J ) 12.1, 3.7 Hz, 1H, H-5′′), 3.61 (m, 1H, H-6), 3.47 (m, 1H, H-11),
2.68 (m, 1H, H-2′), 2.33 (m, 1H, H-2′′), 2.19 (m, 1H, H-7a), 1.64-
1.28 (m, 9H), 0.93 (t, J ) 6.6 Hz, 3H); HRMS (FAB, NBA) m/z calcd
for C19H30N5O6 (M+1) 424.2196, found 424.2181.
3-(2-deoxy-â-D-erythro-pentofuranosyl)-6-(S)-[1-(S)-hydroxyhexyl)]-
5,6,7,8-tetrahydro-8-(R)-(N2-deoxyguanosinyl)-pyrimido[1,2-a]pu-
rin-10(3H)-one (45c). In a glass tube was added 4 (5 mg) and 2′-
deoxyguanosine monohydrate (10 mg) in DMSO (250 µL). The mixture
was stirred in an oil bath at 100 °C for 11 days. The solvent was
removed under vacuum. Purification by HPLC (gradient B) gave
recovered 4 (3 mg) and 45c (0.7 mg, 9%): 1H NMR (DMSO-d6) δ
10.20 (s, 1H), 7.97 (s, 1H), 7.94 (s, 1H), 7.43 (s, 1H), 7.31 (d, J ) 6.8
Hz, 1 H), 6.66 (m, 1H), 6.20 (m, 1H), 6.13 (m, 1H), 5.31 (d, J ) 4.0
Hz, 1H), 5.25 (d, J ) 3.7 Hz), 5.03 (d, J ) 5.5 Hz, 1H), 4.96 (t, J )
5.4 Hz), 4.88 (t, J ) 5.6 Hz, 1H), 4.36 (m, 1H), 3.90 (m, 1H), 3.52
(m, 6H), 2.62 (m, 1H), 2.44 (m, 1H, hidden under solvent peak, visible
in COSY spectrum), 2.20 (m, 3H), 1.65 (m, 1H), 1.38-1.10 (m, 9H),
1
a mixture of diastereomers. H NMR (DMSO-d6) δ 10.37 (br s, 1H,
N1-H), 7.88 and 7.86 (diastereomers, s, s, 1H, H-8), 6.39 (m, 1H,
N2-H), 6.10 (m, 1H, H-1′), 5.26 (m, 1H, 3′-OH), 4.99 and 4.90
(diastereomers, d, d, J ) 4.2 and 4.2 Hz, 1H, 5*-OH), 4.88 (m, 1H,
5′-OH), 4.58 and 4.45-4.38 (diastereomers, d, d, J ) 4.5 and 4.5 Hz,
1H, 2*-OH, the later peak overlapped with 1*-OH), 4.48 (m, 1H,
1*-OH), 4.33 (m, 1H, H-3′), 4.11-4.03 (m, 1H, H-4*), 3.80 (m, 1H,
H-4′), 3.60-3.40 (m, 4H, H-5′, H-5′′, H-2*, H-5*), 3.30-3.15 (m, 2H,
H-1*, H-1**, hidden under water peak but visible in CD3OD), 2.58
(m, 1H, H-2′), 2.16 (m, 1H, H-2′′, 1.72(m, 1H, H-3*), 1.50-1.10 (m,
9H), 0.80 (t, J ) 6.7 Hz, 3H); HRMS (FAB, NBA) m/z calcd for
C20H34N5O7 (M+H) 456.2458, found 456.2471.
3-(2-Deoxy-â-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-6-(R)-
[1-(S)-hydroxyhexyl]pyrimido[1,2-a]purin-10(3H)-one (1). Following
the same procedure described below for 4, 27 was converted to 1 in
80% yield. 1H NMR (CD3OD) δ 7.91 (s, 1H, H-2), 6.42 (m, 1H, H-8),
6.23 (dd, J ) 7.5, 6.5 Hz, 1H, H-1′), 4.51 (m, 1H, H-3′), 3.98 (m, 1H,
H-4′), 3.78 (dd, J ) 12.0, 3.5 Hz, 1H, H-5′), 3.82-3.75 (m, 1H, H-11,
0.84 (t, J ) 6.7 Hz, 1H); MS: LC-ESMS m/z calcd for C29H N10O9
41
(M+1) 673.31, found 673.38.
Synthesis of 5′-d(GCTAGC(27)AGTCC) (34). The oligonucleotide
5′-d(GCTAGC(X)AGTCC) (33, X ) 5a; 10 A260 units) was mixed in
a plastic tube with diisopropylethylamine (50 µL), DMSO (100 µL),
and 1 (0.5 mg). The mixture was stirred at 65 °C for 72 h, adding
additional portions of 1 (0.5 mg) after 24 and 48 h. HPLC showed the
disappearance of 33. The solvent was evaporated under vacuum and
the residue dissolved in 5% acetic acid (500 µL), then stirred for 2 h
at room temperature. The reaction mixture was neutralized to pH 7
(pH paper) with 1 M NaOH and purified by HPLC (gradient A) to
give oligonucleotide 27 (5.4 A260 units, 54%). The modified oligo-
nucleotide (27) was characterized by enzyme digestion followed by
9
J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003 5699

A R T I C L E S
Wang et al.
HPLC analysis and MALDI-TOF MS. MS: MALDI (HPA) m/z calcd
for (M-H) 3832.9, found 3832.2.
Synthesis of 5′-d(GCTAGC(28)AGTCC) (35). Following the
procedure described for 34, 35 was prepared in 57% yield. MS: MALDI
(HPA) m/z calcd for (M-H) 3832.9, found 3833.0.
Synthesis of 5′-d(GCTAGC(29)AGTCC) (36). Following the
procedure described for 34, 36 was prepared in 53% yield. MS: MALDI
(HPA) m/z calcd for (M-H) 3832.9, found 3833.3.
Synthesis of 5′-d(GCTAGC(30)AGTCC) (37). Following the
procedure described for 34, 37 was prepared in 51% yield. MS: MALDI
(HPA) m/z calcd for (M-H) 3832.9, found 3833.7.
Synthesis of 5′-d(GCTAGC(1)AGTCC) (38). Sodium periodate
(100 µL of a 20 mM solution in pH 7 phosphate buffer) was added to
a solution of 34 (3.2 A260 units) in phosphate buffer (300 µL, pH 7.0),
and the reaction stirred at room temperature for 10 min. HPLC
purification afforded 5′-d(GCTAGC(1)AGTCC) (38, 2.5 A260 units,
78%). The structure was confirmed by enzyme digestion followed by
HPLC analysis using nucleoside 1 as an authentic sample and MALDI-
TOF MS. MS: MALDI (HPA) m/z calcd for (M-H) 3800.8, found
3799.9.
Synthesis of 5′-d(GCTAGC(2)AGTCC) (39). Following the pro-
cedure described for 38, 39 was prepared in 82% yield. MS: MALDI
(HPA) m/z calcd for (M-H) 3800.8, found 3799.3.
Synthesis of 5′-d(GCTAGC(3)AGTCC) (40). Following the pro-
cedure described for 38, 40 was prepared in 83% yield. MS (MALDI)
m/z calcd for (M-H) 3800.8, found 3801.8.
Cross-linking Reactions. Equal molar mixtures of the adducted (1.0
A260 units) and complementary (1.0 A260 units) oligonucleotides in 60
µL of potassium phosphate buffer (0.05 M, pH 7.0) containing 1.0 M
KCl were incubated at 37 °C and 46 °C in plastic Eppendorf tubes.
The reactions were analyzed periodically by CZE with UV detetction.
The cross-linked species eluted later than the starting oligonucleotides.
Relative amounts of remaining starting and cross-linked oligonucleotides
were estimated from peak areas. Conversion of peak areas to relative
molar amounts was made using molar absorptivities calculated on the
basis of sequence using the method of Borer.41 The molar absorptivity
of nucleosides 1-4 were assumed to be identical to that of dG and
that of bis-nucleoside 45c to be twice that of dG. The molar absorptivity
contributions of the individual nucleosides were assumed to be identical
for single-stranded and cross-linked oligonucleotides, i.e., no allowance
was made for the hypochromicity of cross-linked duplexes because of
uncertainty as to the extent of denaturation under the CZE conditions.
The consequence of this assumption is that the extent of cross-linking
may be slightly greater than the calculated values. Enzymatic digestion
of the cross-linking reaction and analysis by HPLC showed the presence
of cross-linked nucleoside 45c, which was unambiguously identified
by comparison with an authentic sample.
Melting Studies. Equal amounts of the modified oligonucleotides
and the complementary strands (0.25 A260 units each) were dissolved
in 0.5 mL of melting buffer (10 mM Na2HPO4/NaH2PO4, 1.0 M NaCl,
50 µM Na2EDTA, pH 7.0). UV measurements monitoring the absor-
bance at 260 nm, were taken at 1 min intervals with a 1 °C/min
temperature gradient. The temperature was cycled between 5 and 95
°C. The first derivative of the melting curve was used to establish Tm
values.
Synthesis of 5′-d(GCTAGC(4)AGTCC) (41). Following the pro-
cedure described for 38, 41 was prepared in 80% yield. MS: MALDI
(HPA) m/z calcd for (M-H) 3800.8, found 3800.8.
Enzymatic Digestion. The oligonucleotide (0.2 A260 units) was
dissolved in 30 µL of buffer (pH 7.0, 10 mM Tris-HCl, 10 mM MgCl2)
and incubated with DNase I, snake venom phosphodiesterase I, and
alkaline phosphatase at 37 °C for 90 min. The mixture was then
analyzed by reverse phase HPLC using a YMC ODS-AQ column
(250 × 4.6 mm i.d., 1.5 mL/min) with UV detection monitoring at
260 nm. The following solvent gradient was used: 1-10% acetonitrile
over 15 min, 10-20% acetonitrile over 5 min, hold for 5 min, 20-
100% acetonitrile over 10 min, hold for 5 min and then 100-1%
acetonitrile over 5 min. The modified nucleosides 1-4, 27-30, and
45c were identified by comparison with authentic samples based on
retention times, co-injection, and UV spectra.
Acknowledgment. This work was supported by the National
Institutes of Health (Program Project Grant ES05355 and Center
Grant ES00267) and the American Cancer Society (Research
Grant RPG-96-061-04-CDD).
Supporting Information Available: Copies of MALDI-TOF
spectra of adducted oligonucleotides 34-41. This material is
available free of charge via the Internet at http://pubs.acs.org
JA0288800
(41) Borer, P. N. Handbook of Biochemistry and Molecular Biology, 1st ed.;
CRC Press: Cleveland, 1975. Notes: p 359.
9
5700 J. AM. CHEM. SOC. VOL. 125, NO. 19, 2003