Stereospecific synthesis of oligonucleotides containing crotonaldehyde adducts of deoxyguanosine

Article abstract of DOI:10.1021/tx0100690

Crotonaldehyde reacts with DNA to form two diastereomeric 1,N² cyclic adducts of deoxyguanosine. A synthesis of the two diastereomeric deoxynucleosides has been achieved by reaction of mixed diastereomers of 4-amino-1,2-pentanediol with 2-fluoro-O⁶-(trimethylsilylethyl)-deoxyinosine. The resulting N²-(1-methyl-3,4-dihydroxybutyl)-deoxyguanosine was treated with NaIO₄, cleaving the vicinal diol to the aldehyde. Spontaneous cyclization gave the two diastereomers of the crotonaldehyde-adducted nucleoside that were readily separated by HPLC. The absolute configurations were assigned by an enantiospecific synthesis of one diastereomer from (S)-3-aminobutanoic acid. The synthetic strategy has been extended to preparation of a site-specifically adducted oligonucleotide by reaction of the mixed diastereomers of 4-amino-1,2-pentanediol with an 8-mer oligonucleotide containing 2-fluoro-O⁶-(trimethylsilylethyl)-deoxyinosine. The diastereomeric oligonucleotides were separated by HPLC and absolute configurations of the adducts were established by enzymatic digestion to the adducted nucleosides.

Full text of DOI:10.1021/tx0100690

1506
Chem. Res. Toxicol. 2001, 14, 1506-1512
Ster eosp ecific Syn th esis of Oligon u cleotid es Con ta in in g
Cr oton a ld eh yd e Ad d u cts of Deoxygu a n osin e
Lubomir V. Nechev, Ivan Kozekov, Constance M. Harris, and Thomas M. Harris*
Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University,
Nashville, Tennessee 37235
Received April 4, 2001
Crotonaldehyde reacts with DNA to form two diastereomeric 1,N2 cyclic adducts of
deoxyguanosine. A synthesis of the two diastereomeric deoxynucleosides has been achieved
by reaction of mixed diastereomers of 4-amino-1,2-pentanediol with 2-fluoro-O6-(trimethylsi-
lylethyl)-deoxyinosine. The resulting N2-(1-methyl-3,4-dihydroxybutyl)-deoxyguanosine was
treated with NaIO4, cleaving the vicinal diol to the aldehyde. Spontaneous cyclization gave
the two diastereomers of the crotonaldehyde-adducted nucleoside that were readily separated
by HPLC. The absolute configurations were assigned by an enantiospecific synthesis of one
diastereomer from (S)-3-aminobutanoic acid. The synthetic strategy has been extended to
preparation of a site-specifically adducted oligonucleotide by reaction of the mixed diastereomers
of 4-amino-1,2-pentanediol with an 8-mer oligonucleotide containing 2-fluoro-O6-(trimethylsi-
lylethyl)-deoxyinosine. The diastereomeric oligonucleotides were separated by HPLC and
absolute configurations of the adducts were established by enzymatic digestion to the adducted
nucleosides.
Sch em e 1
In tr od u ction
The R,â-unsaturated aldehyde crotonaldehyde is a
widely dispersed product of pyrolysis and incomplete
oxidation of organic materials (1). It is present in
significant quantities in tobacco smoke (2), is a metabolite
of N-nitrosopyrrolidone (3-5), and arises in living sys-
tems as a product of lipid peroxidation (6). Crotonalde-
hyde is genotoxic, mutagenic, and carcinogenic (7-11).
Like other R,â-unsaturated aldehydes, it reacts with DNA
to form adducts which are believed to be the basis of
genotoxicity. At this juncture, only adducts of dGuo have
been characterized (3, 12, 13).
Adduct formation by the simpler analogue acrolein has
been observed with dGuo, dAdo, and dCyd. It reacts with
deoxyguanosine to form regioisomeric 1,N2-hydroxypro-
pano adducts 1 and 2 as shown in Scheme 1 (8, 14).
Adduct 1 in which the hydroxyl group is proximal to the
N1 position is formed by reaction of guanine N2 with the
â carbon of the aldehyde and N1 with the carbonyl group.
Conversely, isomeric adduct 2 in which the hydroxyl
group is distal to the N1 position is formed by attack of
guanine N1 on the â carbon and attack of N2 on the
carbonyl. With crotonaldehyde, only the diastereomers
of proximal adduct 3 have been detected; two diastere-
omers are created by the stereogenic center bearing the
methyl group. For both diastereomers, the hydroxyl
group on the propano ring has been assigned as being
predominantly trans to the methyl group (8, 15).
unfunctionalized 1,N2-propano adduct is strongly mu-
tagenic (18, 19). It has been proposed that the explana-
tion for this difference is that the hydroxypropano adduct
is able to undergo ring opening to form an N2,3-oxopropyl
adduct (20). An NMR study by de los Santos and
co-workers has confirmed that the hydroxypropano ad-
duct reverts to the 3-oxopropyl species in double-stranded
DNA (21). The lack of mutagenicity of the 3-oxopropyl
adduct at the N2 position of dGuo is consistent with the
recent finding that analogous adducts of butadiene
monoepoxide and diolepoxide are only weakly mutagenic
(22).
It would be useful to examine the structures of DNA
containing the diastereomers of the analogous 1,N2 dGuo
crotonaldehyde adduct to see whether they also assume
the de los Santos structure. The synthesis of DNA
containing adducts of acrolein presented an interesting
problem in that the aldehyde in equilibrium with the
cyclic species is not compatible with the conditions
employed for preparation of phosphoramidite reagents
and assembly of oligodeoxynucleotides. Two synthetic
strategies have been reported, both involving elaboration
of the aldehyde after assembly of the oligodeoxynucle-
otide by periodate cleavage of a 1,2-diol (20, 23). This
paper describes the utilization of one of these methods
for enantiospecific synthesis of the crotonaldehyde adduct
of deoxyguanosine and extension of the synthetic meth-
Site-specific mutagenesis studies of the proximal deoxy-
guanosine adduct of acrolein have revealed it to be
essentially nonmutagenic in point mutation assays al-
though the hydroxypropano adduct blocks the Watson-
Crick face of the nucleoside (16, 17). By contrast, the
* To whom correspondence should be addressed. Phone: (615) 322-
2649. Fax: (615) 322-2649. E-mail: harristm@toxicology.mc.
vanderbilt.edu.
10.1021/tx0100690 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/13/2001

Crotonaldehyde-Deoxyguanosine Adducts
Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1507
Hz, J 2 ) 4.8 Hz), 1.34 (d, 3H, CH3, J ) 6.8 Hz). HRMS (FAB+):
calcd for [M + H]+ C12H16NO3, 222.1130; found, 222.1122.
odology to the preparation of oligodeoxynucleotides con-
taining site-specific crotonaldehyde adducts.
(S)-3-(Ben zoyla m in o)bu tyr a ld eh yd e (5S). Methyl (S)-3-
(benzoylamino)butanoate (0.47 g, 2.12 mmol) was dissolved in
20 mL of anhydrous methylene chloride. The solution was cooled
to -78 °C. Diisobutylaluminum hydride (DIBAL-H) (2.12 mL
of 1 M solution in hexane, 2.12 mmol) was added over 15 min;
the mixture was stirred at -78 °C for 1 h. Additional DIBAL-H
(2.12 mL, 2.12 mmol) was added over 15 min and the mixture
was stirred for 1 h at -78 °C, followed by quenching at -78 °C
with 4 mL of a saturated aqueous solution of NH4Cl. Hydro-
chloric acid (4%, 4 mL) was added and the mixture was warmed
to room temperature. After 0.5 h, the mixture was extracted
several times with CH2Cl2 and the combined organic layers were
dried over MgSO4. The organic solvents were removed and the
residue was purified by chromatography on a silica gel column
(hexane/EtOAc, 1:1) to give 0.25 g (63%) of the substituted
butyraldehyde (5S) (25). 1H NMR (CDCl3) δ 9.85 (bs, 1H, CHO),
7.75 (m, 2H, o-Ph), 7.46 (m, 3H, m- and p-Ph), 6.49 (s, 1H, NH),
4.64 (m, 1H, CH), 2.81 (m, 2H, CH2), 1.40 (d, 3H, CH3, J ) 6.9
Hz). HRMS (FAB+): calcd for C11H14NO2 [M + H]+, 192.1025;
found, 192.1019.
Exp er im en ta l P r oced u r es
Gen er a l Meth od s. Thin-layer chromatography was per-
formed on silica gel glass plates (Merck, Silica Gel 60 F254, layer
thickness 0.25 mm). The chromatograms were visualized under
UV light (254 nm) or by staining with an anisaldehyde/sulfuric
acid solution, followed by heating. Column chromatography was
performed using silica gel (Merck, 70-230 mesh). HPLC analy-
ses and purifications were carried out on a gradient HPLC
(Beckman Instruments; System Gold software) equipped with
pump module 125 and photodiode array detector module 168.
For monitoring reactions and purification a YMC ODS-AQ
column (250 × 4.6 mm, flow rate 1.5 mL/min, or 250 × 10 mm,
flow rate, 5 mL/min) monitored at 260 nm was used with H2O-
acetonitrile for nucleosides, and 0.1 M ammonium formate-
acetonitrile for oligonucleotides. Gradient A: 99% H2O for
initial, 15 min linear gradient to 90% H2O, 5 min linear gradient
to 80% H2O, 3 min linear gradient to 20% H2O, 2 min isocratic
at 20% H2O, 3 min gradient to 99% H2O. Gradient B: 99% H2O
for initial, 5 min linear gradient to 94% H2O, 15 min linear
gradient to 91% H2O, 3 min linear gradient to 20% H2O, 2 min
isocratic at 20% H2O, 3 min gradient to 99% H2O.
In str u m en ta tion . 1H NMR spectra were recorded at 400.13
MHz on a Bruker AM400 NMR spectrometer in D2O or DMSO-
d6. Low and high-resolution FAB mass spectra were obtained
at the Mass Spectrometry Facility at the University of Notre
Dame, Notre Dame, Indiana. Negative ion MALDI-TOF1 mass
spectra of modified oligonucleotides were obtained on a Voyager
Elite DE instrument (Perseptives Biosystems) using a 3-hy-
droxypicolinic acid (3-HPA) matrix containing ammonium hy-
drogen citrate (7 mg/mL) to suppress multiple sodium and
potassium adducts. CD spectra were recorded in water at 25
°C on a J ASCO J -700 spectropolarimeter.
(S)-2-(Ben zoyla m in o)-4-p en ten e (6S). A solution of meth-
ylidenetriphenyl-phosphorane (prepared from methyltriphen-
ylphosphonium bromide (0.56 g, 1.57 mmol) and n-BuLi (0.560
mL of a 2.5 M solution in hexane, 1.4 mmol) in THF (20 mL)
was added to a stirred solution of the above aldehyde (0.25 g,
1.13 mmol) in dry THF (5 mL) at -78 °C. The reaction was
monitored by TLC by adding small aliquots to a mixture of
saturated aqueous (NH4)2SO4 and EtOAc. After the starting
material had been consumed, the mixture was warmed to room
temperature and poured into saturated aqueous (NH4)2SO4,
which was extracted with ether. The extract was dried over
MgSO4, and after evaporation of the solvents, the residue was
purified by chromatography on a silica gel column (hexane/
EtOAc, 2:1) to give 103 mg (42%) of (S)-2-(benzoylamino)-4-
pentene (6S). 1H NMR (CDCl3) δ 7.74 (m, 2H, o-Ph), 7.44 (m,
3H, m- and p-Ph), 5.95 (bs, 1H, NH), 5.84 (m, 1H, ) CH), 5.14
(m, 2H, ) CH2), 4.30 (m, 1H, CH), 2.35 (m, 2H, CH2), 1.26 (d,
3H, CH3, J ) 6.6 Hz). HRMS (FAB+): calcd for C12H16NO [M
+ H]+, 190.1232; found, 190.1227.
4(S)-Am in o-1,2-(R a n d S)-p en ta n ed iol (7S). (S)-2-(Ben-
zoylamino)-4-pentene (6S, 100 mg, 0.53 mmol) was added to a
mixture of water (5 mL), acetone (2 mL), N-methylmorpholine
N-oxide (71 mg, 0.6 mmol) and OsO4 (1-2 mg). The mixture
was stirred overnight at room temperature and the solvents
were evaporated. The residue was purified on a silica gel column
(ether/methanol, 95:5) to give 93 mg (79%) of 4(S)-(benzoyl-
amino)-1,2(R and S)-pentanediol (7S). 1H NMR (DMSO-d6) δ
8.20 (m, 1H, NH), 7.82 (m, 2H, o-Ph), 7.47 (m, 3H, m- and p-Ph),
4.49 (m, 2H, 2× OH), 4.21 (m, 1H, CHN), 3.50 (m, 1H, CHO),
3.29 (m, 2H, CH2O), 1.35-1.73 (m, 2H, CH2C), 1.16 (m, 3H,
CH3). HRMS (FAB+) [M + H]+: calcd, 224.1287; found,
224.1286.
(S)-3-(Ben zoyla m in o)bu ta n oic Acid (24). Benzoyl chloride
(0.50 g, 0.42 mL, 3.58 mmol) was added to a stirred solution of
(S)-3-aminobutanoic acid (4S, 0.50 g, 3.58 mmol, Fluka) in 10
mL of 1 M NaOH. The mixture was cooled (ice bath) during the
addition and stirred at room temperature for 24 h. TLC (ether,
visualized with ninhydrin spray) showed complete consumption
of the starting amino acid. The mixture was acidified (pH ∼6),
extracted with ether and dried over MgSO4. The ether was
evaporated and the residue was purified by a silica gel column
1
(ether) to give 0.48 g (65%) of the product as a white solid. H
NMR (DMSO-d6) δ 12.16 (bs, 1H, COOH), 8.28 (m, 1H, NH),
7.80 (m, 2H, o-Ph), 7.46 (m, 3H, m- and p-Ph), 4.34 (m, 1H, CH),
2.56 (dd, 1H, CH2, J 1 ) 15.3 Hz, J 2 ) 6.8 Hz), 2.38 (dd, 1H, J 1
) 15.3 Hz, J 2 ) 4.3 Hz), 1.17 (d, 3H, CH3, J ) 6.6 Hz). HRMS
(FAB+): calcd for C11H14NO3 [M + H]+, 208.0974; found,
208.0981.
Meth yl Ester of (S)-3-(Ben zoyla m in o)bu ta n oic Acid . (S)-
3-(Benzoylamino)butanoic acid (0.48 g, 2.32 mmol) was dissolved
in 50 mL of methanol containing 4-5 drops of concentrated H2-
SO4. The mixture was refluxed for 1 h. Most of the methanol
was evaporated, water was added and the mixture was extracted
three times with ether. The combined ether layers were washed
with satd. NaHCO3 solution and water and dried over MgSO4.
Evaporation of the solvent gave 0.47 g (92%) of the ester, which
was used in the next step without purification. 1H NMR (CDCl3)
δ 7.78 (m, 2H, o-Ph), 7.46 (m, 3H, m- and p-Ph), 6.95 (bs, 1H,
NH), 4.56 (m, 1H, CH), 3.72 (s, 3H, COOCH3), 2.69 (dd, 1H,
CH2, J 1 ) 15.9 Hz, J 2 ) 5.3 Hz), 2.62 (dd, 1H, CH2, J 1 ) 15.9
The diol (90 mg, 0.4 mmol) in 6 M HCl (10 mL) was refluxed
for 4 h. The solution was evaporated to dryness, water was
added and the mixture was neutralized (pH 6.0). The water were
evaporated again and the 4(S)-amino-1,2(R and S)-pentanediol
was purified on an ion exchange column (DOWEX 50-X8). The
aminodiol was eluted with 1 M NH4OH to give, after evaporation
1
to dryness, 40 mg (83%) of amino alcohol 7S. H NMR (DMSO-
d6) δ 3.75 (m, 1H, CHOH), 3.50 (m, 1H, 1× CH2OH), 3.41 (m,
1H, CH2OH), 3.12 (m, 1H, CHNH), 1.46 (m, 2H, CH2), 1.08 (m,
3H, CH3). HRMS (FAB+) [M + H]+: calcd, 120.1025; found,
120.1002.
N2-(1(S)-m eth yl-3(R a n d S),4-d ih yd r oxybu tyl)d eoxygu a -
n osin e (9S). Amino alcohol 7S (9.8 mg, 0.082 mmol) was added
to a mixture of O6-trimethylsilylethyl-2-fluoro-deoxyinosine (8,
20 mg, 0.054 mmol), DMSO (150 µL), and diisopropylethylamine
(50 µL). The mixture was stirred at 55 °C for 7 h. The reaction
was stopped and the solvents were evaporated under vacuum.
The residue was dissolved in 5% acetic acid (1 mL) and stirred
1
Abbreviations: DIBAL-H, diisobutylaluminum hydride; FAB MS,
fast-atom bombardment mass spectrometry; MALDI TOF MS, matrix-
assisted laser desorption/ionization time-of-flight mass spectrometry;
HRMS, high-resolution mass spectrometry; TMSE, trimethylsilylethyl.
COSY, correlation spectroscopy; HMBC, heteronuclear multibond
correlation spectroscopy (long-range 13C-1H scalar correlated 2D NMR
experiment).

1508 Chem. Res. Toxicol., Vol. 14, No. 11, 2001
Nechev et al.
at room temperature for 1 h. HPLC purification of the reaction
mixture gave 15.5 mg (78%) of N2-(1(S)-methyl-3,4-dihydroxy-
butyl)deoxyguanosine (9S). 1H NMR (DMSO-d6) δ 10.32 (bs, 1H,
N1H), 7.83 (s, 1H, H-8), 6.30, 6.25 (2m, 1H, N2H), 6.08 (m, 1H,
H-1′), 5.21 (m, 1H, 3′-OH), 4.81 (m, 1H, 5′-OH), 4.54 (m, 1H,
γ-OH), 4.49 (m, 1H, δ-OH), 4.28 (m, 1H, H-3′), 4.00 (m, 1H, R-H),
3.74 (m, 1H, H-4′), 3.47 (m, 3H, γ-H, H-5′, H-5′′), 3.23 (m, 2H,
δ-H), 2.40 (m, 1H, H-2′), 2.15 (m, 1H, H-2′′), 1.30-1.60 (m, 2H,
â-H), 1.10 (m, 3H, CH3). HRMS (FAB+) [M+H]+: calcd.
370.1727, found 370.1732.
0.02 units) were added and the solution was incubated at 37 °C
for 3 h.
An aqueous solution of NaIO4 (100 µL, 20 mM) was added to
a solution of N2-(1-methyl-3,4-dihydroxybutyl)guanine-modified
oligodeoxynucleotide (5 A260 units) in 0.05 M phosphate buffer,
pH 7 (300 µL) and the reaction mixture was stirred at room
temperature for 10 min (20). The reaction resulted in two peaks
in the HPLC trace representing the two isomers of the crotonal-
dehyde adduct. The two isomers were of approximately equal
size and were separated by HPLC (gradient B, 0.1 M aqueous
ammonium formate/acetonitrile); retention times of the two
peaks were 18.34 and 18.99 min. The combined yield was 3.8
A260 units (∼80%). The structures were confirmed by enzymatic
hydrolysis as described above and by MALDI-TOF MS: calcd.
for [M - H]-, 2479.5; found for peak 1, 2479.9; found for peak
2, 2479.9. HPLC analysis of the enzyme hydrolysate (gradient
A) showed that the earlier eluting peak (less lipophilic) con-
tained the 6(S) isomer of the crotonaldehyde adduct (compound
3S).
3-(2-Deoxy-â-D-er yth r o-p en tofu r a n osyl)-8-h yd r oxy-6(S)-
m et h yl-5,6,7,8-t et r a h yd r op yr im id o[1,2-a ]p u r in -10(3H )-
on e) (3S). Sodium periodate (500 µL, 20 mM) was added to a
solution of N2-(1(S)-methyl-3(R and S),4-dihydroxybutyl)deoxy-
guanosine (9S, 2 mg, 0.0054 mmol) in 0.05 M phosphate buffer,
pH 7 (300 µL) (20). The reaction mixture was stirred at room
temperature for 5-10 min. HPLC analysis showed complete
disappearance of the peaks for the starting material. The
reaction mixture was purified by HPLC to give 1.60 mg (88%)
of the 6(S) proximal crotonaldehyde adduct (3S). 1H NMR
(DMSO-d6) δ 7.83 (m, 2H, N2-H, H-2), 6.55 (bs, 1H, C8-OH),
6.12 (m, 1H, H-8), 6.03 (dd, 1H, H-1′, J 1 ) J 2 ) 6.8 Hz), 5.17 (d,
1H, 3′-OH, J ) 3.4 Hz), 4.84 (dd, 1H, 5′-OH, J 1 ) J 2 ) 5.5 Hz),
4.26 (bs, 1H, H-3′), 3.73 (m, 1H, H-4′), 3.63 (m, 1H, H-6), 3.45
(m, 2H, H-5′, H-5′′), 2.40 (m, 1H, H-2′), 2.10 (m, 1H, H-2′′), 1.95
(m, 1H, H-7), 1.38 (m, 1H, H-7), 1.15 (m, 3H, CH3). HRMS
(FAB+) [M + H]+: calcd. 338.1464; found, 338.1466.
Resu lts
The 6(S) diastereomer (3S) of the crotonaldehyde
adduct was synthesized enantiospecifically from the
commercially available (S)-3-aminobutanoic acid (4S) as
shown in Scheme 2a. Benzoylation and esterification of
4S followed by reduction with DIBAL gave aldehyde 5S,
which was converted to benzamidopentene 6S by Wittig
olefination with methyltriphenylphosphonium bromide
and n-butyllithium. Glycolation of the olefin with OsO4
and N-methylmorpholine N-oxide followed by hydrolytic
removal of the benzoyl group gave 4(S)-amino-1,2-pen-
tanediol 7S. The introduction of the hydroxy group at C-2
by this procedure is nonstereospecific; however, the
presence of diastereomers at C-2 is of no consequence
since the position is subsequently oxidized. Reaction of
7S with O6-TMSE-protected 2-fluoro deoxyinosine (8) led
to (1S) N2-(1-methyl-3,4-dihydroxybutyl)-2′-deoxygua-
nosine (9S) (20). NMR spectra of 9S recorded in dry
DMSO-d6 showed signals for four types of hydroxyl
groups (two deoxyribosyl and two side chain hydroxyls)
as well as signals for N2-H and N1-H, which indicated
the condensation had occurred via the amino group of
the aminodiol. All signals could be individually assigned
via a COSY spectrum. Oxidation with NaIO4 gave alde-
hyde 10S which cyclized spontaneously to 3S.
Syn th esis of a Mixtu r e of 3R a n d 3S. (() N-Benzoyl
2-amino-4-pentene (6RS) (26) was subjected to oxidation with
OsO4 followed by base hydrolysis to give a mixture of the four
stereoisomers of 4-amino-1,2-pentanediol. Condensation of ami-
nodiol 7RS with O6-trimethylsilylethyl-2-fluoro-deoxyinosine (8)
gave a mixture of the four diastereomers of N2-(1-methyl-3,4-
dihydroxybutyl)-deoxyguanosine (9). Purification was achieved
by HPLC without separation of the diastereomers. The mixture
was oxidized with sodium periodate to form diastereomers 3R
and 3S of the proximal crotonaldehyde adduct. The isomers were
separated using gradient A (retention times of 19.76 and 20.34
min). The earlier eluting diastereomer was identical to the 3S
diastereomer prepared from 4(S)-amino-1,2(R and S)-pen-
tanediol. Accordingly, the late eluting diastereomer is compound
3(R).
Syn th esis of Cr oton a ld eh yd e-Mod ified Oligod eoxyn u -
cleotid e. The preparation of O6-TMSE-2-fluorodeoxyinosine
phosphoramidite and oligonucleotides containing the corre-
sponding nucleobase is described elsewhere (27, 28). The O6-
TMSE-2-fluorodeoxyinosine-modified oligonucleotide was pre-
pared on
a 1 µmol scale using p-tert-butylphenoxyacetyl-
The analogous synthesis of the diastereomer 3R could
not be performed conveniently because (R)-3-aminobu-
tyric acid is not commercially available. As an alternative,
a nonstereospecific synthesis was carried out starting
from racemic 6RS (Scheme 2b) (26). Treatment with
OsO4 gave a mixture of the four diastereomers of 7, which
was condensed with O6-TMSE-protected 2-fluoro deoxyi-
nosine (8) to give a mixture of the four diastereomers of
9. Preparative HPLC was used to isolate 9 without
attempting to separate diastereomers. The diastereomers
of the N2-(1-methyl-3,4-dihydroxybutyl)deoxyguanosine
had nearly superimposable 1H NMR spectra. NMR
spectra of the mixture recorded in dry DMSO-d6 showed
signals for four types of hydroxyl groups (two deoxyri-
bosyl and two side chain hydroxyls) as well as peaks for
N2-H and N1-H, which indicated the condensation had
occurred via the amino group of the aminodiol. All signals
were individually assigned via a COSY spectrum. The
mixture was subjected to oxidation of the diol with
periodate to give diastereomers 3R and 3S, which were
easily separated by reversed-phase HPLC. Circular
dichroism spectra of 3R and 3S are shown in Figure 1;
protected phosphoramidites (PE/Biosystems Expedite reagents).
Cleavage from the solid support and deprotection were carried
out by treatment with 0.1 M NaOH (∼10 h at room tempera-
ture). The neutralized crude material was purified by HPLC
(YMC ODS-AQ column, 250 × 10 mm) using gradient A with
0.1 M aqueous ammonium formate and acetonitrile. The O6-
TMSE-2-fluoroinosine-modified oligodeoxynucleotide (10 A260
units) was mixed in a plastic test tube with diisopropylethyl-
amine (50 µL), DMSO (100 µL), and mixed diastereomers of
4-amino-1,2-pentanediol (0.5 mg). The reaction mixture was
stirred at 55 °C for 3 h. Solvents were evaporated under vacuum.
The residue was dissolved in 5% acetic acid (500 µL) and stirred
for 2 h at room temperature to remove the O6-TMSE group. The
mixture was neutralized with 1 M NaOH and purified by HPLC
to give 8.9 A260 units (∼89%) of the corresponding N2-(1-methyl-
3,4-dihydroxybutyl)guanine-modified oligodeoxynucleotide. The
product was characterized by HPLC analysis of the enzymatic
hydrolysate and MALDI-TOF MS: calcd for [M - H]- 2511.5,
found 2511.3. The enzymatic hydrolysis was carried out in one
step as follows: oligonucleotide (0.5 A260 units) was dissolved
in 20 µL of buffer (pH 7, 0.01 M Tris‚HCl, 0.01 M MgCl2), DNase
I (Promega, 5 units), alkaline phosphatase (Sigma P-4252, 1.7
units), and snake venom phosphodiesterase I (Sigma P-6877,

Crotonaldehyde-Deoxyguanosine Adducts
Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1509
Sch em e 2
and Hecht as the 6(S) enantiomer and the later (peak 5)
as the 6(R) isomer, the elution order on reversed-phase
HPLC being the same as we have found.
The NMR spectra of 3R and 3S were fully in accord
with the assigned structure. The amount of uncyclized
aldehyde present in the samples was below the level of
detection by NMR. The one-dimensional NMR spectra
were in good agreement with reported spectra for cro-
tonaldehyde adducts (8, 15). The earlier studies had
established the hydroxyl group to be trans to the methyl
group in both diastereomers; no attempt was made to
confirm that aspect of the structure. Long-range coupling
between the methine proton (H8, δ 6.12 ppm) and the
carbonyl carbon (C10, δ 157.0 ppm), observed in an
HMBC two-dimensional NMR spectrum (Figure 2), in-
dicated cyclization had occurred via N1 rather than N3
in confirmation of earlier assignments.
F igu r e 1. Circular dichroism spectra of 3S and 3R. Spectra
were obtained in water at 25 °C.
The absolute configuration of the carbon to which the
methyl group is attached in the two diastereomers had
never been established; in the present investigation the
synthesis from aminodiol of known configuration made
it possible to establish that the less lipophilic of the two
diastereomers has the S configuration at the methylated
site (C-6). The configuration at C-8 in this isomer can
also be assigned as S by virtue of the trans relationship
between the methyl group and the hydroxy group.
The synthetic method was extended to preparation of
site-specifically adducted oligonucleotides (Scheme 3).
The 8-mer 5′-d(AGGCXCCT), in which X represents
nucleoside 8, was treated with a mixture of the four
the isomers exhibited opposing bands at 309 nm with the
R isomer having the positive band and the S the negative.
Chung and Hecht in their early work on identification
of the crotonaldehyde adducts of dGuo isolated two
adducts (designated as peak 4 and peak 5); when these
were deglycosylated by acid treatment the resulting
guanine adducts gave mirror image CD spectra with
opposing bands at 280 nm (3). The spectra shown in
Figure 1 are not quite mirror images because the adducts
with the deoxyribose attached are diastereomeric rather
than enantiomeric. Comparison of the spectra reported
in this paper with the earlier spectra allows identification
of the earlier eluting peak (peak 4) isolated by Chung

1510 Chem. Res. Toxicol., Vol. 14, No. 11, 2001
Nechev et al.
Sch em e 3
F igu r e 2. HMBC spectrum of compound 3S. The cross-peak
at the bottom of the spectrum (indicated by the arrow) repre-
sents the correlation between H-8 and C-10 shown in the inset
structure.
F igu r e 3. HPLC profiles of purified adducted oligonucleotides
(A) d(AGGC-3S-CCT) and (B) d(AGGC-3R-CCT). (C and D)
HPLC profiles of enzymatic hydrolysates of the two oligonucle-
otides. In each case, the HPLC trace of an authentic sample of
the corresponding nucleoside adduct is superimposed.
diastereomers of aminodiol 7. The resulting oligonucle-
otide containing nucleosides 9R and 9S was purified as
a single fraction by reversed-phase HPLC and, after
characterization by MALDI-TOF mass spectrometry and
by nuclease digestion to establish the presence of nucleo-
sides 9R and 9S, treated with NaIO4. The resulting
oligonucleotides containing the 3R and 3S diastereomers
of the crotonaldehyde adduct were readily separated from
each other and purified by reversed-phase HPLC (Figure
3). They were characterized by MALDI TOF mass
spectrometry. Enzymatic digestion yielded 3 plus the
normal deoxynucleosides in the expected molar ratios.
The HPLC traces for the deoxynucleoside mixtures
derived from the two oligonucleotides are shown in
Figure 3 along with the chromatograms of the authentic
samples of 3R and 3S. The faster eluting oligonucleotide
yielded deoxynucleoside 3 having the S configuration at
the site of attachment of the methyl group.
Discu ssion
The present synthesis of crotonaldehyde-adducted oli-
godeoxynucleotides will now make it possible to investi-
gate individually the mutagenic properties of the two
diastereomers of the crotonaldehyde adduct. The results
of these studies will be of much interest relative to the
recently reported studies of the corresponding acrolein
adduct which showed negligible mutagenic activity in
point mutation assays (16, 17). The structure and con-
formation of the crotonaldehyde adducts in duplex DNA
may be significantly different from those of the acrolein
adduct. Whereas de los Santos found the acrolein adduct
to undergo ring opening in duplex DNA to form the

Crotonaldehyde-Deoxyguanosine Adducts
Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1511
Sch em e 4
cleavage of this diastereomer again yielded the 6(R)
diastereomer of crotonaldehyde adduct 3. This result
provides support of the hypothesis that both acetalde-
hyde-derived crotonaldehyde adducts arise from a com-
mon intermediate which is formed mainly by nucleophilic
attack on the si face of the Schiff base (see Scheme 4). In
the formation of 3 directly from crotonaldehyde, attack
occurs on the â carbon of the aldehyde with a small
preference for the si face to yield the 6(S) isomer.
Ack n ow led gm en t. We wish to acknowledge techni-
cal assistance by Ms. Pamela Tamura and Amanda
Wilkinson and generous financial support by the Na-
tional Institutes of Health (ES00267, ES05355, and
ES07781).
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