Synthesis of oligonucleotides containing the alkali-labile pyrimidopurinone adduct, M1G

Article abstract of DOI:10.1021/tx990141i

An improved method for the synthesis of oligodeoxyribonucleotides containing the endogenous adduct, pyrimido[1,2-a]purin-10(3H)-one (M₁G), is reported. The key features of the methodology include improved synthesis of the deoxynucleoside of M₁G by transribosylation with deoxycytidine catalyzed by nucleoside 2'-deoxyribosyltransferase and the use of commercially available 4-tert-butylphenoxyacetyl protecting groups for normal nucleotides. Facile deprotection and removal of the M₁G-containing oligomers from the solid support were achieved by treatment with a solution of potassium carbonate in methanol. NMR studies were performed to determine the stability of the oligonucleotides at different pHs.

Full text of DOI:10.1021/tx990141i

90
Chem. Res. Toxicol. 2000, 13, 90-95
Syn th esis of Oligon u cleotid es Con ta in in g th e
Alk a li-La bile P yr im id op u r in on e Ad d u ct, M1G
Nathalie C. Schnetz-Boutaud,† Hui Mao,‡ Michael P. Stone,‡ and
Lawrence J . Marnett*,†,‡
Departments of Biochemistry and Chemistry, Center in Molecular Toxicology, and
Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37235
Received J uly 29, 1999
An improved method for the synthesis of oligodeoxyribonucleotides containing the endogenous
adduct, pyrimido[1,2-a]purin-10(3H)-one (M1G), is reported. The key features of the methodology
include improved synthesis of the deoxynucleoside of M1G by transribosylation with deoxy-
cytidine catalyzed by nucleoside 2′-deoxyribosyltransferase and the use of commercially
available 4-tert-butylphenoxyacetyl protecting groups for normal nucleotides. Facile deprotection
and removal of the M1G-containing oligomers from the solid support were achieved by treatment
with a solution of potassium carbonate in methanol. NMR studies were performed to determine
the stability of the oligonucleotides at different pHs.
However, AMB protecting groups are cumbersome to
incorporate into monomers and are not stable to exten-
sive storage (19). Therefore, we adapted commercially
available protecting groups to the synthesis of M1G-
containing oligonucleotides. In the course of this work,
we also developed a significantly improved synthesis of
the deoxynucleoside of M1G.
In tr od u ction
The synthesis of oligonucleotides containing structur-
ally defined adducts of carcinogens, drugs, and other
xenobiotics is critical to an evaluation of the structural
and genetic effects of DNA damage. Most synthetic
approaches to them include direct reactions of electro-
philes with oligonucleotides (1, 2) or assembly of oligo-
nucleotides from adduct-modified nucleosides (3-6). Our
laboratory is interested in the chemistry and biology of
DNA damage by the endogenous mutagen malondialde-
hyde (MDA)1 (7-9). MDA is formed enzymatically as a
byproduct of arachidonic acid metabolism, as well as
nonenzymatically during lipid peroxidation, and it reacts
with deoxynucleosides to produce a variety of adducts
(10-14). The most abundant adduct at physiological pH
is 3-(2′-deoxy-â-D-erythro-pentofuranosyl)pyrimido[1,2-a]-
purin-10(3H)-one (M1G-dR, 1). The existence of high
levels of M1G adducts in human tissues places a high
priority on studies of its mutagenic potential and repair
(15-18).
The instability of M1G-dR under the strongly alkaline
conditions typically used to deprotect synthetic oligode-
oxynucleotides was a major hurdle to its site-specific
incorporation into appropriate vectors (see Scheme 1).
Previous studies in our laboratory resulted in the first
successful synthesis of an oligonucleotide containing M1G
by using the 2-(acetoxymethyl)benzoyl (AMB) protecting
group on the free amino moieties of dG, dC, and dA (6).
AMB protecting groups are removable from nucleosides
by treatment with potassium carbonate (19), and M1G-
dR proved to be stable under these conditions (6).
Ma ter ia ls a n d Meth od s
Rea gen ts a n d En zym es. HPLC grade solvents were ob-
tained from Fisher (Pittsburgh, PA). Reagent grade chemicals
and deuterated solvents were obtained from Aldrich (Milwau-
kee, WI) and were used as received. Nucleoside 2-deoxyribosyl-
transferase (EC 2.4.2.6, transferase) was a generous gift from
S. Short (Glaxo-Wellcome Inc., Research Triangle Park, NC).
In str u m en ta l An a lysis. HPLC analyses were performed
with a Varian model 9010 solvent delivery system and a
Hewlett-Packard model 1040A diode array detector interfaced
with a Hewlett-Packard model 9000, Series 300 Chemstation.
Samples were injected onto an Ultrasphere ODS reversed phase
column (10 mm × 250 mm) from Beckman (Fullerton, CA) and
eluted at 2.5 mL/min. A gradient from 100 mM ammonium
formate (pH 6.4) to 50% acetonitrile was used as follows: from
0 to 5 min, linear gradient to 10% acetonitrile; from 5 to 20 min,
linear gradient to 15% acetonitrile; from 25 to 35 min, linear
gradient to 25% acetonitrile; from 35 to 40 min, linear gradient
to 35% acetonitrile; and from 40 to 45 min, linear gradient to
50% acetonitrile. The eluant was monitored at 260 and 320 nm.
UV spectra were recorded using a Hewlett-Packard UV/VIS
model 89500 spectrometer. Mass spectra were recorded on a
Finnigan TSQ 7000 triple-quadrupole spectrometer.
Syn th esis of M1G-d R. To a solution of guanine hydrochlo-
ride (1 g, 5.3 mmol) in 1 N HCl (100 mL) at 70 °C was added
dropwise a solution of tetraethoxypropane (1.3 mL, 5.86 mmol)
in methanol (1.25 mL). After addition was complete, the reaction
mixture was further stirred for 0.5 h at 70 °C. The reaction
mixture was allowed to cool to 0 °C and neutralized with K2-
CO3, and the unreacted guanine was filtered. 2-(N-Morpholino)-
ethanesulfonic acid (MES) was added to obtain a final concen-
tration of 0.5 M, followed by the addition of deoxycytidine (1.2
g, 5.3 mmol) to the solution. The pH of the reaction mixture
was adjusted to 6.0, and the transferase (230 µg) was added
(20, 21). The solution was incubated for 2 h at 37 °C with gentle
* To whom correspondence should be addressed: Department of
Biochemistry, Vanderbilt University School of Medicine, Nashville, TN
37232-0146. Phone: (615) 343-7329. Fax: (615) 343-7534. E-mail:
marnett@toxicology.mc.vanderbilt.edu.
† Department of Biochemistry.
‡ Department of Chemistry.
1
Abbreviations: MDA, malondialdehyde; transferase, nucleoside
2-deoxyribosyltransferase; M1G, pyrimido[1,2-a]purin-10(3H)-one; M1G-
dR, M1G-deoxyribose; AMB, 2-(acetoxymethyl)benzoyl; MES, 2-(N-
morpholino)ethanesulfonic acid; t-BPAC, tert-butylphenoxyacetyl.
10.1021/tx990141i CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/25/2000

M1G-Oligonucleotide Synthesis
Chem. Res. Toxicol., Vol. 13, No. 2, 2000 91
Sch em e 1
agitation, after which complete conversion to M1G-dR was
observed. The crude product was purified by chromatography
on silica gel (95:5 CH2Cl2/MeOH). Yield: 220 mg, 13%.
5′-O-(4,4′-Dim eth oxytr ityl)M1G-d R. The synthesis of the
dimethoxytrityl derivative was achieved by slight modification
of the previously reported procedure (22). M1G-dR (0.1 g, 0.33
mmol) was treated with anhydrous pyridine (10 mL), and the
pyridine was evaporated. This procedure was repeated twice,
and the residue was placed under vacuum overnight. Anhydrous
pyridine (10 mL) and diisopropylethylamine (0.34 mL, 1.98
mmol) were added to the flask containing M1G-dR, and the
mixture was stirred under argon. The solution was cooled to 0
°C, and 4,4′-dimethoxytrityl chloride (0.678 g, 1.32 mmol) was
added. The reaction mixture was allowed to warm slowly to
room temperature and was stirred for 18 h. The reaction was
quenched by the addition of methanol (100 µL) and the mixture
stirred for 10 min. The reaction mixture was concentrated under
vacuum, dissolved in CH2Cl2 (50 mL), and washed with 10%
K2CO3 (2 × 50 mL). The organic solution was dried (MgSO4),
filtered, and evaporated under vacuum. Chromatography over
silica gel (99.5:0.5 CH2Cl2/Et3N) yielded the desired product
(0.16 g, 80%). 1H NMR (CDCl3): δ 9.43 (dd, J 6,8 ) 2.3 Hz and
J 7,8 ) 7.2 Hz, 1H, H8), 8.91 (dd, J 6,8 ) 2.3 Hz and J 6,7 ) 3.8 Hz,
1H, H6), 8.12 (s, 1H, H2), 7.41-7.37 (m, 2H, aromatic), 7.30-
7.15 (m, 7H, aromatic), 7.08 (dd, J 6,7 ) 3.8 Hz and J 7,8 ) 7.2
Hz, 1H, H7), 6.80-6.74 (m, 4H, aromatic), 6.64 (t, J 1′,2′ ) 6.3
Hz, 1H, H1′), 4.65-4.62 (m, 1H, H3′), 4.20-4.18 (m, 1H, H4′),
3.75 [s, 6H, (OCH3)2], 3.51-3.34 (m, 2H, H5′ and H5′′), 2.71-
2.48 (m, 2H, H2′ and H2′′).
3′-O-[(N,N-Diisopr opylam in o)(2-cyan oeth yl)ph osph in yl]-
5′-O-(d im eth oxytr ityl)M1G-d R. The phosphoramidite reagent
was synthesized according to the procedure described previously
(22). 5′-O-(Dimethoxytrityl)M1G-dR (100 mg, 0.2 mmol) was
treated with anhydrous pyridine (10 mL), and the pyridine was
evaporated. This procedure was repeated twice, and then the
compound was placed under vacuum overnight. To a solution
of anhydrous 1H-tetrazole (20 mg, 0.26 mmol) was added a
solution of the tritylated compound (100 mg, 0.2 mmol) in dry
CH2Cl2 (3 mL), followed by 2-cyanoethyl-N,N,N′,N′-tetraisopro-
pyl phosphoramidite (95 µL, 0.3 mmol). The reaction mixture
was stirred under argon at room temperature for 3 h. The
mixture was treated with NaHCO3 (20 mL) and extracted with
CH2Cl2 (3 × 20 mL). The combined organic layer was dried
(MgSO4), filtered, and concentrated under vacuum. The residue
was purified by chromatography on silica gel (99.5:0.5 CH2Cl2/
Et3N) to afford pure 3′-O-[(N,N-diisopropylamino)(2-cyanoethyl)-
phosphinyl]-5′-O-(dimethoxytrityl)M1G-dR (120 mg, 76%). 31P
NMR (CDCl3): δ 149.42, 149.45.
Ch r om a togr a p h y Gel Electr op h or esis. Oligonucleotide
purity was evaluated on a Beckman P/ACE 2000 instrument
(Beckman) using “ssDNA 100” gel capillary and “TRIS-borate-
urea buffer” from the manufacturer. Samples were applied at
-10 kV and run at -15 kV (30 °C).
NMR Mea su r em en t s. The purified oligonucleotides were
desalted using a G25 column (Sigma, St. Louis, MO). The
collected fraction was lyophilized and redissolved in 0.5 mL of
buffer solution containing 0.1 M NaCl, 10 mM NaH2PO4, and
50 µM Na2EDTA at pH 6.8. The solutions were lyophilized and
exchanged three times with 99.96% D2O. The final sample was
dissolved in 0.5 mL of 99.996% D2O. The oligonucleotide
concentration was estimated to be 0.5 mM from an extinction
coefficient of 9.31 × 104 M-1 cm-1 calculated at 260 nm.
Experiments were performed on a Bruker DMX500 spectrom-
eter at a 1H frequency of 500.13 MHz at a temperature of 35
°C. One-dimensional spectra were recorded with a sweep width
of 6000 Hz and 32K data points. Two-dimensional spectra were
recorded with 512 real data points in the d1 dimension and 2048
real data points in the d2 dimension for COSY experiments.
Phase-sensitive TOCSY experiments were performed using a
mixing time of 80 ms and MLEV17 spin lock pulse with TPPI
phase cycling. Spectra were processed using FELIX (Biosym
Technologies) on Silicon Graphics Indigo2 workstations. The
data in the d1 dimension were zero-filled to give a matrix of 2K
× 2K real points. A sine-bell apodization function with a 90°
phase shift was used in both dimensions.
In ter con ver sion of M1G-d R a n d N2-(3-Oxo-1-p r op en yl)-
d G. The process of the interconversion between M1G and N2-
(3-oxo-1-propenyl)dG was investigated by recording the NMR
spectra of the sample as a function of pH and time. The pH of
the sample was adjusted by titration with a 10% (by volume)
NaOD or DCl solution. A 3 mm (diameter) micro pH probe
(Ingold, Inc.) was used to measure the pH in the NMR tube.
The spectra at different pHs were recorded using the same
acquisition parameters.
Resu lts a n d Discu ssion
Our previous synthesis of M1G-dR utilized a two-step
enzymatic transribosylation of M1G with the thymine
base of dT (23). The yields in this procedure were limited
by the requirement to purify the M1G base after its
synthesis from dG and tetraethoxypropane. Attempts to
convert the overall procedure to a one-pot reaction were
unsuccessful because of contamination by side products.
However, we found that a one-pot reaction could be
developed by using nucleoside 2′-deoxyribosyltransferase
for the transribosylation. This enzyme exchanges bases
for the cytosine of deoxycytidine, generating the deoxy-
riboside directly (Scheme 2). This enzyme was reported
to form only the N9 isomer of 9-â-D-2′-deoxyribofuranosyl-
1-deazapurine by Betbeder et al., and Muller et al. used
a partially purified enzyme to add deoxyribose to guanine
and modified DNA bases, including M1G, and found that
after 15 h, the coupling yield was 97% and the ratio of
Oligon u cleotid es. Oligonucleotides were synthesized on an
Expedite model 8909 Nucleic Acid Synthesis System (PerSeptive
Biosystems, Framingham, MA) on a 1 µmol scale using the
manufacturer’s standard protocol. The beads from four 1 µmol
cassettes were suspended in a 50 mM solution of K2CO3 in dry
methanol (4 mL) under argon and stirred slowly for 18 h. The
beads were allowed to settle; the supernatant was removed, and
the beads were washed with H2O (2 × 1 mL). The combined
aqueous fractions were neutralized cautiously with 1 M acetic
acid to pH 6 and filtered. The oligonucleotide was purified using
the HPLC system described above.

92 Chem. Res. Toxicol., Vol. 13, No. 2, 2000
Schnetz-Boutaud et al.
Sch em e 2
Sch em e 3a
a
(a) Tetraethoxypropane, 1 N HCl; (b) MES, transferase, dC; (c) DMtr chloride, pyridine; (d) 1H-tetrazole, 2-cyanoethyl-N,N,N′,N′-
tetraisopropyl phosphoramidite, CH2Cl2.
N9 to N7 was 85:15 (24, 25). We used pure nucleoside
2′-deoxyribosyltransferase and observed complete conver-
sion from the M1G base to M1G-dR, with no N7 isomer
formed. Purification by silica gel chromatography (two
times) and reversed phase medium-pressure liquid chro-
matography yielded pure M1G-dR. The modification not
only increased the yield of the synthesis but also de-
creased the time necessary for the synthesis and purifi-
cation.
methanol. To complete the cleavage of the oligonucle-
otide, the beads were washed with water. The same
deprotection procedure was used for the M1G-containing
oligonucleotides synthesized with the AMB protecting
group (6). The fully deprotected oligonucleotide was
analyzed and purified by HPLC injection on a reversed
phase column as described in Materials and Methods.
The purity was confirmed by capillary gel electrophoresis
(Figure 1).
Syn t h esis of M1G-Con t a in in g Oligon u cleot id es.
Cyanoethyl phosphoramidite 4 (Scheme 3) was prepared
by alkylation of the 5′-position of M1G-dR with 4,4′-
dimethoxytrityl chloride, followed by phosphitylation
with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphophoramid-
ite, as described by De Corte et al. (22). The phosphora-
midite 4 was then used to prepare oligonucleotides by
solid phase synthesis. The method used for the synthesis
of the oligodeoxyribonucleotide varied from the one
previously described by the use of the tert-butyl PAC
group instead of the AMB protecting group for unmodi-
fied nucleosides. PAC and alkyl-PAC protecting groups
have previously been used by Sinha et al. and Fujimoto
et al., who showed that they could be removed with K2-
CO3/methanol (26, 27).
NMR Ch a r a ct er iza t ion of d (GGTXTCCG) (X )
M1G). The spectrum of the M1G oligomer had no char-
acteristic resonance from the t-BPAC protecting group,
indicating complete deprotection. Two resonances at 1.58
and 1.71 ppm integrated for three protons each and were
assigned to the methyl groups of the two thymines
adjacent to M1G. A total of 11 resonances were observed
in the downfield region between 7 and 10 ppm that arise
from the base aromatic protons of each nucleotide, in
addition to three exocyclic ring protons of M1G. In a two-
dimensional TOCSY spectrum, two H5-H6 cross-peaks
from two cytosine residues, two CH3-H6 cross-peaks
from two thymine residues, and a total of eight sets of
H1′-H2′ and H1′-H2′′ cross-peaks were observed in
their corresponding regions. Although a complete assign-
ment was not determined, the observed aromatic protons
were assigned as the H6 protons of the two thymines at
An 8-mer, d(GGTXTCCG) where X ) M1G, was pre-
pared and was deprotected with 50 mM K2CO3 in dry

M1G-Oligonucleotide Synthesis
Chem. Res. Toxicol., Vol. 13, No. 2, 2000 93
Ta ble 1. Assign m en ts of th e Exocyclic Rin g P r oton s of M1G
M1G-dR
N2-(3-oxo-1-propenyl)dG
M1G oligomer
(ppm)
N2-(3-oxo-1-propenyl)dG
proton
nucleotidea(ppm)
nucleotidea (ppm)
oligomer (ppm)
∆δb (ppm)
H2c
H6
H7
H8
8.22
8.92
7.20
9.20
7.80
8.21
5.60
8.95
8.42
8.98
7.24
9.23
7.99
8.28
5.71
8.99
0.43
0.70
1.53
0.24
a
b
Obtained from ref 28. Chemical shifts were measured in DMSO-d6. ∆δ values are not calibrated for the pH change. c This proton
was numbered as H8 for deoxyguanosine. For the unmodified oligonucleotide, δH8 ) 8.01 ppm.
F igu r e 2. Expanded plot of a TOCSY spectrum showing the
scalar coupling correlation of exocyclic protons of M1G in 5′-
GGTXTCCG-3′ at pH 6.8 and 35 °C.
F igu r e 1. CGE profile of 5′-GGTXTCCG-3′.
7.42 and 7.47 ppm, the H6 of two cytosines showing as a
doublet at 7.66 and 7.72 ppm, and the H8 of the three
guanines at 7.87, 7.92, and 7.94 ppm, respectively. A
resonance at 8.40 ppm was assigned as H2 of M1G,
corresponding to H8 in guanine. These resonances dem-
onstrated the correct nucleotide composition of the oli-
gonucleotide.
The assignments of exocyclic ring protons of M1G were
determined by examining the scalar coupling correlation
of these protons in a series of COSY experiments. Figure
2 shows the expanded plot of a TOCSY spectrum. The
cross-peaks arising from the three M1G exocyclic ring
protons are labeled, and the assignments are in agree-
ment with a previous report (28). A TOCSY experiment
was also performed at pH 10 in order to assign the
protons of the N2 propenyl group for N2-(3-oxo-1-pro-
penyl)dG, an exocyclic ring-opened form of M1G (Figure
3). Examining the scalar coupling pattern gave the
assignments of 5.71, 8.28, and 7.99 ppm for H7, H6, and
H8, respectively. The chemical shifts of the methyls (1.59
and 1.72 ppm) of the two thymidines adjacent to the
modified base were slightly shifted, and the H6s of both
thymines merged into one resonance upon formation of
N2-(3-oxo-1-propenyl)dG. The chemical shifts of the exo-
cyclic ring protons are listed in Table 1.
F igu r e 3. Expanded plot of a TOCSY spectrum showing the
scalar coupling correlation of exocyclic protons of M1G in 5′-
GGTXTCCG-3′ at pH 10 and 35 °C. Both ring-closed and ring-
opened forms of M1G are apparent.
6.5 to 10, the magnitudes of the signals of the three
exocyclic ring protons, H6-H8, of the closed form (7.4,
8.9, and 9.4 ppm, respectively) decreased as the magni-
tudes of the three resonances corresponding to H6-H8
(5.71, 8.28, and 7.99 ppm, respectively) of the ring-opened
N2-(3-oxo-1-propenyl)dG increased. H2 (8.40 ppm) of M1G
disappeared, and a new resonance arose at 7.99 ppm,
which was assigned as H2 of the N2-(3-oxo-1-propenyl)-
dG oligomer. Figure 4 represents a set of spectra recorded
as a function of time after the pH was adjusted to 10.
The conversion of the M1G oligomer to the N2-(3-oxo-1-
propenyl)dG oligomer was slow; only about 50% of the
M1G oligomer converted to the N2-(3-oxo-1-propenyl)dG
oligomer after 30 min. About 90% of the M1G oligomer
converted after 5 h. However, the resonances from the
In ter con ver sion of M1G a n d N2-(3-Oxo-1-p r op en -
yl)d G. Increasing the pH resulted in M1G converting to
a ring-opened form, N2-(3-oxo-1-propenyl)dG (6). After the
sample was titrated with NaOD to bring the pH from

94 Chem. Res. Toxicol., Vol. 13, No. 2, 2000
Schnetz-Boutaud et al.
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as a function of pH, is slow compared with the ring
opening of a duplex M1G oligomer in which dC is opposite
the adduct and catalyzes the opening of M1G very rapidly
(29).
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Further evidence for the proposed structure of the
M1G-containing oligodeoxynucleotide was obtained by
LC/MS with electrospray ionization. The oligonucleotide
was analyzed by direct loop injection. The peaks corre-
sponding to m/z 823.4 and 1234.8 were observed and
represent 3- and 2- charged ions, respectively. The
theoretical molecular weight of this 8-mer is 2470.6, and
the molecular weight calculated from the ions described
above was 2469.9.
This report describes a straightforward procedure for
the generation of M1G-containing oligonucleotides from
readily available commercial materials. This provides a
convenient method for the generation of oligonucleotides
for a range of structural, biochemical, and genetic studies.
The 8-mer sequence described herein has been used
in in vivo mutagenesis and structural studies (29, 30),
but we also have synthesized 19-mers and 31-mers
for in vitro replication and repair experiments. Thus,
the synthetic operations described can be readily
adapted for different oligonucleotide lengths and
amounts.
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Crystal structures of nucleoside 2-deoxyribosyltransferase in
native and ligand-bound forms reveal architecture of the active
site. Structure 4, 97-107.
Ack n ow led gm en t. We thank Dr. G. R. Reddy for
valuable discussions, P. Horton for her technical as-
sistance with the oligonucleotide synthesis, and Dr. S.
Short for providing nucleoside 2-deoxyribosyltransferase.
This work was supported by a research grant from the
National Institutes of Health (CA47479).

M1G-Oligonucleotide Synthesis
Chem. Res. Toxicol., Vol. 13, No. 2, 2000 95
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