Synthesis and characterization of DNA containing O6-carboxymethylguanine

Article abstract of DOI:10.1016/S0040-4020(00)00556-1

O⁶-Carboxymethylguanine was formed in DNA treated with N-nitrosoglycocholic acid and believed to be implicated in human gastrointestinal and colorectal tumour. An efficient method is presented here for synthesis of oligodeoxynucleotides containing O⁶-carboxy-methylguanine at pre-determined positions. The synthetic protocol also allows for production of oligomers containing O⁶-aminocarbonyl-methylguanine. These guanine-modified oligomers have been fully characterized and could provide a useful tool for biological studies of these modified bases. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00556-1

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 6075±6081
Synthesis and Characterization of DNA
Containing O6-Carboxymethylguanine
*
Yao-Zhong Xu
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
Received 15 February 2000; revised 17 May 2000; accepted 8 June 2000
AbstractÐO6-Carboxymethylguanine was formed in DNA treated with N-nitrosoglycocholic acid and believed to be implicated in human
gastrointestinal and colorectal tumour. An ef®cient method is presented here for synthesis of oligodeoxynucleotides containing O6-carboxy-
methylguanine at pre-determined positions. The synthetic protocol also allows for production of oligomers containing O6-aminocarbonyl-
methylguanine. These guanine-modi®ed oligomers have been fully characterized and could provide a useful tool for biological studies of
these modi®ed bases. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
methods to prepare DNA containing modi®ed bases at pre-
determined positions and successfully used these modi®ed
DNA for repair assays and other biological studies.5 Here
we wish to report an ef®cient method for chemical prepa-
ration of oligodeoxynucleotides containing O6-CMG.
Chemical alkylation of DNA bases is believed to play an
important role in the induction of cancer. For instance the
formation of O6-methylguanine or O4-methylthymine has a
causative role in carcinogenesis by N-nitroso compounds.1
A recent work showed that N-nitrosoglycocholic acid
(NOGC), a nitrosated bile acid conjugate, reacted with
DNA to give rise to several base-adducts including O6-
methylguanine and O6-carboxymethylguanine (O6-CMG).2
It has been suggested in a recent paper3 that O6-methyl-
guanine observed as a product of the reaction of NOGC
and DNA may rise through decarboxylation of O6-CMG,
however the mechanism remains to be clari®ed. Recently
studies of the cytotoxicity of azaserine in human cells4
showed that it produced DNA containing carboxymethyl-
ated bases (including O6-CMG) and O6-methylguanine and
that the carboxymethylated bases are the more signi®cant
contributor to azaserine lethality in human cells.4 O6-
Methylguanine is well established as a toxic and promuta-
genic lesion and can be repaired by O6-alkylguanine-DNA
alkyltransferase (MGMT). However, O6-CMG is not a
substrate for MGMT.2,4 The O6-CMG analogue, S6-
carboxymethylthioguanine in a synthetic DNA was not
recognized by proteins of post-replication DNA mismatch
repair system.4 However, it is not yet known whether O6-
CMG in DNA can be recognized by other DNA repair
systems since DNA containing O6-CMG was not available.
To understand the mechanism of the toxicity of O6-CMG
towards human cells, the availability of DNA containing
O6-CMG at pre-determined positions would be of great
use. In the past decade, we have developed various chemical
Results and Discussion
Preparation of N2-phenylacetyl-O6-methoxycarbonyl-
methyl-20-deoxyguanosine (4) and its phosphoramidite
monomer (5)
The synthetic route is shown in Scheme 1 and primarily
based on the preparation of O6-methoxycarbonylmethyl-
20-deoxyguanosine [i.e. O6-carboxymethyl-20-deoxyguano-
sine, methyl ester].6 However, acetyl groups instead of
methoxyacetyl groups6 were used in our synthesis for the
protection of the hydroxyl groups of the nucleoside, and our
choice of protecting groups is probably more cost-ef®cient.
Therefore 30,50-diacetyl-N2-phenylacetyl-20-deoxyguano-
sine (1) was ®rst converted into 30,50-diacetyl-N2-phenyl-
acetyl-6-mesitylenesulphonyl-20-deoxyguanosine (2). 2 has
been previously prepared, but not isolated.7 In this work, the
intermediate (2) was ®rst puri®ed and then converted into its
6-quinuclidinium salt, which in turn was replaced by methyl
glycolate. The step for the puri®cation of 2 did provide a
better yield of the product 3. The acetyl groups protecting
the sugar of the nucleoside were selectively removed by
triethylamine in methanol. This treatment did not remove
the phenylacetyl group protecting the N2-amino group of
the guanine and the desired product (4) was obtained by
simple evaporation of the reagents and crystallisation from
methanol. This procedure is preferred over the alternative
deprotection using aqueous NaOH for a short period,7
because the de-protection with triethylamine in methanol
Keywords: O6-carboxymethylguanine; O6-methylguanine; modi®ed
guanine; DNA synthesis.
* Tel.: 120-7679-2327; fax: 120-7679-7193; e-mail: ucbcyzx@cl.ac.uk
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00556-1

6076
Y.-Z. Xu / Tetrahedron 56 (2000) 6075±6081
Scheme 1. i. 2-Mestiylsulphonyl chloride. ii. (a) Quinuclidine, (b) Methyl glycolate and DBU. iii. 0.5 M Triethylamine in MeOH. iv. (a) DMT-Cl,
(b) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.
is chemically speci®c and there is less the risk of removing
the phenylacetyl group on the base. The product (4) was
then reacted with DMT-Cl and phosphitylated by standard
protocols7b to give the required monomer (compound 5).
agents including 0.05 M K2CO3 in methanol, concentrated
ammonia and 0.5 M aqueous NaOH.10 Table 1 shows the
distribution of nucleoside derivatives after overnight treat-
ment of 4 at room temperature with each of these agents.
With K2CO3 in methanol,11 the deprotection was not
complete and it gave rise to O6-methyldeoxyguanosine
(8), an unwanted product. Further exposure to this depro-
tecting agent would lead to formation of even greater
amounts of O6-methyldeoxyguanosine. Deprotection with
concentrated ammonia produced mainly an amide,
N2-phenylacetyl-O6-aminocarbonylmethyldeoxyguanosine
(9b).12 The conversion of the ester (4) into the amide form
(9) was rapid while the removal of phenylacetyl group from
4 by ammonia was very slow due largely to the poor solu-
bility of the resultant amide (9b) in the aqueous ammonia.
However both amides (9a and 9b) could be readily and
quantitatively converted into their carboxylate forms (7)
by the treatment with aqueous alkaline solution. Deprotec-
tion of the amides with aqueous NaOH gave the desired
product, O6-carboxymethyl deoxyguanosine (7a) in very
good yields.
Synthesis and deprotection of oligomers containing
O6-CMG
Oligomers containing O6-CMG were synthesized by an ABI
391 DNA synthesizer with the manufacturer's standard
protocol except the modi®ed monomer (5) was added
manually and the coupling time for the modi®ed monomer
increased to 3 min to ensure a better coupling yield. Based
upon the measurement of DMT-cleaving solution, the
coupling yield of the modi®ed base was no different from
those of unmodi®ed monomers.
In the conventional synthesis of DNA, an isobutyryl group
is generally used for the protection of the N2-amino of
guanine, however it has been documented that an acyl
group at the N2-position of O6-substituted guanine is
much more resistant to removal by ammonia than that at
N2-position of unmodi®ed guanine.7,8 For this reason base-
labile groups such as the phenylacetyl group9 have been
used for N2 protection of O6-alkylguanine7 and therefore
this was chosen for the protection of O6-CMG in this syn-
thesis. Prior to synthesis of DNA containing O6-CMG, we
examined deprotection of 4 with various deprotecting
We then examined the deprotection step at the oligomer
level. A pentamer containing O6-CMG (i.e. CGXAT,
where X is O6-CMG) was deprotected in 0.5 M aqueous
NaOH overnight at room temperature and more than 90%
of the product eluted as a single peak on HPLC. This
product, after puri®cation, was enzymatically digested and
Table 1. Distribution of nucleosides when 4 was treated with deprotecting agents10
6a16b
4515
7a17b
30115
.95
8a18b
,5
9a19b
10190
K2CO3/MeOH
NH3
0.5 M NaOH

Y.-Z. Xu / Tetrahedron 56 (2000) 6075±6081
6077
Figure 1. Reverse phase HPLC pro®les of the nucleosides from enzymatic digestion of synthetic pentamers: (a) CGXAT, XˆO6-carboxymethyl-20-deoxy-
guanosine; (b) CGYAT, YˆO6-aminocarbonylmethyl-20-deoxyguanosine. See Ref. 13 for the analysis conditions.
found to have O6-CMG nucleoside and four standard
nucleosides (Fig. 1a), con®rming the pentamer has the
right composition. It is worth noting that the alkaline depro-
tection did not remove the carboxymethyl group from the
6-position of the guanine. A possible explanation is that the
alkaline solution ®rst hydrolyses the ester and resultant
negatively charged carboxylate anion prevents further
nucleophilic attack by hydroxide on the 6-position of the
guanine.
could be converted readily into O6-CMG by alkaline treat-
ment, and Fig. 2 shows a typical time course of such a
conversion.
As shown above, deprotection with aqueous alkali satisfac-
torily produced oligomer containing O6-CMG. This result is
agreed with our early ®nding14 that aqueous alkaline
solution can be used as a simple deprotecting agent when
base-labile groups such as the phenoxyacetyl group are used
to protect guanine and adenine and the isobutyryl group to
protect cytosine. A possible risk with the alkaline solution is
that it might transform N6-acyladenine and N4-acylcytosine
into hypoxanthine and uracil respectively. We have exam-
ined this possibility by deprotection of a synthetic 12 mer
[CGC XAG CTC GCG (X: O6-CMG)] with 0.5 M aqueous
NaOH for 1 day, 2 days or 3 days,15 and found that all
deprotection methods produced the desired oligomer and
that there was no substantial difference between these pro-
cedures. Form our data, deprotection with 0.5 M aqueous
NaOH for 2 days should be suf®cient and without the risk to
damage the bases of the oligomers. With this protocol for
deprotection, longer oligomers (such as 34 mer) containing
O6-CMG were also successfully prepared. These successful
results probably depended upon our use of base-labile
groups for the protection of the amino groups of all naturally
occurring base monomers (from Glen research) as the
Deprotection of synthetic oligomers is commonly done by
using concentrated ammonia. As mentioned above, depro-
tection of the modi®ed nucleoside (4) was limited by its
poor solubility in ammonia. However the oligomer was
soluble in aqueous ammonia, but removal of the phenyl-
acetyl group on N2-position of O6-methoxycarbonylmethyl-
guanine was still slow. The result is consistent with previous
reports7,8 that acyl groups in O6-alkylguanine are more
resistant to ammonia deprotection. Complete removal by
aqueous ammonia of the protecting groups from the
oligomer was achieved by incubation for 5 days at room
temperature. However this had converted the modi®ed
oligomer into the oligomer containing O6-aminocarbonyl-
methylguanine (Fig. 1b). This may provide a simple method
to produce oligomers containing O6-aminocarbonylmethyl-
guanine. O6-Aminocarbonylmethylguanine in oligomers
Figure 2. The time course of conversion of pentamer A [CGYAT, YˆO6-aminocarbonylmethylguanine) into pentamer B [CGXAT, XˆO6-carboxymethyl-
guanine]: Oligomer A was dissolved in 0.5 M aqueous NaOH. The solution at the speci®ed time was analyzed by reversed phase HPLC (for conditions, see
Ref. 19).

6078
Y.-Z. Xu / Tetrahedron 56 (2000) 6075±6081
guanine. Deprotection of modi®ed guanines with various
agents were thoroughly examined at the both nucleoside
and oligomer levels. Treatment of the synthetic oligomers
with aqueous alkali gave the desired modi®ed oligomers
while treatment with concentrated ammonia gave oligomers
containing O6-aminocarbonylmethylguanine. The latter
could be easily converted to O6-carboxymethylguanine.
These guanine-modi®ed oligomers have been fully charac-
terized and are now being used for biological studies.
Experimental
Chemicals and general methods
Syntheses of oligodeoxynucleotides were carried out by
ABI 391 DNA synthesizer (Applied Biosystems), using
Ultramild monomers (from Glen research) and supports in
which the amino group of dA is protected with the phenoxy-
acetyl group, that of dG protected with the iso-propyl-
phenoxyacetyl group and that of dC protected with the
acetyl group. All other chemicals were from either Aldrich
or Sigma and used directly without further puri®cation
unless stated otherwise. General methods such as puri®-
cation with Nensorb Prep cartridges (Du Pont) or Fast
protein liquid chromatography (FPLC) on a Mono Q 5/5
column (Pharmacia),16 nucleoside composition analysis by
reversed phase HPLC were carried as described before.17
Figure 3. Liquid chromatogram on a NucleoPac column at pH 12 of
synthetic 12 mers with the sequence CGC XAG CTG GCG, where X is
O6-methylguanine in P1; X is O6-carboxymethylguanine in P2; X is
guanine in P3 respectively. These identical oligomers with one base
being different were well separated (for conditions, see Ref. 20).
base-labile groups were removed very rapidly and resultant
adenine and cytosine in the oligomer are more stable
towards the alkaline solution.
Characterization of oligomers containing O6-CMG
Oligomers containing O6-CMG, synthesized and depro-
tected as above, were recovered with a Nensorb cartridge
(from Dupont) and then puri®ed with liquid chroma-
tography. Short oligomers (e.g. pentamers) were puri®ed
with reversed phase HPLC19 which can effectively separate
a pentamer containing O6-CMG from the pentamer con-
taining O6-aminocarbonylmethylguanine (see Fig. 2). For
oligomers of medium or longer length (e.g. 12 mer or 34
mer), anion exchange liquid chromatography is preferred.16
An eluent of pH 12 gave a good separation of a 12 mer
containing O6-CMG from the same 12 mer containing O6-
methylguanine or the parent containing guanine (Fig. 3).
This separation of the O6-CMG oligomer from the O6-
methylguanine oligomer could be ascribed to the fact that
the former has an extra charge from the carboxylate anion.16
At pH 12 the oligomer containing O6-CMG has the same
number of charges as its parent containing guanine.
However the later elution of the parent oligomer may be
due to its relatively high hydrophobicity compared to the
O6-CMG oligomer as we noted before that oligomers
containing thioguanine (which is believed to be more hydro-
phobic than guanine) were always eluted later that their
parents.16 These good separations should be very useful
for monitoring possible de-carboxylation and further de-
methylation of O6-CMG in DNA repair processes.
30,50-Diacetyl-N2-phenylacetyl-6-mesitylenesulphonyl-
20-deoxyguanosine (2). 30,50-Diacetyl-N2-phenyl acetyl-
20-deoxyguanosine (3 g, 6.4 mmol), prepared as described
before7 was dissolved in dichloromethane (100 mL), to
which triethylamine (3 mL), dimethylaminopyridine
(30 mg) and 2-mesitylenesulphonyl chloride (1.5 g,
6.8 mmol) were added sequentially. TLC (10% CH3OH/
CHCl3) was used to monitor the reaction. After 30 min,
about two thirds of the starting material (Rfˆ0.55) was
converted to a new spot (Rfˆ0.85). Then 0.5 g of 2-mesityl-
enesulphonyl chloride was supplemented and the solution
was left stirred for further a 30 min by which period the
starting material was almost consumed. Then the solution
was concentrated to a small volume and applied to a silica
gel column and eluted with chloroform. The fractions
containing the product were pooled together and concen-
trated to a small volume for crystallization. The crystals
formed were washed with diethylether and collected by
®ltration and dried to give crystals of 3.9 g (yield: 93%).
UV lmaxˆ279 nm. 1H NMR Data (in DMSO-d6): 2.14 (3H,
s, acetyl at 50), 2.21 (3H, s, CH3 at 4-position of mesityl-
sulphonyl), 2.29 (3H, s, acetyl at 30), 2.62 (6H, s, 2£CH3 at 2
and 6 positions of mesitylsulphonyl), 2.63±3.11 (2H, m, 20-
H and 200-H), 3.93 (2H, s, CH2 of phenylacetyl), 4.31±4.38
(3H, m, 40-H and 50-H), 5.44 (1H, d, 30-H), 6.36 (1H, t, 10-
H), 7.36±7.47 (7H, m, C6H5 of phenylacetyl and 2£H at 2
and 5 positions of mesitylsulphonyl), 8.48 (1H, s, 8-H) and
12.10 (1, s, N2±H, ex).
Puri®ed modi®ed oligomers were enzymatically digested
and analyzed by reversed phase HPLC in which the peak
for modi®ed nucleoside was observed and co-eluted with
authentic sample. These procedures establish a method to
produce oligomers containing the desired modi®ed
guanines.
30,50-Diacetyl-N2-phenylacetyl-O6-methoxycarbonylmeth-
yl-20-deoxyguanosine (3). Compound 2 (2.9 g, 4.46 mmol)
was dissolved in acetonitrile (20 mL, pre-dried over molec-
ular sieves), to which quinuclidine (0.77 g, 6 mmol) was
added. The solution was stirred at room temperature.
In this paper, an ef®cient method is reported for chemical
synthesis of oligomers containing O6-carboxymethyl-

Y.-Z. Xu / Tetrahedron 56 (2000) 6075±6081
6079
After 30 min, TLC (10% CH3OH/CHCl3) showed all of the
starting material was converted into a new spot with its Rf
being near zero. Then, methyl glycolate (2 mL, 25 mmol)
and 1,8-diazabicyclo(5,4,0)undec-7-ene [DBU] (0.6 mL,
3.9 mmol) were added. After 2 h, about 80% of the inter-
mediate was converted into another new spot (Rfˆ0.8).
Then additional methyl glycolate (0.5 mL) and DBU
(0.3 mL) were added and the solution was left stirred over-
night. Then the solution was concentrated to a small volume
and diluted with ethyl acetate (100 mL), washed with
saturated aqueous NaCl (3£100 mL). The organic layer
was dried over Na2SO4, ®ltered and concentrated to an
oily residue. The residue was co-evaporated with toluene
and puri®ed by silica gel column chromatography. The
desired product was eluted with chloroform, collected and
dried to give the title compound 3 (in foam, 1.8 g, 75%). UV
lmaxˆ267 nm; nma1x(KBr) 3446 (br) (N±H), 1743, 1616,
1456, 1234 cm21; H NMR Data (in DMSO-d6): 1.98 (3H,
s, acetyl at 50), 2.08 (3H, s, acetyl at 30), 2.56±3.18 (2H, m,
20-H and 200-H), 3.67 (3H, s, OCH3), 3.79 (2H, s, CH2 of
phenylacetyl), 4.20±4.31 (3H, m, 40-H and 50-H), 5.19 (2H,
s, CH2 of O6-CH2O), 5.40 (1H, m, 30-H), 6.34 (1H, t, 10-H),
7.20±7.35 (5H, m, C6H5), 8.48 (1H, s, 8-H) and 10.67 (1H,
s, N2±H, ex). MS m/z (FAB positive ion) 542 (45, MH1),
342 (100, MH12sugar fragment), 224 (25, MH12sugar
fragment2Pac group); HRMS (FAB positive ion): MH1,
found 542.1906. C25H28O9N5 requires 542.1887.
and the solution was cooled in an ice-bath. 10 mg of
dimethylaminopyridine and 340 mg of dimethoxytrityl
chloride (DMT-Cl) were added. TLC (ethyl ether, then
5% CH3OH/CHCl3) was used to monitor the reaction, and
when required, additional amount of DMT-Cl was added to
complete the reaction. The reaction was then quenched by
addition of 0.5 mL of methanol. After work-up, the product
was separated by silica gel column chromatography (0±
1.5% methanol in chloroform) and dried to give a foam.
The foam was precipitated with toluene in cold pentane
and dried to give a slightly yellowish powder (480 mg,
65%). The DMT-derivative was then converted into the
title compound 5 by a standard protocol described before.18
In brief: the above product 50-O-(4,40-dimethoxytriphenyl-
methyl)-N2-phenylacetyl-O6-methoxycarbonylmethyl-20-
deoxyguanosine was dissolved in 5 mL of dried THF and
0.6 mL of N,N-diisopropylethylamine. Under stirring,
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.2
mL) was added. The mixture was stirred for half a hour at
room temperature by which period the starting material was
almost completely converted to two higher Rf products as
checked by TLC (dichloromethane/ethyl acetate/diiso-
propylethylamine 75:25:2). The mixture was diluted with
30 mL of ethyl acetate and washed with saturated
NaHCO3 (3£30 mL). The organic phase was dried over
Na2SO4 and evaporated to a small volume, then co-evapo-
rated with toluene to an oily residue. The residue
was puri®ed by chromatography on silica gel eluting
with dichloromethane/ethyl acetate/diisopropylethylamine
(75:25:2). The fractions containing the desired products
were combined, evaporated and precipitated with toluene
into cold pentane, and freeze-dried with benzene to give a
while powder (120 mg, 46%).
N2-Phenylacetyl-O6-methoxycarbonylmethyl-20-deoxy-
guanosine (4). Compound 3 (1.7 g, 3.15 mmol) was
dissolved in 25 mL of 0.4 M triethylamine in methanol.
TLC (10% CH3OH/CHCl3) followed the deacylation
course. The acetyl groups of the starting material 3
(Rfˆ0.8) were gradually removed to form three new spots
(Rfˆ0.73 and Rfˆ0.62 for the 30-deacetylated and 50-
deacetylated products, and Rfˆ0.35 for the 30,50-dideacety-
lated product). Overnight, all the starting material was
converted into a single spot (Rfˆ0.35). The solution was
concentrated to a small volume, then co-evaporated with
absolute alcohol (once) and toluene (twice) to give a
white foam. The residue was crystallized from methanol
to give white crystals (650 mg from ®rst crop and 250 mg
from second crop, yield: 62.5%). Mp 129±1318C; [found:
C, 54.47; H, 5.18; N, 14.78. C21H23O7N5 requires C, 55.14;
H, 5.04; N, 15.32]; UV lmaxˆ269 nm; nmax(KBr) 3420±
Modi®ed nucleosides: 6, 7, 8 and 9
Compound 6a [O6-methoxycarbonylmethyl-20-deoxyguano-
sine] was prepared by a similar procedure as reported
before6 except that in our synthesis acetyl groups instead
of methoxyacetyl groups6 were employed for the protection.
In brief: 30,50-diacetyl-20-deoxyguanosine, prepared as
described before7a, was converted into 30,50,-diacetyl-O6-
methoxycarbonylmethyl-20-deoxyguanosine by reacting
the starting material with 2-mesitylsulphonyl chloride and
then with methyl glycolate/DBU in a similar protocol to that
used to prepare compound 3 (see above). Then the resultant
product 30,50,-diacetyl-O6-methoxycarbonylmethyl-20-deoxy-
guanosine (200 mg) was dissolved in 5 mL of 1 M triethyl-
amine in methanol. After 20 min at room temperature,
besides the starting material (Rfˆ0.75), there were three
UV spots (Rfˆ0.7, 0.6 and 0.3) as shown by TLC (10%
CH3OH/CHCl3). The de-acylation was complete overnight
as TLC showed a single UV spot (Rfˆ0.3). Thus, the reac-
tion solution was evaporated under reduced pressure and
lyophilized with benzene to give a white powder 6a. The
UV and NMR data of the product 6a are in agreement with
those published6 for O6-methoxycarbonylmethyl-20-deoxy-
guanosine. Compound 6b [N2-phenylacetyl-O6-methoxy-
carbonylmethyl-20-deoxyguanosine] is the same compound
as named 4.
1
3354 (br) (O±H, N±H), 1705,1620, 1450, 1234 cm21; H
NMR Data (in DMSO-d6): 2.30±2.70 (2H, m, 20-H and 200-
H), 3.57 (2H, m, 50-H), 3.64 (3H, s, OCH3), 3.80 (2H, s, CH2
of phenylacetyl), 3.84 (1H, m, 40-H), 4.41 (1H, d, 30-H),
4.91 (1H, t, 50-OH, ex), 5.18 (2H, s, CH2 of O6-CH2O),
5.32 (1H, d, 30-OH, ex), 6.34 (1H, t, 10-H), 7.23±7.33
(5H, m, C6H5), 8.51 (1H, s, 8-H) and 10.67 (1H, s, N2±H,
ex). MS m/z (FAB positive ion) 458 (47, MH1), 342 (100,
MH12sugar fragment), 224 (30, MH12sugar fragment2
Pac group); HRMS (FAB positive ion): MH1, found
458.1692. C21H24O7N5 requires 458.1676.
50-O-(4,40-Dimethoxytriphenylmethyl)-N2-phenylacetyl-
O6-methoxycarbonylmethyl-20-deoxyguanosine-30-O-(2-
cyanoethyl-N,N-diisopropylamino)-phosphoramidite (5).
Compound 4 was converted to its DMT derivative by a
standard procedure18. In brief: 450 mg of the protected
nucleoside 4 was dissolved in 10 mL of anhydrous pyridine
Compound 7a [O6-carboxymethyl-20-deoxyguanosine] was
prepared either by hydrolysis of 6a (Rmˆ7.2 min, in

6080
Y.-Z. Xu / Tetrahedron 56 (2000) 6075±6081
HPLC21) with 4 mM Ca(OH)2 as reported before6 or by
enzymatic de-esteri®cation with esterase [EC 3.1.1.1 from
Porcine liver from Sigma] in 0.55 M phosphate buffer (pH
8.0). Both protocols gave the same compound (Rmˆ4 min)
as analyzed by HPLC21 Compound 7b [N2-phenylacetyl-
O6-carboxymethyl-20-deoxyguanosine, Rmˆ8.2 min in
HPLC21) was produced by the same enzymatic de-esteri®-
cation of 4 (Rmˆ12 min in HPLC21).
manufacturer's standard protocol except the modi®ed
monomer was added manually and its coupling time
increased to 3 min to ensure a better coupling yield. The
CPG support bearing the synthesized oligomers was treated
with 0.5 M aqueous NaOH (2 days at RT) for production of
oligomer containing O6-carboxymethylguanine or with
concentrated ammonia (5 days at RT) for production of
O6-aminocarbonylmethylguanine respectively. The resul-
tant oligomers were ®rst recovered by Nensorb Prep
cartridges according to the manufacturer's instruction. For
further puri®cation, short oligomers (e.g. 5 mer) was puri-
®ed by reversed phase HPLC with 0.05 M KH2PO4 buffer at
pH 6.6 and acetonitrile,19 and longer oligomers (12 mer and
34 mer) were puri®ed by FPLC with 0.4 to 1.2 M aqueous
NaCl (pH 12) similar to that reported before.16
Compound 8a [O6-methyl-20-deoxyguanosine] and 8b
[N2-phenylacetyl-O6-methyl-20-deoxy guanosine] were
prepared as reported before.7
Compound 9a [O6-aminocarbonylmethyl-20-deoxyguano-
sine] and compound 9b [N2-phenylacetyl-O6-aminocarbonyl-
methyl-20-deoxyguanosine] were prepared by ammonolysis
of 4. Compound 4 (45 mg) was placed in 5 mL of concen-
trated aqueous ammonia and the solution vigorously stirred
at room temperature for 10 min during which time the clear
solution became a white suspension. TLC (20% CH3OH/
CHCl3) showed that the starting material (Rfˆ0.7) were
almost completely converted to a major spot (Rfˆ0.5, for
9b) and a minor spot (Rfˆ0.3 for 9a). The suspension was
spun with centrifuge. The supernatant was removed, and the
white precipitate was washed with water (5 times) and dried
under reduced pressure to give white powder compound 9b
(35 mg, 80%). Compound 9a was prepared in the same way
as above except that the suspension was vigorously stirred
overnight to remove the phenylacetyl group from N2-
position of the nucleoside. By this protocol 40 mg of 9a
(white powder) was obtained from the ammonolysis of
60 mg of 4. Data for 9a: Rfˆ0.33 (in 20% MeOH/CHCl3);
Rmˆ5.5 min (HPLC21); UV: lmaxˆ284 and 247 nm;
Conversion of O6-aminocarbonylmethylguanine
oligomers to O6-carboxymethylguanine oligomers
O6-Aminocarbonylmethylguanine oligomers prepared as
above were dissolved in 0.5 M aqueous NaOH, and the
conversion was monitored either by reversed phase HPLC
(for 5 mer)19 or by FPLC (for 12 mer).16
Nucleoside composition analysis
Oligomers, after puri®cation, were digested by incubation
with nuclease P1 in a solution of pH 4.5 overnight at 378C,
then with alkaline phosphatase in a solution of pH 8 for 2 h
at 378C. The digest was analyzed with reversed phase
HPLC13 as reported before.14,18
n
max(KBr) 3420 (br) (O±H, N±H), 1693, 1624,1245 cm21
;
Acknowledgements
1H NMR Data (in DMSO-d6): 2.29±2.73 (2H, m, 20-H and
200-H), 3.47±3.56 (2H, m, 50-H), 3.81 (1H, m, 40-H), 4.34
(1H, d, 30-H), 4.82 (2H, s, CH2 of O6-CH2O), 4.99 (1H, t, 50-
OH, ex), 5.28 (1H, d, 30-OH, ex), 6.20 (1H, t, 10-H), 6.41
(2H, br, NH2 at N2, ex), 7.34±7.39 (2H, br, NH2 of CONH2,
ex) and 8.12 (1H, s, 8-H). MS m/z (FAB positive ion): 325
(35, MH1) 209 (70 MH12sugar fragment), 136 (88);
HRMS (FAB positive ion): MH1, found 325.1266.
C12H17O5N6 requires 325.1260. Data for 9b: Rfˆ0.5 (in
The author is most grateful to Prof. Peter Swann for his
encouragement and support. The author wishes to thank
Dr Timothy Waters for his valuable discussion and Dr
Peter Karran for his interest in the work. This project is
funded generously by the Cancer Research Campaign.
References
20% MeOH/CHCl3); Rmˆ8.7 min (HPLC21); UV: lmax
ˆ
268 nm; nmax(KBr) 3446±3225 (br) (O±H, N±H), 1695,
1. Swann, P. F. Mutat. Res. 1990, 233, 81±94.
2. Shuker, D. E. G.; Margison, G. P. Cancer Res. 1997, 57, 366±
369.
1
1579, 1251 cm21; H NMR Data (in DMSO-d6): 2.29±
2.73 (2H, m, 20-H and 200-H), 3.57±3.60 (2H, m, 50-H),
3.84 (3H, m, 40-H and CH2 of phenylacetyl), 4.42 (1H, d,
30-H), 4.92 (1H, t, 50-OH, ex), 4.94 (2H, s, CH2 of O6-
CH2O), 5.32 (1H, d, 30-OH, ex), 6.33 (1H, t, 10-H), 7.23±
7.52 (7H, m, C6H5 and NH2 of CONH2), 8.49 (1H, s, 8-H)
and 10.67 (1H, s, N2±H, ex). MS m/z (FAB positive ion):
443 (40, MH1), 327 (100, MH12sugar fragment) 270 (30,
MH12sugar fragment2alkyl), 209 (15, MH12sugar frag-
ment2Pac group); HRMS (FAB positive ion): MH1, found
443.1694. C20H23O6N6 requires 443.1679.
3. Harrison, K. L.; Jukes, R.; Cooper, D. P.; Shuker, D. E. G.
Chem. Res. Toxicol. 1999, 12, 106±111.
4. O'Driscoll, M.; Macpherson, P.; Xu, Y.-Z.; Karran, P. Carcino-
genesis 1999, 20, 1855±1862.
5. (a) Grif®n, S.; Branch, P.; Xu, Y.-Z.; Karran, P. Biochemistry
1994, 33, 4787±4793. (b) Sibghat-Ullah; Xu, Y.-Z.; Day, R. S.
Biochemistry 1995, 34, 7438±7442. (c) Swann, P. F.; Waters,
T. R.; Moulton, D. C.; Xu, Y.-Z.; Zheng, Q.-G.; Edwards, M.;
Mace, R. Science 1996, 273, 1109±1111. (d) Sibghat-Ullah;
Gallinari, P.; Xu, Y.-Z.; Goodman, M. F.; Bloom, L. B.; Jiricny,
J.; Day, R. S. Biochemistry 1996, 35, 12926±12932.
6. Harrison, K. L.; Fairhurst, N.; Challis, B. C.; Shuker, D. E. G.
Chem. Res. Toxicol. 1997, 10, 652±659.
Synthesis and puri®cation of oligodeoxynucleotides
containing O6-carboxymethylguanine or O6-amino-
carbonylmethylguanine
7. (a) Li, B. F. L.; Swann, P. F. Biochemistry 1989, 28, 5779±
5786. (b) Smith, C. A.; Xu, Y.-Z.; Swann, P. F. Carcinogenesis
1990, 11, 811±816.
Oligodeoxynucleotides containing modi®ed bases were
synthesized by an ABI DNA synthesizer with the

Y.-Z. Xu / Tetrahedron 56 (2000) 6075±6081
6081
8. Borowy-Borowski, H.; Chambers, R. W. Biochemistry 1987,
26, 2465±2471.
14. Xu, Y.-Z.; Zheng, Q.; Swann, P. F. J. Org. Chem. 1992, 57,
3839±3845.
9. Reese, C. B.; Skone, P. A. J. Chem. Soc. Perkin Trans. 1 1984,
1263±1271.
15. After synthesis, the CPG support bearing synthesized 12 mer
[CGC XAG CTC GCG (X: modi®ed G)] was divided into three
parts. Each part was placed in an Eppendorf tube, to which 1 mL of
0.5 M aqueous NaOH was added and the solution was left at room
temperature for a speci®ed period (e.g. 1 day, 2 days or 3 days)
respectively. The oligomer was recovered with Nensorb Prep
puri®cation cartridge and then analyzed by FPLC with alkaline
solution (pH 12) [85% of 0.4 M NaCl and 15% of 1.2 M NaCl]
(also see Ref. 16).
10. The general protocol for deprotection was carried out
as follows: N2-phenylacetyl-O6-methoxycarbonylmethyldeoxy-
guanosine (4) [2 mg] was placed in an Eppendorf tube, to which
a deprotecting agent (200 mL of 0.05 M K2CO3 in methanol, or of
concentrated ammonia or of 0.5 M aqueous NaOH) was added.
The reaction solution was left at room temperature overnight and
analyzed by reversed phase HPLC (for conditions, see Ref. 13) and
by TLC (in 20% MeOH/CHCl3).
16. Xu, Y.-Z.; Swann, P. F. Anal. Biochem. 1992, 204, 185±189.
17. Xu, Y.-Z. Tetrahedron 1998, 54, 187±196.
11. The producer of this agent (Glen Research, USA) recom-
mended that the agent could completely remove the protecting
groups from the oligomers made by Ultramild monomers (where
guanine is protected with isopropylphenylacetyl group) within 2 h
at room temperature. We also used 10% DBU in methanol for
deprotection of the modi®ed nucleoside (4) and found that about
one third of the product was an unwanted O6-methyldeoxyguano-
sine (8a).
12. The product, N2-phenylacetyl-O6-aminocarbonylmethyldeoxy-
guanosine (9b) was not soluble in aqueous ammonia and therefore
precipitated out. However, when the solution was vigorously
stirred, the product 9b was further deprotected to give O6-amino-
carbonylmethyldeoxyguanosine (9a) as the major product.
13. Analysis conditions: Column: 8NVC184m Radial-Pac
Cartridge from Waters, 1 mL/min, 260 nm. The column was eluted
by buffer A (0.05 M KH2PO4, pH 4.5) and buffer B (consisting of
67% buffer A and 33% CH3CN) with the following gradient: Time
(buffer B): 0 min (2%), 4 min (2%), 15 min (15%), 20 min (50%),
25 min (75%).
18. Xu, Y.-Z.; Zheng, Q.; Swann, P. F. Tetrahedron 1992, 48,
1729±1740.
19. Analysis conditions: Column: 8NVC184m Radial-Pac
Cartridge from Waters, 1 mL/min, 260 nm. The column was eluted
by buffer C (0.05 M KH2PO4, pH 6.6) and buffer D (consisting of
67% buffer C and 33% CH3CN) with the following gradient: Time
(buffer D): 0 min (10%), 30 min (40%).
20. Analysis conditions: NucleoPac PA-100 column from Dionex;
1 mL/min, 260 nm. The column was eluted by Eluent E (0.2 M
NaCl, pH 1.2) and Eluent F (1.2 M NaCl, pH 12) with the
following gradient: Time (Eluent F): 0 min (15%), 5 min (15%),
20 min (18%).
21. Analysis conditions: Column: 8NVC184m Radial-Pac
Cartridge from Waters, 1 mL/min, 260 nm. The column was eluted
by buffer A (0.05 M KH2PO4, pH 4.5) and buffer B) with the
following gradient: Time (buffer B): 0 min (15%), 5 min (50%),
8 min (70%), 15 min (70%), 17 min (15%).