Synthesis of 1,N6-ethano-2'-deoxyadenosin, a metabolite product of 1,3- bis(2-chloroethyl)nitrosourea, and its incorporation into oligomeric DNA

Article abstract of DOI:10.1021/jo980170i

1,N⁵-Ethano-2'-deoxyadenosine (1) is one of the adducts formed during DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)nitrosourea (BCNU), and was synthesized and incorporated into a site-specific deoxyoligonucleotide for the first time. The product 6-chloropurine-2'- deoxyriboside (11) was prepared in high yield by the reaction of 2'- deoxyinosine (6) with SOCl₂ which then was derivatized to give compound 12 using tert-butyldimethylsilyl chloride, which was reacted with 2- hydroxyethylamine to produce compound 13 in 86% yield. Reaction of 13 with (PhO)₃P⁺MeI^- in DMF gave the cyclized 1,N⁶-ethano derivative 10 in 67% yield. Desilylation of 10 with triethylamine trihydrofluoride in THF gave 1,N⁶-ethano-dA (1) in 91% yield. Tritylation of compound 1 with DMT⁺BF₄^- gave the 5'-O-DMT product 14 in 62% yield, which then was phosphitylated with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, which yielded a 1:1 mixture of the diastereoisomers 15 in 71% yield. This fully protected compound 15 was incorporated site-specifically into a 25-mer oligonucleotide. The coupling efficiency of ethano-dA phosphoramidite was 93%. Enzymatic hydrolysis and analysis by HPLC confirmed the incorporation of ethano-dA and base composition of the DNA oligomer. The latter is now under investigation for its biochemical and physical properties.

Full text of DOI:10.1021/jo980170i

J . Org. Chem. 1998, 63, 4385-4389
4385
Syn th esis of 1,N6-Eth a n o-2′-d eoxya d en osin e, a Meta bolic P r od u ct
of 1,3-Bis(2-ch lor oeth yl)n itr osou r ea , a n d Its In cor p or a tion in to
Oligom er ic DNA
H. Maruenda,† A. Chenna, L.-K. Liem,‡ and B. Singer*
Donner Laboratory, Lawrence Berkeley National Laboratory, University of California,
Berkeley, California 94720
Received J anuary 30, 1998
1,N6-Ethano-2′-deoxyadenosine (1) is one of the adducts formed during DNA reaction with the
antitumor agent, 1,3-bis(2-chloroethyl)nitrosourea (BCNU), and was synthesized and incorporated
into a site-specific deoxyoligonucleotide for the first time. The product 6-chloropurine-2′-
deoxyriboside (11) was prepared in high yield by the reaction of 2′-deoxyinosine (6) with SOCl2,
which then was derivatized to give compound 12 using tert-butyldimethylsilyl chloride, which was
then reacted with 2-hydroxyethylamine to produce compound 13 in 86% yield. Reaction of 13 with
(PhO)3P+MeI- in DMF gave the cyclized 1,N6-ethano derivative 10 in 67% yield. Desilylation of
10 with triethylamine trihydrofluoride in THF gave 1,N6-ethano-dA (1) in 91% yield. Tritylation
-
of compound 1 with DMT+BF4 gave the 5′-O-DMT product 14 in 62% yield, which then was
phosphitylated with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, which yielded a 1:1
mixture of the diastereoisomers 15 in 71% yield. This fully protected compound 15 was incorporated
site-specifically into a 25-mer oligonucleotide. The coupling efficiency of ethano-dA phosphoramidite
was 93%. Enzymatic hydrolysis and analysis by HPLC confirmed the incorporation of ethano-dA
and base composition of the DNA oligomer. The latter is now under investigation for its biochemical
and physical properties.
Ch a r t 1
In tr od u ction
1,3-Bis(2-chloroethyl)nitrosourea (BCNU) is one of a
family of therapeutic nitrosoureas compounds used in
cancer treatment. Reaction with DNA leads to several
adducts, including saturated exocyclic adducts of adenine,
cytosine, and guanine (1,N6-ethano-A; 3,N4-ethano-C;
N2,3-ethano-G; and 1,O6-ethano-G),1 which resemble the
exocyclic etheno adducts formed from the reaction of the
chemical carcinogen, vinyl chloride, with DNA.2 In
addition, a cross-linked DNA is formed by BCNU between
the N-1 of G and the N-3 of C (1-(3-cytosinyl)-2-(1-
guanyl)ethane),3 which is believed to be the chemostatic
event.4
All of the possible cyclic etheno derivatives have been
synthesized (1,N6-etheno-dA; 3,N4-etheno-dC; 1,N2-etheno-
dG; and N2,3-etheno-dG) and studied for their biochemi-
cal effects in defined oligonucleotides (reviewed by Le-
onard5). Of the possible cyclic ethano compounds, all
* To whom correspondence should be addressed. Tel.: (510) 642-
0637. Fax: (510) 486-6488.
† Present address: Pontificia Universidad Catolica del Peru. Seccion
Quimica. Apartado Postal 1761 Lima 100 Peru. E-mail: hmaruen@
pucp.edu.pe.
‡ Present address: Clinical Research Center, Singapore General
Hospital, Outram Road, Singapore 169608.
(1) Ludlum, D. B. Mutat. Res. 1990, 233, 117-126.
(2) Singer, B.; Holbrook, S. R.; Fraenkel-Conrat, H.; Kusmierek, J .
T. In The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and
Mutagenesis; Singer, B., Bartsch, H., Eds.; IARC Scientific Publication,
Oxford University Press: New York, 1986; pp 45-58.
(3) Ludlum, D. B. In The Role of Cyclic Nucleic Acid Adducts in
Carcinogenesis and Mutagenesis; Singer, B., Bartsch, H., Eds.; IARC
Scientific Publication, Oxford University Press: New York, 1986; pp
137-146.
have been characterized from the reaction of DNA with
BCNU.1 However, the first synthesis of an ethano
deoxynucleoside was only recently described by Zhang
et al., who synthesized and incorporated 3,N4-ethano-dC
(3) (Chart 1) into a site-specific oligonucleotide,6 which
enabled them to investigate how this type of adduct
affects replication. They reported that in vitro replication
(4) Buckley, N.; Brent, T. P. J . Am. Chem. Soc. 1988, 110, 7520-
7529.
(5) Leonard, N. J . ChemtractssBiochem. Mol. Biol. 1992, 3, 273-
297.
(6) Zhang, W.; Rieger, R.; Iden, C.; J ohnson, F. Chem. Res. Toxicol.
1995, 8, 148-156.
S0022-3263(98)00170-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/11/1998

4386 J . Org. Chem., Vol. 63, No. 13, 1998
Maruenda et al.
Sch em e 1
was completely blocked, indicating this adduct would be
lethal.7 However, no synthetic route has been described
for the synthesis of 1,N6-ethano-dA (1) and its incorpora-
tion into a site-specific oligonucleotide.
In this paper, we report for the first time the large-
scale synthesis of 1,N6-ethano-dA (1), its conversion to
the phosphoramidite 15 (Scheme 2), and site-specific
incorporation into a 25-mer deoxyoligonucleotide. This
oligonucleotide is currently being studied for its possible
mutagenesis and enzymatic repair as well as physical
properties.
of anhydrous potassium carbonate. However, only a very
low yield of the desired product (1-2%) was obtained.
Although later Zhang et al.6 successfully synthesized
3,N4-ethano-dC (3) (Chart 1), the procedure was not
suitable as a synthetic route to prepare the 1,N6-ethano-
dA (1). In using this procedure, it was observed that
when the silylated deoxyinosine 7 was treated with
4-chlorophenyl phosphorodichlorate in the presence of
1,2,4-triazole in pyridine (Scheme 1), a fluorescent mate-
rial was obtained as the main product, whereas the
desired 6-(3-nitro-1,2,4-triazol-1-yl) derivative 8 was
recovered only as a trace component.10 An earlier report11
suggested the formation of fluorescent N-(purin-6-yl)py-
ridinium salts, such as 9, by phosphorylations in pyri-
dine. It was also found that the substitution of the
3-nitro-1,2,4-triazol moiety of 8 by 2-chloroethylamine did
not proceed as expected, and upon heating only traces of
the cyclized product 10 in a complex mixture were
detected.10 Since our results showed that the spontane-
ous ring closure is not as easily attainable as in the case
of ethano-dC6, we did not continue this route.
However, using a new and different synthetic route,
the desired compound 1 was obtained in high yield and
purity. The 6-chloropurine-2′-deoxyriboside 11 was pre-
pared according to the procedure published by Robins and
Basom.12 This compound 11 was then derivatized to give
compound 12 in 86% yield using tert-butyldimethylsilyl
chloride (Scheme 2), which was reacted with 2-hydroxy-
ethylamine to produce compound 13 in 64% yield. Iodi-
nation of the â-hydroxyl group of 13 with methyltri-
phenoxyphosphonium iodide13 in DMF at room temper-
ature for 30 min led to the spontaneous cyclization of the
Resu lts a n d Discu ssion
To study the biochemical role of the 1,N6-ethano-2′-
deoxyadenosine (1) adduct, it was necessary to synthe-
size, on a large scale and in high yield, compound 1
(Scheme 1), to use as a precursor for the preparation of
the corresponding phosphoramidite. This was success-
fully incorporated in a satisfactory yield, site-specifically
in a defined 25-mer oligonucleotide.
Syn th esis of 1,N6-Eth a n o-2′-d eoxya d en osin e (1).
Ludlum’s laboratory has isolated and identified several
adducts resulting from the reaction of BCNU with
nucleosides and DNA.8 Among them is 1,N6-ethano-dA.
However, the yields and the quantities obtained by this
approach were very low. Therefore, a total synthetic
method with high yield was necessary for biological and
biophysical studies.
Initially, we attempted the synthesis of this compound
by modifying Tong and Ludlum’s9 procedure in which
3′,5′-O-bis(tert-butyldimethylsilyl)-2-deoxyadenosine was
heated with 1,2-dibromoethane in DMSO in the presence
(10) Low-resolution mass spectroscopy on purified material was
consistent with structure.
(11) Adamaiak, R. W.; Biala, E. New. Nucleic Acids Res. 1985, 13
(8), 2989-3003.
(12) Robins, M. J . and Basom, G. L. Can. J . Chem. 1973, 51, 3161-
3169.
(7) Zhang, W.; J ohnson, F.; Grollman, A. P.; Shibutani, S. Chem.
Res. Toxicol. 1995, 8, 157-163.
(8) Ludlum, D. B. Pharmac. Ther. 1987, 34, 145-153.
(9) Tong, W. P.; Ludlum, D. B. Biochem. Pharmacol. 1979, 28, 1175-
1179.

Sunthesis of 1,N6-Ethano-2′-deoxyadenosine
J . Org. Chem., Vol. 63, No. 13, 1998 4387
Sch em e 2. Syn th esis of th e P h osp h or a m id ite of 1,N6-Eth a n o-d A Ad d u ct
resulting N6-(2-iodoethyl) derivative, producing the 1,N6-
ethano-derivative 10 in 67% yield. Desilylation of the
latter was accomplished by a treatment with triethy-
lamine trihydrofluoride14 in THF. This protocol allowed
almost quantitative recovery of 1,N6-ethano-dA (1) in 91%
yield, whereas the typical tetrabutylammonium fluoride
silyl deprotection procedure6 necessitated several puri-
fication steps to free the product from tetrabutylammo-
nium fluoride residue. The 1,N6-ethano-dA (1) synthe-
5′-O-DMT product 14 in 62% yield in 1 h. Phosphityla-
tion of 5′-O-DMT-1,N6-ethano-dA (14) with 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite19 yielded a 1:1
mixture of the expected diastereoisomers 15 in 71% yield.
This product 15 was coevaporated three times from dry
benzene and kept in a vacuum desiccator over P2O5 and
NaOH pellets before being used to make the desired
oligonucleotide. The fully protected compound 15 was
used to synthesize a 25-mer oligonucleotide as shown in
the Experimental Section. The commercially available
normal phenoxyacetyl phosphoramidite nucleotides (PAC)
were used in the synthesis of this DNA oligomer and
followed standard phosphoramidite chemistry. The over-
all yield was 87%, and the coupling efficiency of 1,N6-
ethano-dA phosphoramidite was 93%. The oligonucle-
otide was then deprotected under aqueous conditions by
using 28-30% ammonia for 1.5 h at 65 °C and then
purified by HPLC. The use of the PAC protecting group
allowed the deprotection of DNA oligomer in a shorter
time (1.5 h) than the standard phosphoramidite, which
requires several hours (8-16 h).
1
sized by this method was analyzed by TLC, HPLC, H
NMR, 13C NMR, UV, electrospray/MS, FAB/MS, and
high-resolution MS (see the Experimental Section). The
UV of this compound 1 showed the same UV spectral
characteristics as that described by Tong and Ludlum:
3,9
pH 7 (λmax 262, λmin 235), pH 1 (λmax 262, λmin 235),
and pH 13 (λmax 269, λmin 239). The FAB/MS of product
1 showed a protonated molecular ion at m/z 580 MH+
+
and protonated base at m/z 162 BH2
. All the data
collected were in agreement with the structure of 1,N6-
ethano-dA (1). This compound proved to be stable to all
the reagents and conditions used in the automated DNA
synthesis, and therefore its phosphoramidite was pre-
pared.
The composition of the DNA oligomer was confirmed
after enzymatic digestion to nucleosides, followed by
HPLC analysis, which showed that the modified base
survived the conditions used in the DNA synthesizer and
the deprotection procedure (Figure 1).
This new oligonucleotide-containing 1,N6-ethano-2′-
deoxyadenosine is being used to obtain information about
the repair, mutagenesis, and structural properties of this
Syn th esis of th e P h osp h or a m id ite of 1,N6-Eth a n o-
2′-d eoxya d en osin e (15) a n d Its Site-Sp ecific In cor -
p or a tion in to a DNA Oligon u cleotid e. Compound 1
has two hydroxy groups (3′- and 5′-OH) on the sugar
moiety. To incorporate this adduct in an oligonucleotide,
all reactive hydroxy groups must be protected. The first
attempt to tritylate compound 1 according to the stan-
dard methods with DMTCl16,17 failed. However, the
treatment of compound 1 with DMT+BF4
yielded the
- 18
(16) Schaller, H.; Weimann, G.; Lerch, B.; Khorana, H. G. J . Am.
Chem. Soc. 1963, 85, 3821-3827.
(13) Saito, T.; Murakami, M.; Inada, T.; Hayashibara, H.; Fujii, T.
Purines. LIV. Chem. Pharm. Bull. 1993, 41 (3), 453-457.
(14) Pirrung, M. C.; Shuey, S. W.; Lever, D. C.; Fallon, L. A. Bioorg.
Med. Chem. Lett. 1994, 4, 1345-1346.
(15) Ogilvie, K. K.; Beaucage, S. L.; Schifman, A. L.; Theriault, N.
Y.; Sadana, K. L. Can. J . Chem. 1978, 56, 2770-2780.
(17) Chaudhary, S. K.; Hernandez, O. Tetrahedron Lett. 1979, 2,
99-102.
(18) Bleasdale, C.; Ellswood, S. B.; Golding, B. T. J . Chem. Soc.,
Perkins. Trans. 1 1990, 803-805.
(19) Bodepudi, V.; Shibutani, S.; J ohnson, F. Chem. Res. Toxicol.
1992, 5, 608-617.

4388 J . Org. Chem., Vol. 63, No. 13, 1998
Maruenda et al.
with solvent A linearly increased to 40% over 30 min at a flow
rate of 2 mL/min. System 2. Analysis of the enzyme digest
of the oligonucleotide was performed using a Supelcosil LC-
18-DB column (2.5 cm × 0.46 cm, 5 µm, Supelco, Inc.) with
0% solvent A and 100% solvent C. Solvent A was maintained
isocratic for 10 min and then was increased linearly to 12%
over 30 min, at a flow rate of 1 mL/min.
6-Ch lor o-9-[3′,5′-O-bis(ter t-bu tyld im eth ylsilyl)-2′-d eox-
yp en t a fu r a n osyl]p u r in e (12). 6-Chloro-9-(2′-deoxypen-
tafuranosyl)purine12 (11) (0.97 g, 3.6 mmol), tert-butyldimeth-
ylsilyl chloride (2.39 g, 4.4 equiv), and imidazole (2.16 g, 8.8
equiv) were dissolved in dry DMF (3 mL), and the mixture
was stirred at room temperature for 3 h. The solution was
then poured into 60 mL of water and extracted with CH2Cl2
(3 × 30 mL). The combined organic extracts were washed with
brine (30 mL) and dried over MgSO4, and the residue, obtained
after evaporation of the organic solvent, was purified by flash
column chromatography (CH2Cl2/MeOH/Et3N 20:0.1:0.1). The
product was obtained as a light oil in 86% yield (1.53 g): 1H
NMR δ 0.05 (s, 6H, Si(CH3)2), 0.07 (s, 6H, (CH3)2), 0.86 (s, 9H,
SiC(CH3)3), 0.90 (s, 9H, SiC(CH3)3), 2.45 (m, 1H, H-2′), 2.60
(m, 1H, H-2′), 3.81 (dd, 2H, H-5′), 4.02 (m, 1H, H-4′), 4.65 (m,
1H, H-3′), 6.45 (t, 1H, H-1′), 8.56 (s, 1H, H-8), 8.78 (s, 1H, H-2);
13C NMR δ -5.32, -5.52, -4.89, -4.71, 17.95, 18.39, 25.69,
25.91, 41.57, 43.53, 71.76, 84.87, 88.17, 132.31, 143.77, 150.81,
151.22, 151.81; HRMS calcd for C22H40N4O3Si2Cl [M + H+]
499.232 751, found 499.233 010; LRMS (FAB+) m/e 499 [M +
H]+, 155 [base + 2H]+; Rf 0.35 (CH2Cl2/MeOH/Et3N 20:0.1:
0.1).
F igu r e 1. HPLC profile of 2′-deoxynucleosides obtained as a
result of enzymatic digestion of the oligonucleotide that
contains 1,N6-ethano-dA (Experimental Section). HPLC was
performed using system 2. The retention times of the deoxy-
nucleosides are dC, 16.5 min; 1,N6-ethano-dA, 19.3; dG, 21.3
min; dT, 22.6 min; and dA, 24.9 min. The UV spectrum of 1,N6-
ethano-dA at pH 4.5 is shown in the inset.
6-N-Hyd r oxyla m in o-9-[3′,5′-O-b is(ter t-b u t yld im et h yl-
silyl)-2′-d eoxyp en ta fu r a n osyl]p u r in e (13). Compound 12
(0.77 g, 1.5 mmol) was dissolved in anhydrous methanol (7
mL). Ethanolamine (0.47 g, 5 equiv, 0.46 mL) was added
dropwise and the solution allowed to reflux for 2 h. The
solvent was evaporated under reduced pressure, and the
resulting oil was dissolved in CH2Cl2 (25 mL) and washed with
saturated brine (3 × 10 mL). The organic extract was dried
over MgSO4, and after filtration and concentration the residue
was purified by flash column chromatography (CH2Cl2/MeOH/
Et3N 19.25:0.75:0.1) to give 0.5 g (64%) of 13 as an off-white
oil: 1H NMR δ 0.05 (s, 6H, Si(CH3)2), 0.07 (s, 6H, Si(CH3)2),
0.86 (s, 9H, SiC(CH3)3), 0.90 (s, 9H, SiC(CH3)3), 2.43 (m, 1H,
H-2′), 2.58 (m, 1H, H-2′), 3.77 (m, 2H, H-5′), 3.86 (m, 2H, CH2-
NH), 3.89 (m, 2H, CH2OH), 3.99 (m, 1H, H-4′), 4.59 (m, 1H,
H-3′), 5.85 (br, 1H, CH2OH), 6.42 (t, 1H, H-1′), 6.94 (br, 1H,
CH2NH), 8.16 (s, 1H, H-8), 8.35 (s, 1H, H-2); 13C NMR δ -5.60,
-5.52, -4.94, -4.76, 17.87, 18.29, 25.63, 25.84, 41.32, 43.60,
61.89, 62.48, 71.42, 84.23, 87.67, 110.39, 119.63, 138.04,
152.76, 154.80; HRMS calcd for C24H46N5O4Si2 [M + H]+
524.308 837, found 524.308 490 LRMS (FAB+) m/e 524 [M +
H]+, 180 [base + 2H]+; Rf 0.26 (CH2Cl2/MeOH/Et3N 19.25:0.75:
0.1).
adduct. This should lead to a better understanding of
the cytotoxic and carcinogenic mechanisms of the effect
of BCNU.
In summary, 1,N6-ethano-dA, which is one of many
adducts formed from the reaction of the chemotherapeu-
tic agent 1,3-bis(2-chloroethyl)nitrosourea (BCNU) and
DNA, was synthesized on a large scale for the first time
and converted to its phosphoramidite. This compound
was then incorporated into a site-specific deoxyoligo-
nucleotide to be used for biochemical and physical stud-
ies.
Exp er im en ta l Section
Gen er a l P r oced u r es. 1H, 13C, and 32PNMR spectra were
recorded in CDCl3, unless stated otherwise. Fast atom bom-
bardment (FAB) mass spectra were run in glycerin or thioglyc-
erin as matrix. Ultraviolet spectra were recorded in a spec-
trophotometer using 0.5 cm cuvettes. TLC was performed on
EM 5735/7 silica gel 60, F254 plates. Flash column chroma-
tography was carried out using silica gel 60, eluting with the
solvents indicated. Compounds characterized in this manner
were homogeneous by TLC analysis and gave NMR spectra
indicative of their purity. 6-Chloro-9-(2′-deoxypentafuranosyl)-
purine12 and 4,4′-dimethoxytrityl tetrafluoroborate18 were
prepared following published procedures. 2′-Deoxyinosine was
purchased from Sigma. Methylene chloride, benzene, pyridine,
and triethylamine were dried by distilling over CaH2. All other
anhydrous reagents and remaining chemicals were purchased
from Aldrich Chemical Co. (Milwaukee, WI) and used without
further purification.
1,N6-E t h a n o-9-[3′,5′-O-b is(ter t-b u t yld im et h ylsilyl)-2′-
d eoxyp en ta fu r a n osyl]p u r in e (10). Compound 13 (0.8 g, 1.5
mmol) was dissolved in anhydrous DMF (6 mL) containing
Et3N (5 equiv, 1.0 mL). The solution, cooled in ice bath, was
treated with methyltriphenoxyphosphonium iodide (1.6 g, 2.5
equiv) and stirred at room temperature for 45 min. The
mixture was then treated with anhydrous MeOH (2 mL) to
quench the residual phosphonium reagent. All solvents were
evaporated, and the oily residue was dissolved in CH2Cl2 (10
mL) and washed with NaHCO3 (5%, 5 mL) and saturated brine
(3 × 5 mL). The organic phase was then dried over MgSO4,
concentrated to dryness, and purified by column chromatog-
raphy (CH2Cl2/MeOH/Et3N 18:1:0.1) with a yield of 67% (0.52
g): 1H NMR (DMSO-d6) δ 0.06 (s, 6H, Si(CH3)2), 0.07 (s, 6H,
Si(CH3)2), 0.81 (s, 9H, SiC(CH3)3), 0.85 (s, 9H, SiC(CH3)3), 2.38
(m, 1H, H-2′), 2.78 (m, 1H, H-2′), 3.63 (dd, 1H, H-5′), 3.77 (dd,
1H, H-5′′), 3.87 (m, 1H, H-3′), 4.07 (t, 2H, J ) 9.5 Hz, CH2N-),
4.58 (m, 1H, H-4′), 4.68 (t, 2H, J ) 9.5 Hz, CH2Nd), 6.35 (t,
HP LC. Solvent systems included solvent A (acetonitrile),
solvent B (triethylammonium acetate; 0.1 M, pH 7.0), and
solvent C (potassium phosphate buffer; 0.01 M, pH 4.5).
System 1. The oligonucleotide was purified using a PRP-1
C-18 semipreparative reversed-phase column (Hamilton Co.).
The initial concentration was 10% solvent A, 90% solvent B,
1H, J ) 6.5 Hz, H-1′), 8.65 (s, 1H, H-8), 8.76 (s, 1H, H-2); 13
C
NMR δ -5.44, -5.33, -4.84, -4.63, 17.92, 18.38, 25.69, 25.94,
41.78, 44.17, 47.08, 62.69, 71.63, 85.30, 88.44, 117.01, 142.50,
143.52, 148.13, 151.11; HRMS calcd for C24H44N5O3Si2 [M +

Sunthesis of 1,N6-Ethano-2′-deoxyadenosine
J . Org. Chem., Vol. 63, No. 13, 1998 4389
H]+ 506.298 273, found 506.298 020; LRMS (FAB+) m/e 506
[M + H]+, 162 [base + 2H]+; Rf 0.30 (CH2Cl2/MeOH/Et3N 16:
1:0.1).
LRMS (FAB+) m/e 780 [M + H]+, 162 [base + 2H]+; Rf 0.24
(CH2Cl2/MeOH/Et3N 19:0.5:0.1).
Solid -P h a se Syn th esis of th e Oligon u cleotid e. The
phosphoramidite of compound 15 (65 mg) was coevaporated
three times from dry benzene then was dried over P2O5 and
NaOH pellets in a vacuum desiccator for 48 h at room
temperature. This material was dissolved under dry nitrogen
in 2 mL of dry acetonitrile and used to synthesize the DNA
oligomer. Synthesis of the oligonucleotide was performed on
an Applied Biosystem 392 automated DNA synthesizer on a
1 µmol scale using phosphoramidite chemistry on a controlled
pore glass (CPG) support with an aminopropyl succinate
linker. Phenoxyacetyl phosphoramidites (PAC) from Phar-
macia were used to synthesize the DNA oligomer. The
synthesis followed standard protocol of the DNA synthesizer
for phosphoramidite chemistry, except for the two steps in
which the modified deoxynucleoside and the base after were
inserted. In the steps where the modified nucleotide and the
following unmodified nucleotide were incorporated, the time
of coupling was increased to 16 min and 10 min, respectively,
to maximize the coupling efficiency, which was 93%. The
overall yield of the synthesis of the oligonucleotide was 87%,
which was recovered as the 5′-dimethyloxytritylated derivative
and deprotected with 28-30% aqueous ammonia for 1.5 h at
65 °C to cleave off the nucleobase protecting groups. The 5′-
DMT-oligomer was purified by HPLC and eluted at 20 min
using a PRP-1 C-18 semipreparative reversed-phase column
(Hamilton Co.) using system 1. Detritylation of the pure
oligomer was accomplished by the treatment with 80% acetic
acid for 20 min at room temperature and then was desalted
on a Sep-pack C-18 column. Finally, the product was lyoph-
ilized on a Speed-Vac evaporator and dissolved in 1 mL of
deionized water. The amount of DNA measured using UV
spectrometry was 19.6 A260 units/1 mL. The resulting fully
deprotected DNA containing the modified base was reanalyzed
by HPLC and found to be a single product that has the
following sequence:
1,N6-E t h a n o-9-(2′-d eoxyp en t a fu r a n osyl)p u r in e (1).
Compound 10 (0.26 g, 0.52 mmol), dissolved in anhydrous THF
(2 mL), was treated with triethylamine trihydrofluoride (332
µL, 2.01 mmol) and stirred at room temperature for 1 h. The
white slurry formed was evaporated to dryness, dissolved in
water, and neutralized with NaHCO3. The water was evapo-
rated, and the white solid was purified by flash column
chromatography (CH2Cl2/MeOH/Et3N 15:5:0.2) with a yield of
91% (0.13 g): 1H NMR (DMSO-d6) δ 2.31 (m, 1H, H-2′), 2.58
(m, 1H, H-2′), 3.56 (m, 2H, H-5′), 3.86 (q, 1H, H-4′), 4.41 (m,
1H, H-3′), 3.99 (t, 2H, J ) 10 Hz, CH2N-), 4.45 (t, 2H, J ) 9.6
Hz, CH2Nd), 5.02 (br, 1H, 5′- OH), 5.37 (br, 1H, 3′-OH), 6.31
(t, 1H, H-1′), 8.49 (s, 1H, H-8), 8.50 (s, 1H, H-2); 13C NMR (D2O;
TMS, ext standard) δ 39.03, 44.66, 48.22, 61.27, 70.78, 84.91,
87.51, 116.57, 143.42, 144.48, 148.96, 151.37; HRMS calcd for
C
12H16N5O3 [M + H]+ 278.125 315, found 278.125 920; LRMS
(ES+) m/e 278 [M + H]+, 162 [base + 2H]+; Rf 0.28 (CH2Cl2/
MeOH/Et3N 15:5:0.2).
1,N6-Eth a n o-9-[5′-O-(4,4′-d im eth oxytr ityl)-2′-d eoxyp en -
ta fu r a n osyl]p u r in e (14). Nucleoside 1 (25 mg, 0.09 mmol),
DMT+BF4- (35 mg, 1 equiv), and Li2CO3 (14 mg, 2 equiv) were
combined and dried under vacuum for 30 min. 2,6-Lutidine
(1 mL) was added, and the solution was allowed to stir at room
temperature for 3 h. The reaction was monitored by TLC,
which indicated the presence of starting material. More
DMT+BF4- (35 mg, 1 equiv) was then added, and the reaction
mixture was stirred at room temperature for another 3 h
period. The mixture was diluted with CH2Cl2 (10 mL) and
filtered over MeOH (1 mL). The solvents were evaporated,
and the residue was purified by flash column chromatography
(CH2Cl2/MeOH/Et3N 18.5:1.5:0.1) to yield 32 mg (62%) of a
colorless oil: 1H NMR δ 2.50 (m, 1H, H-2′), 2.83 (m, 1H, H-2′),
3.28 (m, 2H, H-5′), 3.69 (s, 6H, OCH3), 3.97 (m, 1H, H-4′), 4.10
(m, 1H, H-3′), 4.14 (t, 2H, J ) 4.6 Hz, CH2N-), 4.60 (t, 2H, J
) 5.4 Hz CH2Nd), 5.37 (br, 3′-OH), 6.28 (t, J ) 6.3 Hz, 1H,
H-1′), 6.71 (dd, J ) 8.9, 2.0 Hz, 4H, H ortho to OCH3), 7.09-
7.35 (m, 9H, ArH), 7.66 (s, 1H, H-8), 7.76 (s, 1H, H-2); 13C NMR
δ 40.59, 46.56, 52.28, 55.06, 63.83, 71.19, 84.24, 86.09, 86.23,
112.89, 119.64, 126.59, 127.60, 127.99, 129.92, 129.96, 135.65,
137.56, 143.17, 144.57, 151.46, 158.27; HRMS calcd for
5′-CCGCTXGCGGGTACCGAGCTCGAAT-3′
X ) 1,N6-Ethano-A
This sequence was originally taken from the polylinker region
of M13 phage. It has been used extensively in this laboratory
for mutagenesis and repair studies.
C
33H34N5O5 [M + H]+ 580.255 995, found 580.255 410; LRMS
To confirm the compostion of the oligomer, about 0.45 A260
units was dissolved in 44 µL of freshly deionized water, 0.8
µL of 1 M MgCl2, and 3.5 µL 0.5 M Tris-HCl buffer (pH 7.5)
and digested with snake venom phosphodiesterase (2.5 µL) and
bacterial alkaline phosphatase (4.0 µL) at 37 °C for 16 h. The
digested mixture was analyzed on a C-18 reversed-phase
HPLC Supelcosil column (250 × 4.5 mm) using system 2.
Quantitation of the deoxynucleosides was on the basis of
integration of the peak areas at 260 nm, which confirmed the
base composition and the incorporation of the adducts, as well
as the spectrum of 1,N6-ethano-dA (Figure 1).
(FAB+) m/e 580 [M + H]+, 162 [base + 2H]+, Rf 0.27 (CH2Cl2/
MeOH/Et3N 18.5:1.5:0.1).
1,N6-E t h a n o-9-[5′-O-(4,4′-d im et h oxyt r it yl)-3′-O-[(N,N-
d iisop r op yla m in o)(â-cya n oeth oxy)p h osp h in yl]-2′-d eoxy-
p en ta fu r a n osyl]p u r in e (15). The 5′-DMT-protected nucle-
oside 14 (68 mg, 0.12 mmol), dried over P2O5 and KOH over
high vacuum for 12 h, was dissolved in anhydrous CH2Cl2 (2
mL) and anhydrous Et3N (218 µL, 12 equiv) under N2
atmosphere. 2-Cyanoethyl N,N-diisopropylchlorophosphora-
midite (87 µL, 3 equiv) was added dropwise and the reaction
mixture stirred at room temperature for 1.5 h. It was then
treated with anhydrous MeOH (200 µL), diluted with CH2Cl2
(5 mL), and washed with brine (2 × 3 mL). The organic layer
was dried over MgSO4, evaporated to dryness, and purified
by flash column chromatography (CH2Cl2/MeOH/Et3N 19:0.5:
0.1) to give the product as a light yellow oil (65 mg) in 71%
yield: 31P NMR δ 149.37, 149.43 (diastereomeric pair); HRMS
calcd for C42H51N7O6P [M + H+] 780.363 846, found 780.365 110;
Ack n ow led gm en t. This work was supported by
Grant No. CA47723 from the National Institutes of
Health to B.S. and was administered by the Lawrence
Berkeley National Laboratory under Department of
Energy Contract No. DE-AC03-76SF00098.
J O980170I