Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine

Article abstract of DOI:10.1021/tx9501795

Improved methodology has been developed for preparation of oligodeoxynucleotides bearing adducts on the N² position of guanine in which the adduction reaction is carried out in homogeneous solution rather than while the oligonucleotide is immobilized on a solid matrix. The methodology utilizes a new synthon, 2-fluoro-O⁶-(trimethylsilylethyl)-2'-deoxyinosine (3). Nucleoside 3 is stable to the conditions of oligonucleotide synthesis, but the O⁶ protection is eliminated under very mild conditions following displacement of the 2-fluoro group by amine nucleophiles. Oligonucleotides containing 3 could be removed from the solid support by treatment with 0.1 M NaOH (8 h, rt) without disruption of 3. Reaction of the crude, partially deprotected oligonucleotide with (R)-2-amino-2-phenylethanol in homogeneous solution, followed by removal of the remaining protective groups with NH₄OH (60 °C, 8 h) and then 0.1% acetic acid, gave the adducted oligonucleotide in good purity and yield. Alternatively, fully deprotected oligonucleotide containing 3 could be prepared by use of labile phenoxyacetyl-type protecting groups on the exocyclic amino groups.

Full text of DOI:10.1021/tx9501795

630
Chem. Res. Toxicol. 1996, 9, 630-637
Im p r oved Str a tegies for P ostoligom er iza tion Syn th esis
of Oligod eoxyn u cleotid es Bea r in g Str u ctu r a lly Defin ed
Ad d u cts a t th e N2 P osition of Deoxygu a n osin e
Bart L. DeCorte,† Dimitrios Tsarouhtsis, Satyanarayan Kuchimanchi,‡
Monica D. Cooper, Pamela Horton, Constance M. Harris,* and
Thomas M. Harris*
Chemistry Department and Center in Molecular Toxicology, Vanderbilt University,
Nashville, Tennessee 37235
Received October 18, 1995X
Improved methodology has been developed for preparation of oligodeoxynucleotides bearing
adducts on the N2 position of guanine in which the adduction reaction is carried out in
homogeneous solution rather than while the oligonucleotide is immobilized on a solid matrix.
The methodology utilizes a new synthon, 2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (3).
Nucleoside 3 is stable to the conditions of oligonucleotide synthesis, but the O6 protection is
eliminated under very mild conditions following displacement of the 2-fluoro group by amine
nucleophiles. Oligonucleotides containing 3 could be removed from the solid support by
treatment with 0.1 M NaOH (8 h, rt) without disruption of 3. Reaction of the crude, partially
deprotected oligonucleotide with (R)-2-amino-2-phenylethanol in homogeneous solution, followed
by removal of the remaining protective groups with NH4OH (60 °C, 8 h) and then 0.1% acetic
acid, gave the adducted oligonucleotide in good purity and yield. Alternatively, fully deprotected
oligonucleotide containing 3 could be prepared by use of labile phenoxyacetyl-type protecting
groups on the exocyclic amino groups.
oligomers containing styrene oxide adducts at the N6
position of deoxyadenosine. Extension of the strategy to
In tr od u ction
Oligodeoxynucleotides containing structurally defined
adducts of carcinogens, drugs, and other xenobiotics have
recently attracted much interest for structural and
biological studies. Major synthetic approaches to them
include direct reactions of electrophiles with oligonucle-
otides (1,2) and assembly of oligonucleotides from adduct-
modified nucleosides (3-5). Our interest in adducted
oligonucleotides involves mutagenic substances. In 1991,
we reported a postoligomerization strategy for the syn-
thesis of oligonucleotides containing styrene oxide ad-
ducts at the N6 position of adenine. This strategy
involves reversal of the customary electrophile-nucleo-
phile relationship of mutagen and DNA, i.e., with an
amino alcohol as a replacement for the epoxide in a
reaction with a 6-halopurine as a replacement for adenine
(6). Similar postoligomerization strategies using other
reactive nucleosides have been used by others for differ-
ent purposes (7,8). This method offers the advantage of
being completely regio- and stereospecific and is adapt-
able to any DNA sequence including those containing
multiple deoxyadenosines. The condensation reaction
was carried out with the oligonucleotide still attached to
the solid support used in its synthesis and with exocyclic
amino groups and phosphodiesters fully protected since
conditions needed to deprotect the oligonucleotide seemed
likely to lead to loss of the halo substituent. The reaction
of 6-chloropurine deoxynucleoside, within the immobi-
lized oligonucleotide, worked well for the preparation of
weaker nucleophiles required the use of a leaving group
better than chloride ion. Fluorine is more reactive than
chlorine in aromatic nucleophilic substitution reactions,
and 6-fluoropurine was found to have significantly higher
reactivity than the 6-chloro species (9). Oligonucleotides
bearing adenine N6 adducts of the carcinogenic bay-
region diol epoxides of benzo[a]pyrene were prepared by
reaction of the 6-fluoropurine oligonucleotides with ami-
notriols derived from the diol epoxides (10).
Our original publication also reported the preparation
of oligonucleotides adducted at the N2 position of guanine
using 2-fluorodeoxyinosine as the deoxyguanosine syn-
thon (6). However, the procedure proved to be somewhat
inferior to that for adenine adducts because the rate of
reaction with 2-amino-2-phenylethanol was significantly
reduced over the corresponding reaction of 6-halonucleo-
sides, and substantial quantities of byproducts were
formed. In spite of these difficulties, pure samples of
adducted oligonucleotides could be prepared. However,
extensive purification was required due to side reactions,
the principal one being nucleophilic attack by the amine
at the C4 position of N-acylated cytosine to give N4-
styrene oxide adducts of deoxycytidine. This observation
is in accord with earlier reports on the reactivity of
acylated cytosine with amine nucleophiles (7,11). With
the more reactive 6-halopurines, the cytosine side reac-
tion was not quantitatively significant. In the present
paper we describe synthetic strategies which improve
both the yield and purity of oligonucleotides containing
guanine adducts.
* Author to whom correspondence and reprint requests should
addressed at the Department of Chemistry, Vanderbilt University,
Nashville, TN 37235.
† Present address: J anssen Research Foundation, Spring House, PA
19477.
Ma ter ia ls a n d Meth od s
Gen er a l Meth od s. 1H NMR spectra were recorded at 200.13
‡ Present address: ChemGenes, Waltham, MA 02154.
X Abstract published in Advance ACS Abstracts, March 15, 1996.
and 300.13 MHz on
a Bruker AC 200 or AC 300 NMR
0893-228x/96/2709-0630$12.00/0 © 1996 American Chemical Society
+
+

Postoligomerization Synthesis of Oligodeoxynucleotides
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 631
spectrometer with CD3CN, CDCl3, or MeOH-d4 as solvent. 13C
NMR spectra were recorded at 75.47 MHz on a Bruker AC 300
NMR spectrometer with CDCl3 or DMSO-d6 as solvent and
tetramethylsilane or CDCl3 as an internal standard. 31P NMR
spectra were recorded at 81.0 MHz on a Bruker AC 200 NMR
spectrometer with CD3CN as solvent; 85% H3PO4 was used as
the external standard. 19F NMR spectra were recorded at
282.40 MHz on a Bruker AC 300 NMR spectrometer with CH3-
CN-d3 and MeOH-d4 as solvents; CFCl3 was used as the internal
standard. Methylene chloride, pyridine, triethylamine, and
diisopropylethylamine were distilled from calcium hydride under
a nitrogen atmosphere. 1,4-Dioxane was distilled from sodium
under a nitrogen atmosphere. Methanol was distilled from
sodium methoxide (5 mol %) under a nitrogen atmosphere.
Other chemicals were used as purchased without further
purification. Melting points are uncorrected.
2-F lu or o-(O6-t r im et h ylsilylet h yl)-2′-d eoxyin osin e (3).
This compound was prepared essentially as described previously
for the synthesis of the O6-benzyl (3,6) and O6-(2-nitrophenyl-
ethyl) (3) analogues. O6-(Trimethylsilylethyl)-2′-deoxyguanosine
(2) (0.53 g, 1.44 mmol), dried in vacuo in an Abderhalden drying
apparatus at 64 °C for 20 h, was transferred to a polypropylene
conical tube (50 mL) equipped with a stirring bar and a septum
under an argon atmosphere. Anhydrous pyridine (5.42 mL) was
added to a second polypropylene conical tube (50 mL) equipped
with a stirring bar and septum, under an argon atmosphere.
Both tubes were placed in a cooling bath (dry ice-acetone), and
the temperature was lowered to -35 to -40 °C. Hydrogen
fluoride-pyridine (70% solution, 9.65 mL, Aldrich Chemical Co.)
was added to the tube containing pyridine over a period of 3
min to generate a 45% hydrogen fluoride-pyridine solution (17).
The yield in this reaction is highly dependent upon the quality
of the HF/pyridine reagent; deeply colored reagent gave con-
siderable depurination. This solution was stirred under an
argon atmosphere for 15 min at -35 to -40 °C. The clear
solution was then added slowly to 2 and stirred at -35 to -40
°C for 5 min. tert-Butyl nitrite (0.43 mL, 36 mmol) was added
over 5 min to the reaction mixture while maintaining the bath
temperature of -35 °C. The red reaction mixture was stirred
under argon for 25 min. The solution was cautiously transferred
to a K2CO3 solution (23 g in 34 mL H2O) at 0 °C over a 2 min
period. The solution was extracted with EtOAc (5 × 40 mL),
and the combined organic portions were dried over K2CO3,
filtered, and concentrated in vacuo. The red oily residue was
placed under high vacuum overnight. Flash chromatography,
using a gradient of CH2Cl2/MeOH/Et3N (95:4:1 to 90:9:1)
provided 2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (3, 0.51
Thin layer chromatography was performed with silica gel
F254 (Merck) as the adsorbent on glass plates. The chromato-
grams were visualized under UV (254 nm) or by staining with
an anisaldehyde/sulfuric acid solution, followed by heating.
Column chromatography was performed using silica gel 60, 70-
230 mesh (Merck).
O6-(Tr im et h ylsilylet h yl)-2′-d eoxygu a n osin e (2). This
compound, first described by Gaffney and J ones (12), was
prepared via
a
Mitsunobu reaction (3,14,15). N2,3′,5′-Tri-
acetyldeoxyguanosine (16) (1, 5.11 g, 13 mmol) was dried in
vacuo in a Abderhalden drying apparatus for 24 h at 78 °C. A
1-L, three-necked flask equipped with a reflux condenser was
charged with 1, triphenylphosphine (6.82 g, 26.01 mmol), and
anhydrous dioxane (430 mL). The suspension was maintained
under an argon atmosphere and heated to 100 °C (oil bath
temperature). The pale orange solution was stirred for 5 min,
and trimethylsilylethanol (3.08 g, 26.0 mmol) and diethyl
azodicarboxylate (4.1 mL, 26.0 mmol) were simultaneously
added to the solution. The reaction mixture was stirred for 15
min at 100 °C, and allowed to cool to room temperature over a
2 h period. The orange reaction mixture was concentrated in
vacuo to obtain a viscous red oil. The oily residue was dissolved
in anhydrous methanol (120 mL) and transferred to a solution
of sodium methoxide in methanol (0.35 M, 200 mL). The
solution was stirred at room temperature under an argon
atmosphere for 7 h. Glacial acetic acid (6.23 mL, 109 mmol)
dissolved in water (30 mL) was added, and the reaction mixture
was concentrated in vacuo to give a red slurry. The residue
was suspended in methylene chloride (100 mL) and washed with
water (40 mL). The aqueous portion was extracted with CH2-
Cl2 (5 × 100 mL); the combined organic portions were dried with
Na2SO4 and concentrated in vacuo. The red oil was purified by
flash chromatography using a gradient of CH2Cl2/MeOH/Et3N
(97.8:2.0:0.2 to 94.8:5.0:0.2) to yield 2 as a pale yellow powder
(4.23 g, 89%): mp 66-68 °C, TLC Rf 0.41 (CH2Cl2/MeOH, 9:1).
1H NMR (MeOH-d4) δ 8.00 (s, 1H, H8), 6.30 (dd, 1H, J ) 6.1
Hz, J ) 2.1 Hz, H1′), 4.61-4.53 (m, 3H, OCH2CH2Si, H3′), 4.03
(q, 1H, J ) 2.7 Hz, H4′), 3.83 (dd, 1H, J ) 3.3, 12.2, H5′), 3.73
(dd, 1H, J ) 12.2, 2.9, H5′′), 2.76 (m, 1H, H2′), 2.34 (m, 1H,
H2′′), 1.21 (m, 2H, CH2Si), 0.08 (s, 9H, [(CH3)3Si]. 1H NMR
(CDCl3) δ 7.64 (s, 1H, H8), 6.16 (dd, 1H, J ) 8.8 Hz, J ) 5.7
Hz, H1′), 5.04 (s, 2H, NH2), 4.56 (d, 1H, J ) 4.8 Hz, H3′), 4.39
(t, 2H, J ) 8.6 Hz, OCH2CH2Si), 4.07 (br, 1H, H4′), 3.70 (m,
2H, H5′, H5′′), 2.73 (m, 1H, H2′), 2.21 (m, 1H, H2′′), 1.02 (t, 2H,
J ) 8.6 Hz, CH2Si), -0.10 (s, 9H, (CH3)3Si). 13C NMR (CDCl3)
δ 161.18 (C aromatic), 158.46 (C aromatic), 151.86 (C aromatic),
138.49 (C aromatic), 116.23 (C aromatic), 88.71 (C4′), 86.58
(C1′), 72.15 (C3′), 64.62 (OCH2CH2Si), 62.90 (C5′), 39.85 (C2′),
17.09 (CH2Si), -1.83 [(CH3)3Si]. HRMS-FAB1 (glycerol-TFA-
DMSO matrix) calcd for C15H25N5O4Si 368.1754, found 368.1749
(M + H)+.
1
g, 95%): mp 120-122 °C, TLC Rf 0.36 (CH2Cl2/MeOH, 9:1). H
NMR (MeOH-d4) δ 8.46 (s, 1H, H8), 6.41 (t, 1H, J ) 6.7 Hz,
H1′), 4.67-4.72 (m, 2H, OCH2CH2Si), 4.56 (m, 1H, H3′), 4.02
(q, 1H, J ) 3.6 Hz, H4′), 3.81 (dd, 1H, J ) 3.6, H5′′), 3.73 (dd,
1H, J ) 2.9, H5′), 2.77 (m, 1H, H2′′), 2.44 (m, 1H, H2′), 1.26 (m,
2H, CH2Si), 0.1 (s, 9H, [(CH3)3Si]. 1H NMR (CDCl3) δ 8.56 (s,
1H, H8), 6.31 (t, 1H, J ) 6.7 Hz, H1′), 5.35 (d, 1H, J ) 4.2 Hz,
CHOH), 4.94 (t, 1H, J ) 5.5 Hz, CH2OH), 4.64 (t, 2H, J ) 8.3
Hz, OCH2CH2Si), 4.40 (m, 1H, H3′), 3.87 (q, 1H, J ) 4.0 Hz,
H4′), 3.55 (m, 2H, H5′ and H5′′), 2.68 (m, 1H, H2′), 2.32 (m,
1H, H2′′), 1.20 (t, 2H, J ) 8.3 Hz, CH2Si), 0.08 (s, 9H, (CH3)3-
Si). 13C NMR (CDCl3) δ 161.8 (d, J C,F ) 18 Hz, C6), 156.0 (d,
J C,F ) 209 Hz, C2), 152.7 (d, J C,F ) 18 Hz, C4), 142.4 (d, J C,F
)
2 Hz, C8), 119.5 (d, J C,F ) 5 Hz, C5), 88.0 (C4′), 83.8 (C1′),
70.5 (C3′), 66.1 (OCH2CH2Si), 61.4 (C5′), 39.3 (C2′), 16.9
(CH2Si), -1.4 [(CH3)3Si)] 19F NMR (MeOH-d4) δ -51.55. 19F
NMR (CDCl3) δ -50.40. HRMS-FAB (glycerol-TFA-DMSO
matrix) calcd for C15H23N4O4SiF 371.1551, found 371.1551
(M + H)+.
5′-O-(4,4′-Dim et h oxyt r it yl)-2-flu or o-O6-(t r im et h ylsilyl-
eth yl)-2′-d eoxyin osin e (4). The dimethoxytritylation of 3 was
carried out by standard procedures (18). 2-Fluoro-O6-(trimeth-
ylsilylethyl)-2′-deoxyinosine (3) (200 mg, 0.54 mmol) was dried
by treatment with anhydrous pyridine (3 × 10 mL) and placed
under vacuum for 90 min. Anhydrous pyridine (20 mL) and
diisopropylethylamine (0.56 mL, 3.24 mmol) were added to the
flask containing (3), and the mixture was stirred under an argon
atmosphere. The solution was cooled to 10 °C, and 4,4′-
dimethoxytrityl chloride (728 mg, 2.15 mmol) was added. The
reaction mixture was allowed to warm slowly to room temper-
ature, and the progress of the reaction was monitored by TLC.
After 15 h the reaction mixture was treated with methanol
(0.052 mL, 1.3 mmol) and stirred for 10 min to quench the
unreacted trityl chloride. The reaction mixture was concen-
trated in vacuo, and the orange compound was taken up in CH2-
Cl2 (125 mL) and extracted with 10% aqueous K2CO3 solution
(2 × 50 mL). The combined organic layers were dried over K2-
CO3, filtered, and evaporated. Flash chromatography (CH2Cl2/
MeOH/Et3N, 98:1.5:0.5 to 94.5:5.0:0.5) yielded 5′-O-(dimeth-
oxytrityl)-2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (4) (305
mg, 84%): TLC Rf 0.29 (CH2Cl2/MeOH, 9.8:0.2). 1H NMR
(MeOH-d4) δ 8.29 (s, 1H, H8), 7.34-7.31 (m, 2H, aromatic),
1
Abbreviations: DMTr, 4,4′-dimethoxytrityl; FAB, fast atom bom-
bardment mass spectrometry; MALDI-FT, matrix-assisted laser-des-
orption Fourier transform mass spectroscopy; NPE, p-nitrophenethyl;
PAC, 4-tert-butylphenoxyacetyl; SO, styrene oxide; TEAA, triethylam-
monium acetate; TMSE, trimethylsilylethyl.
+
+

632 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
DeCorte et al.
7.22-7.12 (m, 7H, aromatic), 6.75-6.69 (m, 4H, aromatic), 6.38
(t, 1H, J ) 4.8 Hz, H1′), 4.69-4.61 (m, 3H, OCH2CH2Si and
H3′), 4.13 (m, 1H, H4′), 3.73 (s, 3H, CH3O), 3.72 (s, 3H, CH3O),
3.41-3.37 (m, 1 H, H5′′) 3.27-3.24 (m, 1H, H5′), 2.97-2.92 (m,
1H, H2′′), 2.50-2.45 (m, 1H, H2′), 1.28-1.24 (m, 2H, CH2Si), 0.08
(9H, s, (CH3)3Si). 13C NMR (CDCl3) δ 162.52 (d, J C,F ) 18 Hz,
C6), 158.45 (C aromatic), 157.87 (d, J C,F ) 214 Hz, C2), 152.4
(d, J C,F ) 19 Hz, C4), 144.60 (C aromatic), 140.83 (d, J C,F ) 3
Hz, C8), 135.61 and 135.55 (C aromatic), 129.92 (C aromatic),
127.99 and 127.74 (C aromatic), 126.79 (C aromatic), 120.11 (d,
J C,F ) 5 Hz, C5), 113.06 (C aromatic), 86.46 (C4′), 86.40 (CPh-
(PhOMe)2), 84.57 (C1′), 72.00 (C3′), 66.68 (OCH2CH2Si), 63.77
(C5′), 55.05 (CH3O)2), 40.15 (C2′), 17.46 (CH2Si), -1.55[(CH3)3-
Si]. 19F NMR (MeOH-d4) δ -51.13. 19F NMR (CDCl3) δ -50.4.
HRMS-FAB (glycerol-TFA-DMSO matrix) calcd for C36H41N4O6-
SiF 673.2858, found 673.2847 (M + H)+.
standard protocol. Initially, the TCA concentration was reduced
from 3% to 0.3%; subsequently syntheses have been performed
with the standard 3% trichloroacetic acid solution without
significant decrease in yield.
Capillary gel electrophoresis was performed on a Beckman
P/ACE 2000 instrument using the manufacturer’s ssDNA 100
gel capillary and Tris-borate-urea buffer. Samples were applied
at -5 kV and run at -15 kV at 30 °C.
Enzymatic digestions were carried out under conditions
described by Borowy-Borowski et al. (21) (0.2-0.4 ODs of
oligodeoxynucleotide, 0.1 M Tris-HCl, pH 8.2, 2.0 mM MgCl2, 6
µg of snake venom phosphodiesterase, 6 µg of alkaline phos-
phatase at 37 °C). The digests were analyzed by HPLC (C-18
column, 4.6 × 250 mm) with the following gradient: (A) 0.1 M
triethylammonium acetate (TEAA),1 pH 6.5 and (B) CH3CN,
1-10% B over 15 min; 10-20% B over 5 min; hold for 5 min,
and then to 100% B over 10 min at a flow rate of 1.5 mL/min.
3′-O-[(N,N-Diisopr opylam in o)-(2-cyan oeth yl)ph osph in yl]-
5′-O-(d im eth oxytr ityl)-2-flu or o-O6-(tr im eth ylsilyleth yl)-2′-
d eoxyin osin e (5). 5′-O-(dimethoxytrityl)-2-fluoro-O6-(trimeth-
ylsilylethyl)-2′-deoxyinosine (4, 180 mg, 0.26 mmol) was dried
by treatment with anhydrous pyridine (2 × 10 mL) and placed
under vacuum for 3 h. Anhydrous 1H-tetrazole (21 mg, 0.31
mmol) was added to a flame-dried 25-mL round-bottomed flask
and kept under an argon atmosphere. A solution of the
tritylated compound in anhydrous CH2Cl2 (5 mL) was injected
into the reaction flask followed by 2-cyanoethyl-N,N,N′,N′-
tetraisopropyl phosphoramidite (125 mg, 0.39 mmol) (19,20).
The reaction mixture was stirred under an argon atmosphere
at room temperature for 2 h. The mixture was transferred into
a saturated solution of NaHCO3 (30 mL) and extracted with
CH2Cl2 (5 × 40 mL). The organic layer was dried over Na2SO4
and concentrated in vacuo. The yellow residue was purified by
flash chromatography (CH2Cl2/EtOAc/pyridine, 69:30:1) to yield
3′-O-[(N,N-diisopropylamino)-(2-cyanoethyl)phosphinyl]-5′-O-
(dimethoxytrityl)-2-fluoro-O6-(trimethylsilylethyl)-2′-deoxy-
inosine (5, 200 mg, 88%). TLC Rf 0.88 (CH2Cl2-MeOH, 9.8:0.2).
1H NMR (MeOH-d4) δ 8.09 (s, 1H, H8), 7.36-7.32 (m, 2H, H
aromatic), 7.27-7.16 (m, 7H, H aromatic), 6.79-6.73 (m, 4H,
H aromatic), 6.32 (m,1H, H1′), 4.82 (m, 1H, H3′), 4.65 (m, 2H,
OCH2CH2Si), 4.18 (m, 1H, H4′), 3.72 (m, 8H, (CH3O)2 and H5′,
H5′′), 3.63-3.56 (m, 2H, N(CHMe2)2), 3.31-3.27 (m, 2H, POCH2),
2.97-2.92 (m, 1H, H2′), 2.63 (t, 1H, J ) 4.4 Hz, CH2CN), 2.58
(m, 1H, H2′′), 2.53 (t, 1H, J ) 4.4 Hz, CH2CN), 1.23 (m, 2H,
CH2Si), 1.16 (m, 12H, N(CH(CH3)2)2), 0.09 (9H, (CH3)3Si). 1H
NMR (CDCl3) δ 7.98 and 7.96 (s, 1H, H8), 7.40-6.60 (m, 13H,
H aromatic), 6.29 (t, 1H, J ) 6.5 Hz, H1′), 4.67 (m , 1H, H3′),
4.58 (t, 2H, J ) 8.5 Hz, OCH2CH2Si), 4.20 (m, 1H, H4′), 3.75
and 3.60 (m, 2H, H5′ and H5′′), 3.67 and 3.66 (s, 6H, (CH3O)2),
3.55 (m, 2H, N(CHMe2)2), 3.50 and 3.30 (m, 2H, POCH2), 2.76
and 2.53 (m, 2H, H2′ and H2′′), 2.37 (t, 2H, J ) 6.3 Hz, CH2-
CN), 1.18 (t, 2H, J ) 8.5 Hz, CH2Si), 1.09 (m, 12H, (CH(CH3)2)2),
0.04 (9H, (CH3)3Si). 13C NMR (CDCl3) δ 162.40 (d, J C,F ) 18
Hz, C6), 158.30 (s, C aromatic), 157.71 (d, J C,F ) 215 Hz, C2),
152.34 (d, J C,F ) 19 Hz, C4), 144.31 (s, C aromatic), 140.74 (d,
J C,F ) 3 Hz, C8), 135.42 and 135.37 (s, C aromatic), 129.85 and
129.81 (s, C aromatic), 127.92 and 127.87 (s, C aromatic), 127.61
(s, C aromatic), 126.66 and 126.62 (s, C aromatic), 120.10 (d,
J C,F ) 5 Hz, C5), 117.39 and 117.25 (s, CN), 112.89 (s, C
aromatic), 86.27 (s, PhC(PhOMe)2), 85.80 (d, J C,P ) 6 Hz, C4′)
Kin et ic St u d y of Ad d u ct ion of (S)-2-Am in o-2-p h en yl-
eth a n ol to Ma tr ix-Bou n d Mod ified 11-Mer 6. Individual
samples containing 0.2 µmol of beads, 40 µL of solvent (DMSO
or dimethylacetamide), and (S)-2-amino-2-phenylethanol (1.37
mg, 10 µmol) in sealed glass tubes were heated in an oil bath
at 65, 75, or 85 °C. Tubes were removed daily for 4 days and
cooled, and the contents were transferred to a small vial. The
supernatant was removed with a pipette, and the beads were
washed with methanol (4 × 1 mL) and diethyl ether (4 × 1 mL).
After drying the beads were treated with 1 mL of conc. NH4OH
for 12 h at 60 °C. The vial was cooled to room temperature
and opened, and the contents were evaporated to dryness with
a Speed-Vac. HPLC analysis was carried out on an analytical
C-18 column, using a gradient of (A) 0.1 M TEAA, pH 7.0, and
(B) CH3CN, from 5% to 20% B over 10 min and 20% to 30% B
over 20 min with a flow rate of 1.5 mL/min.
Ad d u ction of (R)-2-Am in o-2-p h en yleth a n ol to Mod ified
11-Mer 6 (Solu tion Syn th esis). Following synthesis of modi-
fied 11-mer 6 by the standard protocol (except for the use of
0.3% trichloroacetic acid for detritylation) the beads from four
1-µmol cassettes were suspended in 0.1 M NaOH (5 mL) and
stirred slowly for 8 h at room temperature. The beads were
allowed to settle, the supernatant was removed, and the solid
residue was washed with H2O (5 × 5 mL). The combined
aqueous fractions were neutralized cautiously with dilute acetic
acid. The solution was lyophilized, and the residual solid
(partially deprotected oligonucleotide 7) was transferred into a
conical vial (3 mL). DMSO (2.5 mL) and (R)-2-amino-2-
phenylethanol (0.58 mmol, 80 mg) were added, and the suspen-
sion was heated for 2 days at 75 °C with occasional stirring.
The reaction vessel was allowed to cool to room temperature,
the contents were transferred to a 20 mL vial, and diethyl ether
(10 mL) was added. The supernatant was removed, and the
residual solid was washed with diethyl ether (4 × 10 mL). The
solid was allowed to dry, concentrated NH4OH (10 mL) was
added, and the tightly closed vial was heated for 8 h at 60 °C.
After cooling to room temperature, the vial was cautiously
opened and the excess ammonia was allowed to evaporate for 4
h. The solution was transferred into a conical plastic test tube
(50 mL) and lyophilized. The dry material was dissolved in H2O
(8 mL) and filtered, and the components were separated by
HPLC to give O6-protected oligomer 8. The HPLC separation
was carried out on a PRP-1 column (7 × 305 mm, Hamilton) at
40 °C using a gradient of (A) 0.1 M TEAA, pH 7.0, and (B) CH3-
CN, from 5% to 40% B over 30 min, with a flow rate of 3.0 mL/
and 85.58 (d, J C,P ) 6 Hz, C4′), 84.47 (s, C1′), 73.79 (d, J C,P
)
17 Hz, C3′) and 73.33 (d, J C,P ) 17 Hz, C3′), 66.50 (s, OCH2-
CH2Si), 63.28 and 63.13 (s, C5′), 58.18 (d, J C,P ) 20 Hz, POCH2)
and 58.09 (d, J C,P ) 19 Hz, POCH2), 54.97 and 54.94 (s, (CH3O)2),
43.05 (d, J C,P ) 12 Hz, N(CHMe2)2) and 42.73 (d, J C,P ) 13 Hz,
N(CHMe2)2), 39.17 and 39.15 s, C2′), 24.50-24.25 (m, (CH-
(CH3)2)2), 20.19 (d, J C,P ) 7 Hz, CH2CN) and 20.01 (d, J C,P ) 7
Hz, CH2CN), 17.32 (s, CH2Si), -1.64 (s, (CH3)3Si). 19F NMR
(CH3CN-d3) δ -51.13 and -51.17. 19F NMR (CDCl3) δ -50.0
and -50.1. 31P NMR (CH3CN-d3) δ 149.96 and 149.82. 31P
NMR (CDCl3) δ 146.6 and 146.5.
min. The fractions containing oligomer
8 were collected,
lyophilized, treated with 0.1% acetic acid (5 mL) for 2 h3 and
relyophilized. Loss of the O6-TMSE group from the adducted
oligonucleotide occurred slowly in neutral aqueous solution even
in the absence of acetic acid. O6-Deprotected oligodeoxynucle-
otide 9 was separated under the same conditions except a
gradient from 5% to 20% acetonitrile over 20 min was used.
After collection of the peak of interest, the solvent was re-
moved on a lyophilizer, and the samples were desalted on a
Bio-Gel P-2 (Bio-Rad Laboratories) column (1.5 × 40 cm) by
elution with H2O to give 80 ODs (18%) of oligomer 9. Final
Oligon u cleotid es. Oligodeoxynucleotides were synthesized
on a Model 391 PCR-Mate DNA Synthesizer (Applied Biosys-
tems) on
a 1- or 10-µmol scale using the manufacturer’s
+
+

Postoligomerization Synthesis of Oligodeoxynucleotides
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 633
F igu r e 1. Scheme for synthesis of 2-fluoro-O6-TMSE-deoxyinosine (3) and derivatives.
purification was accomplished with denaturing polyacrylamide
gel electrophoresis. The purified oligomer was characterized
by capillary gel electrophoresis, enzymatic digestion, and mass
spectroscopy.
Syn th esis of F u lly Dep r otected Oligon u cleotid e 11. The
beads from 1.5 1-µmol cassettes of 6, synthesized with phos-
phoramidites having 4-tert-butylphenoxyacetyl protecting groups
(Perseptives/Biosystems), were stirred with 1.5 mL of 0.1 M
NaOH for 20 h at rt. The supernatant was removed from the
beads and neutralized with 0.1 M acetic acid to pH 7.0. After
desalting on a Sephadex G-25 column (H2O elution), 88 ODs of
crude 11 was obtained, which was purified by reverse-phase
HPLC on an ODS-AQ column (YMC, Inc.), 10 × 250 mm, using
a gradient of (A) 0.1 M ammonium formate, pH 6.39, (B) CH3-
CN, from 10% to 20% B over 30 min, with a flow rate of 3.0
F igu r e 2. Kinetics of adduction of (S)-2-amino-2-phenyletha-
mL/min, to give 28 ODs of purified 11.
nol to matrix-bound 5′-d(GGCAG-X-TGGTG)-3′, where X )
2-fluoro-O6-TMSE-deoxyinosine (75 °C). Samples were removed
from the beads and deprotected with conc. NH4OH (8 h,
60 °C) before HPLC analysis (see Materials and Methods for
details).
Syn th esis of Oligon u cleotid es 8 a n d 9 fr om 11. Oligo-
nucleotide 11 (8 ODs) and (R)-2-amino-2-phenylethanol (1.6 mg,
0.012 mmol) were dissolved in dry DMSO (0.05 mL) in a conical
vial. The solution was heated at 75 °C. Aliquots were removed
periodically and analyzed by HPLC. Analysis showed no 11
remained after 24 h; 8 and 9 were present in a ratio of 86:14.
After removal of the DMSO in vacuo, the residue was dissolved
in a small volume of H2O and dialyzed against H2O to remove
excess amino alcohol. Some conversion of 8 to 9 occurred during
dialysis, but complete conversion to 9 was accomplished by
treatment with 0.1% acetic acid at rt for 2 h. The identity of
the product was confirmed by coinjection with 9, prepared by
the first procedure described above.
preparation of N2 adducts of aryl amines (22,23); how-
ever, a low yield was obtained in the reaction with the
hindered benzo[a]pyrene triol amine (22). We decided
to investigate the use of O6-protected 2-fluoro-2′-deoxy-
inosine in our postoligomerization strategy. Protection
at O6 enhances the susceptibility of the 2 position to
nucleophilic attack and improves the solubility charac-
teristics of the synthetic intermediates. A number of O6-
protecting groups have been examined previously (12-
14); of these, the p-nitrophenethyl has been most widely
used in oligonucleotide synthesis (3,14,24,25). Schmid
and Behr (26) have recently carried out a postoligomer-
ization synthesis of a spermine-modified oligonucleotide
by treatment of matrix-bound oligonucleotide containing
2-fluoro-O6-(p-nitrophenethyl)-2′-deoxyinosine with sper-
mine in methanol at 50 °C; this treatment simultaneously
cleaved the oligonucleotide from the beads, removed the
protecting groups, and introduced the spermine residues.
However, we found the p-nitrophenethyl group to be
Resu lts a n d Discu ssion
In the search to improve the postoligomerization
strategy for guanine adducts, we initially sought ways
to increase the reactivity of the guanine synthon. Others
also have attempted to find better leaving groups in order
to prepare N2-adducted deoxyguanosine. Steinbrecher et
al. have examined the use of triflate as the leaving group
for synthesis of deoxyguanosine nucleosides; it appears
to be more reactive with unhindered amines than the
fluoro compound and has been used successfully for the
+
+

634 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
DeCorte et al.
O6-TMSE-2′-deoxyguanosine (2) was prepared by Mit-
sunobu condensation of N2,3′,5′-triacetyldeoxyguanosine
(1) with trimethylsilylethanol (Figure 1) (14,15). After
removal of the acetyl groups by alkaline hydrolysis, the
2-amino group was replaced with fluoro using tert-butyl
nitrite in HF/pyridine (17). Cleavage of the TMSE group
was avoided by the use of low temperature; the reaction
gave fluoro nucleoside 3 in 95% yield. When the stability
of nucleoside 3 in the standard DNA synthesizer detri-
tylation solution (3% trichloroacetic acid in methylene
chloride) was examined, slow depurination, without loss
of the TMSE group, was observed over a period of hours.
Inasmuch as the deblocking cycle on the synthesizer is
of the order of minutes (2.5-3.0 min for a 10 or 15 µmol
synthesis), we decided to continue toward oligonucleotide
synthesis with the expectation that depurination would
not be a serious problem, at least with reasonably short
sequences. Cyanoethyl phosphoramidite 5 was prepared
by alkylation of the 5′ position of 3 with 4,4′-dimethoxy-
trityl chloride (18) followed by phosphitylation with
2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite
(19,20).
F igu r e 3. HPLC trace of products from reaction of matrix-
bound oligonucleotide
6 with (S)-2-amino-2-phenylethanol
(DMSO, 3 days, 75 °C) after deprotection with conc. NH4OH (8
h, 60 °C). A′ and A contain cytosine side product with and
without O6-TMSE on dG at the primary adduction site.
somewhat sensitive to base-catalyzed elimination during
the extended treatment required for displacement of
fluorine with hindered amines.
The phosphoramidite was then used to prepare
oligonucleotides by solid-phase synthesis. An 11-mer,
d(GGCAGXTGGTG) where X ) 3, was prepared and,
while still fully protected and immobilized on the control-
pore glass beads, was treated with 50 equiv of (S)-2-
amino-2-phenylethanol in DMSO at 65 °C. Aliquots were
removed daily, treated with conc. NH4OH to remove the
oligonucleotide from the beads and all protecting groups
except the TMSE group, and assayed by HPLC. The
results are shown in Figure 2, and a representative
HPLC trace is shown in Figure 3. The starting material
was two-thirds gone after day 1 and had fully disap-
peared by day 3. The desired guanine adduct (8) repre-
sented ∼50% of the mixture after day 1 and peaked at
Ultimately, we chose the trimethylsilylethyl group; this
protective group is stable to base but readily cleaved by
fluoride ion. Gaffney and J ones had previously explored
TMSE for protection of O6 in deoxyguanosine during
solid-phase synthesis of DNA but found the group to have
poor stability, undergoing facile acid-catalyzed cleavage
(13). Cleavage occurs by protonation of the ring which
facilitates fragmentation of the TMSE group. We an-
ticipated the group would be more satisfactory as a
protective group for 2-fluoro-2′-deoxyinosine because the
fluoro-substituted purine would be more stable to acid
due to its decreased basicity.
F igu r e 4. Scheme for solution synthesis of 5′-d(GGCAG-GN2-SO-GGGTG)-3′ (9). SO ) (R) adduct of styrene oxide.
+
+

Postoligomerization Synthesis of Oligodeoxynucleotides
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 635
F igu r e 6. Capillary gel electrophoresis of 9.
F igu r e 5. (A) HPLC trace of mixture of partially deprotected
oligonucleotide (7) after removal from the solid support with
0.1 M NaOH (room temperature, 8 h). (B) HPLC trace of 7 after
treatment with (R)-2-amino-2-phenylethanol (DMSO, 2 days, 75
°C) and conc. NH4OH (8 h, 60 °C).
57% after day 2 dropping to 15% by day 4. The drop was
due in part to loss of the TMSE group from the adducted
oligonucleotide to give fully deprotected 9, a process
which is in no way deleterious.
F igu r e 7. Enzymatic digest of 9. SO ) (R) adduct of styrene
oxide. Deoxyinosine arises from contamination of the snake
venom phosphodiesterase with adenosine deaminase. See Mate-
rial and Methods for HPLC conditions.
It is noteworthy that loss of TMSE from unadducted
oligonucleotide was not detected. The sum of the pro-
tected and deprotected species was ∼70% of the mixture
at day 3. The major side product contained both cytosine
and guanine adducts and also underwent deprotection
with time.2 The experiment was repeated at 75 and 85
°C where both processes occurred faster and in dimethyl-
acetamide where the reactions were slightly slower. In
no case were conditions found which provided any better
differentiation of the rates of the desired and the un-
desired adduction reactions. The sequence under inves-
tigation contained only one cytosine; cytosine adduction
would be an even more serious problem in oligonucle-
otides containing several residues. The conclusion to be
drawn from the above experiments is that O6 protection
does not enhance the rate of 2-fluoro-2′-deoxyinosine
adduction sufficiently to avoid undesirably large amounts
of cytosine adduction.
As an alternative approach for circumventing the
cytosine problem, the possibility was examined that the
protective groups could be removed from cytosine without
concurrent destruction of 3. Nucleoside 3 was found to
be quite stable in the presence of aqueous 0.1 M NaOH
at 25 °C; even after 12 h, decomposition was minimal.
However, these conditions were sufficient to deprotect
N4-benzoylcytosine. Significantly, hydroxide attack oc-
curred exclusively on the benzoyl group; no evidence
could be found for formation of deoxyuridine. The 11-
mer oligonucleotide containing 3 was freshly synthesized,
and the beads were treated with 0.1 M NaOH for 8 h at
25 °C, followed by neutralization to pH 7 (Figure 4).
HPLC analysis of the supernatant showed the product
to be a complex mixture of oligodeoxynucleotides (7) due
to partial removal of protective groups from the exocyclic
amino groups of the bases. The mixture (panel A in
Figure 5) was too complex to warrant separation of the
individual components. Without any attempts at puri-
fication, the oligonucleotide mixture was treated with (R)-
2-amino-2-phenylethanol in dry DMSO (2 days, 75 °C),
followed by concentrated NH4OH to complete removal of
protective groups to give 8; panel B of Figure 5 shows
the HPLC trace of unpurified 8 in which there is no
evidence of contamination by the side product oligomers
seen in the matrix-bound synthesis (Figure 3). After
HPLC purification, 8 was treated with 0.1% acetic acid
(2 h, rt) to remove residual TMSE groups3 to give
adducted 11-mer 9 in good yield and high purity. Ad-
ducted oligonucleotide
9 was further purified by
PAGE before evaluation by capillary gel electrophore-
sis (Figure 6) which showed it to be homogeneous and
by enzyme digestion with snake venom phosphodi-
esterase and alkaline phosphatase (Figure 7) to give the
appropriate nucleosides: dC (1.0); dG (5.65); T (1.93), dA
(0.81, sum of dA + dI); dGN2-SO (1.08); theory: dC (1.0);
dG (6), T(2), dA (1.0), dGN2-SO (1.0). Adducted cytidine
was not detected. Mass spectroscopic analysis (MALDI-
FT, 3-hydroxypicolinic acid matrix, Mr ) 3574.4, mea-
sured mass ) 3573.8) confirmed the constitution of the
oligonucleotide.
2
3
Enzymatic digestion of side products showed loss of cytosine and
The trimethylsilylethyl protecting group can also be removed with
appearance of a new peak coincident with authentic dCN4-SO
.
1.0 M tetrabutylammonium fluoride in THF.
+
+

636 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
DeCorte et al.
F igu r e 8. Scheme for solution synthesis of 5′-d(GGCAG-GN2-SO-GGGTG)-3′ (9) via fully deprotected oligonucleotide 11. SO ) (R)
adduct of styrene oxide.
The solution-phase reaction procedure provided an
unexpected benefit over the previously described solid-
phase adduction reaction; the solution reaction of 2-amino-
2-phenylethanol was faster than that with immobilized
oligonucleotides, a consideration which becomes particu-
larly important with more hindered amines. NMR
quantities (150-200 ODs from 10-µmol reactions) of
several related styrene oxide-adducted oligonucleotides
have been prepared by this route. These yields can be
compared to the 250-300 ODs of nonadducted 11-mer
that have been obtained from 10 µmol syntheses per-
formed by commercial suppliers; the yields are at least
10-fold better than those obtained from our initial
synthetic strategy.
In a third strategy (Figure 8), we have found that the
fully deprotected 11-mer containing 3 can be prepared if
the oligonucleotide is synthesized using phosphoramid-
ites having the exocyclic amino groups protected with the
more labile PAC-type acyl groups (27-31). As others
have found (30,31), it is important that the capping step
on the synthesizer be carried out with a PAC-type
anhydride rather than the normal acetic anhydride;
otherwise, acetyl groups can be introduced on guanine
N2; the N-acetyl groups are not as easily removed as the
PAC and can remain after removal of PAC protection.
Treatment of the matrix-bound PAC-protected oligo-
nucleotide (10) with 0.1 M NaOH at rt for 8-24 h
released the oligonucleotide from the matrix and depro-
tected both the phosphates and the exocyclic amino
groups. The 2-fluoro substituent remained intact as well
as the O6-TMSE group. Fully deprotected oligonucleotide
11 was isolated and purified before further reaction;
during the process slight loss of the O6-TMSE group was
observed. Mass spectroscopic analysis of 11 confirmed
its constitution (MS (electrospray) calculated Mr ) 3556.5;
observed ions 1777.3 (M - 2H)/2z, 1184.4 (M - 3H)/3z,
and 888.2 (M - 4H)/4z representing a measured mass )
3556.5). When purified 11 was reacted with (R)-2-amino-
2-phenylethanol (DMSO, 75 °C) for 24 h, only O6-TMSE
adducted oligonucleotide 8 and fully deprotected product
9 were observed; no 11 was detected. This is in contrast
to the matrix-bound reaction where 30% starting mate-
rial remained after 24 h at 75 °C.
In the case of the N2-styrene oxide adducts, this
procedure has no significant advantage since the adducts
are stable to the hot NH4OH treatment needed to remove
the traditional acyl protecting groups. However, the
procedure could be very useful for preparing labile
guanine N2 adducts and also for preparing oligonucle-
otides containing 2-fluorodeoxyinosine residues (for 19F
NMR use, for example). This methodology could also be
used for preparation of [2-N15] dG-containing oligonucle-
otides; the expensive isotopic label could be introduced
at the final step of the synthesis by treatment of the fully
deprotected oligonucleotide with 15NH4OH. Furthermore,
reactions carried out in solution using fully deprotected,
purified oligonucleotides are far easier to monitor than
those utilizing matrix-bound or the soluble partially-
protected oligonucleotides. In work reported elsewhere
(32), we have used this methodology for preparation of
oligonucleotides containing N2-dG-N2-dG cross-links.
In summary, we feel that these new strategies of
carrying out oligonucleotide adduction reactions in solu-
tion, on either partially or fully deprotected oligonucle-
otides, should find many useful applications.
Ack n ow led gm en t. We thank Dr. Robert Hettich
(Oak Ridge National Laboratory) for obtaining MALDI-
FT mass spectra on oligonucleotide 9 and Dr. Ajai
Chaudhary and Brian Nobes (Center for Mass Spectrom-
etry, Department of Pharmacology, Vanderbilt Univer-
sity, USPHS RR-05805) for the electrospray mass spec-
+
+

Postoligomerization Synthesis of Oligodeoxynucleotides
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 637
(16) Gaffney, B. L., Marky, L. A., and J ones, R. A. (1984) Synthesis
and characterization of a set of four dodecadeoxyribonucleoside
undecaphosphates containing O6-methylguanine opposite ad-
enine, cytosine, guanine, and thymine. Biochemistry 23, 5686-
5691.
trum of oligonucleotide 11 and FAB mass spectra.
Generous support of this project by USPHS Grants
ES00267, ES05355, and ES05509 is gratefully acknowl-
edged.
(17) Robins, M. J ., and Uznanski, B. (1981) Nucleic acid related
compounds. 34. Non-aqueous diazotization with tert-butyl nitrite.
Introduction of fluorine, chlorine and bromine at C-2 of purine
nucleosides. Can. J . Chem. 59, 2608-2611.
(18) J ones, R. (1984) In Oligonucleotide Synthesis, A Practical Ap-
proach; Gait, M. J ., Ed.; IRL Press, Washington, DC, pp 22-34.
(19) Barone, A. D., Tang, J .-Y., and Caruthers, M. H. (1984) In situ
activation of bis-dialkylaminophosphinessA new method for
synthesizing deoxyoligonucleotides on polymer supports. Nucleic
Acids Res. 12, 4051-4061.
(20) Bannwarth, W., and Trzeciak, A. (1987) A simple and effective
chemical phosphorylation procedure for biomolecules. Helv. Chim.
Acta 70, 175-186.
(21) Borowy-Borowski, H., Lipman, R., Chowdary, D., and Tomasz,
M. (1990) Duplex oligodeoxyribonucleotides crosslinked by mito-
mycin C at a single site: synthesis, properties, and crosslink
reversibility. Biochemistry 29, 2992-2999.
(22) Steinbrecher, T., Wameling, C., Oesch, F., and Seidel, A. (1993)
Activation of the C-2 position of purine by the trifluoromethane-
sulfonate group: Synthesis of N2-alkylated deoxyguanosines.
Angew. Chem., Int. Ed. Engl. 32, 404-406.
(23) Scheer, S., Steinbrecher, T., and Boche, G. (1994) A selective
synthesis of 4-aminobiphenyl-N2-deoxyguanosine adducts. Envi-
ron. Health Perspect. (Suppl. 6) 102, 151-152.
(24) Eritja, R., Acedo, M., Avino, A., and Fabrega, C. (1995) Prepara-
tion of oligonucleotides containing non-natural base analogues.
Nucleosides Nucleotides 14, 821-824.
(25) Avino, A., Garcia, R. G., Marquez, V. E., and Eritja, R. (1995)
Preparation and properties of oligodeoxynucleotides containing
4-O-butylthymine, 2-fluorohypoxanthine and 5-azacytosine. Bioorg.
Med. Chem. Lett. 5, 2331-2336.
(26) Schmid, N., and Behr, J .-P. (1995) Recognition of DNA sequences
by strand replacement with poly-amino-oligonucleotides. Tetra-
hedron Lett. 36, 1447-1450.
(27) Ko¨ster, H., Kulikowski, K., Liese, T., Heikens, W., and Kohli, V.
(1981) N-Acyl protecting groups for deoxynucleosides. Tetrahedron
37, 363-369.
(28) Schulhof, J . C., Molko, D., and Teoule, R. (1987) The final
deprotection step in oligonucleotide synthesis is reduced to a mild
and rapid ammonia treatment by using labile base-protecting
groups. Nucleic Acids Res. 15, 397-416.
(29) Wu, T., and Ogilvie, K. K. (1988) N-Phenoxyacetylated guanosine
and adenosine phosphoramidites in the solid phase synthesis of
oligoribonucleotides: Synthesis of a ribozyme sequence. Tetra-
hedron Lett. 29, 4249-4252.
(30) Chaix, C., Molko, D., and Teoule, R. (1989) The use of labile base
protecting groups in oligoribonucleotide synthesis. Tetrahedron
Lett. 30, 71-74.
(31) Sinha, N. D., Davis, P., Usman, N., Perez, J ., Hodge, R., Kremsky,
J ., and Casale, R. (1993) Labile exocyclic amine protection of
nucleosides in DNA, RNA and oligonucleotide analog synthesis
facilitating N-deacylation, minimizing depurination and chain
degradation. Biochimie 75, 13-23.
(32) Tsarouhtsis, D., Kuchimanchi, S., DeCorte, B. L., Harris, C. M.,
and Harris, T. M. (1995) Synthesis of oligonucleotides containing
interchain cross-links of bifunctional pyrroles. J . Am. Chem. Soc.
117, 11013-11014.
Refer en ces
(1) Cosman, M., Ibanez, V., Geacintov, N. E., and Harvey, R. G. (1990)
Preparation and isolation of adducts in high yield derived from
the binding of two benzo[a]pyrene-7,8-dihydroxy-9,10-oxide ste-
reoisomers to the oligonucleotide d(ATATGTATA). Carcinogenesis
11, 1667-1672.
(2) Baertschi, S. W., Raney, K. D., Stone, M. P., and Harris, T. M.
(1988) Preparation of the 8,9-epoxide of the mycotoxin aflatoxin
B1: the ultimate carcinogenic species. J . Am. Chem. Soc. 110,
7929-7931.
(3) Lee, H., Hinz, M., Stezowski, J . J ., and Harvey, R. G. (1990)
Syntheses of polycyclic aromatic hydrocarbon-nucleoside and
oligonucleotide adducts specifically alkylated on the amino func-
tions of deoxyguanosine and deoxyadenosine. Tetrahedron Lett.
31, 6773-6776.
(4) Zhou, Y., and Romano, L. J . (1993) Solid-phase synthesis of
oligonucleotides containing site-specific N-(2′-deoxyguanosin-8-
yl)-2-(acetylamino)fluorene adducts using 9-fluorenylmethoxy-
carbonyl as the base-protecting group. Biochemistry 32, 14043-
14052.
(5) Lakshman, M. K., Sayer, J . M., Yagi, H., and J erina, D. M. (1992)
Synthesis and duplex-forming properties of a nonanucleotide
containing an N6-deoxyadenosine adduct of a bay-region diol
epoxide. J . Org. Chem. 57, 4585-4590.
(6) Harris, C. M., Zhou, L., Strand, E. A., and Harris, T. M. (1991)
New strategy for the synthesis of oligodeoxynucleotides bearing
adducts at exocyclic amino sites of purine nucleosides. J . Am.
Chem. Soc. 113, 4328-4329.
(7) MacMillan, A. M., and Verdine, G. L. (1991) Engineering tethered
DNA molecules by the convertible nucleoside approach. Tetrahe-
dron 47, 2603-2616.
(8) Matteucci, M. D., and Webb, T. R. (1987) Synthesis and crosslink-
ing properties of a deoxyoligonucleotide containing N6,N6-etha-
nodeoxyadenosine. Tetrahedron Lett. 28, 2469-2472.
(9) Lakshman, M., and Lehr, R. E. (1990) Synthesis of polycyclic
aromatic hydrocarbon substituted 2′-deoxyadenosine analogs.
Tetrahedron Lett. 31, 1547-1550.
(10) Kim, S. J ., Stone, M. P., Harris, C. M., and Harris, T. M. (1992)
A postoligomerization synthesis of oligodeoxynucleotides contain-
ing polycyclic aromatic hydrocarbon adducts at the N6 position
of deoxyadenosine. J . Am. Chem. Soc. 114, 5480-5481.
(11) Weber, H., and Khorana, H. G. (1972) CIV. Total synthesis of
the structural gene for an alanine transfer ribonucleic acid from
yeast. Chemical synthesis of an icosadeoxynucleotide correspond-
ing to the nucleotide sequence 21 to 40. J . Mol. Biol. 72, 219-
249.
(12) Gaffney, B. L., and J ones, R. A. (1982) Synthesis of O6-alkylated
deoxyguanosine nucleosides. Tetrahedron Lett. 23, 2253-2256.
(13) Gaffney, B. L., and J ones, R. A. (1982) A new strategy for the
protection of deoxyguanosine during oligonucleotide synthesis.
Tetrahedron Lett. 23, 2257-2260.
(14) Himmelsbach, F., Schulz, B. S., Trichtinger, T., Charubala, R.,
and Pfleiderer, W. (1984) The p-nitrophenylethyl (NPE) group.
A versatile new blocking group for phosphate and aglycone
protection in nucleosides and nucleotides. Tetrahedron 40, 59-
72.
(15) Mitsunobu, O. (1981) The use of diethyl azodicarboxylate and
triphenylphosphine in synthesis and transformation of natural
products. Synthesis 1-28.
TX9501795
+
+