Preparation and analysis of oligonucleotides containing the C4′-oxidized abasic site and related mechanistic probes

Article abstract of DOI:10.1021/jo0512249

The C4′-oxidized abasic site (C4-AP) is produced by a variety of DNA damaging agents. This alkali labile lesion can exist in up to four diastereomeric cyclic forms, in addition to the acyclic ketoaldehyde. Synthetic oligonucleotides containing the lesion

Full text of DOI:10.1021/jo0512249

Preparation and Analysis of Oligonucleotides Containing the
C4′-Oxidized Abasic Site and Related Mechanistic Probes
Jaeseung Kim,† Cortney R. Kreller, and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University,
3400 North Charles Street, Baltimore, Maryland 21218
mgreenberg@jhu.edu
Received June 15, 2005
The C4′-oxidized abasic site (C4-AP) is produced by a variety of DNA damaging agents. This alkali
labile lesion can exist in up to four diastereomeric cyclic forms, in addition to the acyclic keto-
aldehyde. Synthetic oligonucleotides containing the lesion were prepared from a stable photochemi-
cal precursor. Chemical integrity of the lesion containing oligonucleotides was probed using
phosphodiesterase lability. Analysis of the 3′,5′-phosphate diester of the monomeric lesion released
from single diastereomers of photolabile precursors by 1H NMR indicates that isomerization of the
hemiacetal and/or hemiketal is rapid. The syntheses and characterization of oligonucleotides
containing configurationally stable analogues of C4-AP, which serve as mechanistic probes for
deciphering the structural basis of the biochemical and biological effects of the C4′-oxidized abasic
lesion, are also described.
Introduction
ing oligonucleotides containing C4-AP at a defined site
were reported.9,10 The results of biochemical and biologi-
cal experiments using these oligonucleotides have pro-
vided motivation to prepare related molecules designed
to probe the structural basis of C4-AP’s effects on DNA
repair and replication. The synthesis and physical char-
acterization of oligonucleotides containing the C4-AP
lesion and related probes are described herein.
The C4′-hydrogen atom of a nucleotide forms one
of the two weakest carbon-hydrogen bonds in the
2′-deoxyribose component of DNA.1,2 This relatively labile
atom is readily accessible to diffusible species and minor
groove binding molecules that oxidize DNA.3 Conse-
quently, the C4′-oxidized abasic site (C4-AP) is often
formed from abstraction of the C4′-hydrogen atom.4,5
However, the lesion is formed in highest yield (∼40%)
when DNA is damaged by the antitumor agent bleo-
mycin.6-8 It was difficult to examine the effects of the
C4-AP lesion on DNA replication and its repair until
recently. In the past two years, two methods for prepar-
* Corresponding author. Phone: 410-516-8095. Fax: 410-516-7044.
† Current address: Institut Pasteur Korea, Division of Chemistry,
39-1 Hawolgok-dong Seongbuk-gu, Seoul, 136-791 Korea.
(1) Miaskiewicz, K.; Osman, R. J. Am. Chem. Soc. 1994, 116, 232-
238.
(2) Colson, A. O.; Sevilla, M. D. J. Phys. Chem. 1995, 99, 3867-
3874.
Studies using synthetic oligonucleotides have shown
that C4-AP incision by phosphodiesterases present in
Escherichia coli is as efficient as that of a typical abasic
site (AP).11 Replication of C4′-oxidized abasic site by the
Klenow exo- fragment of DNA polymerase I is also
(3) Balasubramanian, B.; Pogozelski, W. K.; Tullius, T. D. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9738-9743.
(4) Greenberg, M. M. In DNA and Aspects of Molecular Biology; Kool,
E. T., Ed.; Comprehensive Natural Products Chemistry; Elsevier:
Amsterdam, 1999; Vol. 7, pp 371-426.
(5) Pogozelski, W. K.; Tullius, T. D. Chem. Rev. 1998, 98, 1089-
1107.
(6) Stubbe, J.; Kozarich, J. W. Chem. Rev. 1987, 87, 1107-1136.
(7) Rabow, L. E.; Stubbe, J.; Kozarich, J. W. J. Am. Chem. Soc. 1990,
112, 3196-3203.
(8) Rabow, L. E.; McGall, G. H.; Stubbe, J.; Kozarich, J. W. J. Am.
Chem. Soc. 1990, 112, 3203-3208.
(9) Kim, J.; Gil, J. M.; Greenberg, M. M. Angew. Chem., Int. Ed.
2003, 42, 5882-5885.
(10) Chen, J.; Stubbe, J. Biochemistry 2004, 43, 5278-5286.
(11) Greenberg, M. M.; Weledji, Y. N.; Kim, J.; Bales, B. C.
Biochemistry 2004, 43, 8178-8183.
10.1021/jo0512249 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/03/2005
8122
J. Org. Chem. 2005, 70, 8122-8129

C4′-Oxidized Abasic Site Containing Oligonucleotides
TABLE 1. Effect of Opposing Nucleotide on the
Photochemical Conversion of 1 in 2
SCHEME 1. Isomerization of the C4′-Oxidized
Abasic Site
% cleavage upon NaOH treatmenta
opposing nucleotide
before hν
after hνb
dA
dG
dC
dT
1.1 ( 0.1
1.1 ( 0.3
1.0 ( 0.1
2.1 ( 0.6
94.2 ( 0.6
96.2 ( 0.9
94.5 ( 1.9
93.8 ( 0.9
a NaOH (0.1 M) at 37 °C for 30 min. b Rayonet photoreactor
(λmax ) 350 nm), 30 min.
from the thermally stable precursor (1) upon UV irradia-
tion (350 nm). The lesion is released using the venerable
ortho-nitrobenzyl photoredox reaction. This photochemi-
cal reaction enjoys wide application ranging from organic
synthesis to biomolecule caging and plays a prominent
role in the preparation of oligonucleotide microarrays.17-22
We typically employ a Rayonet Photoreactor equipped
with lamps that have maximum emission at 350 nm
(2.4 mW/cm2) and less than 1% relative fluence at
300 nm to carry out these reactions. The photochemical
conversion of 1 to a NaOH (0.1 M) labile lesion in a
30-nucleotide duplex (2a-d) is independent of the op-
posing nucleotide (Table 1). Greater than 93% conversion
is achieved in 30 min. This is not surprising, for although
the mechanistic details describing the release of mol-
ecules following the photochemical reaction have been
discussed in the literature, the initial photochemical
process is believed to involve a singlet excited state with
a several nanosecond-long lifetime.23-25 Quenching of
short-lived singlet excited states is inefficient, which is
consistent with the independence of the reaction on
opposing nucleotide.
similar to an AP site.12 However, single-stranded shuttle
vector studies in E. coli revealed that replication of the
C4′-oxidized abasic template site is unique.13 Replication
of a template containing this lesion, which is smaller than
a native nucleotide, produces three nucleotide deletions.
The three nucleotide deletion products are formed ex-
clusively when the E. coli are exposed to conditions that
up regulate the three DNA polymerases typically associ-
ated with bypassing DNA damage. Replication of tem-
plates containing other abasic lesions (AP, L) does not
give rise to the formation of three nucleotide deletion
products.14,15 In general, this is an unusual occurrence,
which is seldom observed in the replication of even bulky
DNA lesions, and then only in limited sequence contexts.
A structural explanation for the formation of three
nucleotide deletions from the replication of plasmids
containing C4-AP has not been formulated. However, the
C4-AP and AP lesions differ only by the substitution of
a hydrogen atom by a hydroxyl group in the former. We
suggest that the 4-hydroxyl group must play a role in
replication, perhaps via hydrogen bonding with the
polymerase(s). The additional hydroxyl group may also
affect replication indirectly by introducing configurational
lability at the C4-position (Scheme 1). C4-AP can exist
as a mixture of four cyclic isomers and the acyclic keto-
aldehyde. However, studies on AP suggest that the latter
should be present in very small (e1%) amounts.16 The
ability of C4-AP to epimerize at C4 raises the possibility
that the lesion could significantly alter the trajectory of
the DNA backbone. We describe the synthesis and
characterization of oligonucleotides designed to probe the
role of the C4-hydroxyl group in the replication of the
C4′-oxidized abasic lesion.
(17) McCray, J. A.; Trentham, D. R. Annu. Rev. Biophys. Biophys.
Chem. 1989, 18, 239-270.
Results and Discussion
(18) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes,
C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-
5026.
Photochemical Generation of the C4′-Oxidized
Abasic Lesion (C4-AP). The C4-AP lesion is generated
(19) McGall, G. H.; Barone, A. D.; Diggelmann, M.; Fodor, S. P. A.;
Gentalen, E.; Ngo, N. J. Am. Chem. Soc. 1997, 119, 5081-5090.
(20) Beier, M.; Hoheisel, J. D. Nucleic Acids Res. 2000, 28, e11.
(21) Pirrung, M. C. Angew. Chem., Int. Ed. 2002, 41, 1276-1289.
(22) Wender, P. A.; Zercher, C. K.; Beckham, S.; Haubold, E. M.
J. Org. Chem. 1993, 58, 5867-5869.
(12) Greenberg, M. M.; Weledji, Y. N.; Kroeger, K. M.; Kim, J.;
Goodman, M. F. Biochemistry 2004, 43, 2656-2663.
(13) Kroeger, K. M.; Kim, J.; Goodman, M. F.; Greenberg, M. M.
Biochemistry 2004, 43, 13621-13627.
(14) Kroeger, K. M.; Jiang, Y. L.; Kow, Y. W.; Goodman, M. F.;
Greenberg, M. M. Biochemistry 2004, 43, 6723-6733.
(15) Kroeger, K. M.; Goodman, M. F.; Greenberg, M. M. Nucleic
Acids Res. 2004, 32, 5480-5485.
(16) Wilde, J. A.; Bolton, P. H.; Mazumder, A.; Manoharan, M.;
Gerlt, J. A. J. Am. Chem. Soc. 1989, 111, 1894-1896.
(23) Schupp, H.; Wong, W. K.; Schnabel, W. J. Photochem. 1987,
36, 85-97.
(24) Il’ichev, Y. V.; Schwo¨rer, M. A.; Wirz, J. J. Am. Chem. Soc. 2004,
126, 4581-4595.
(25) Corrie, J. E. T.; Barth, A.; Munasinghe, V. R. N.; Trentham,
D. R.; Hutter, M. C. J. Am. Chem. Soc. 2003, 125, 8546-8554.
J. Org. Chem, Vol. 70, No. 20, 2005 8123

Kim et al.
TABLE 2. Probing the Integrity of the Photochemical
Conversion of 1 (2a) to C4-AP Using Endonuclease IV
SCHEME 2. Synthesis of a Bis-phosphate Diester
Model for C4-AP Generationa
% cleavage
treatment
before hν
after hνb
background (none)
NaOHa
endonuclease IV
0.6 ( 0.2
2.7 ( 2.8
1.3 ( 0.7
6.2 ( 0.4
95.6 ( 3.1
95.0 ( 0.7
a NaOH (0.1 M) at 37 °C for 30 min. b Rayonet photoreactor
(λmax ) 350 nm), 30 min.
The integrity of C4-AP containing oligonucleotides
prepared in this way has been examined using
ESI-MS.9,13 Two species that differ by 18 atomic mass
units are detected in a ratio that is dependent upon the
temperature of the inlet source. These ions are attributed
to the cyclic and acyclic (keto-aldehyde) forms of C4-AP
(Scheme 1). The integrity of a 30-nucleotide-long oligo-
nucleotide duplex containing C4-AP (3a) was also probed
using the type II E. coli base excision repair enzyme
endonuclease IV (Endo IV) (Table 2). We previously
reported that C4-AP is efficiently incised by endonuclease
IV.11 In the current situation, we used the enzyme as a
reagent to probe the integrity of the C4-AP containing
DNA. Treatment of 5′-32P-2a (10 nM) with Endo IV
(50 nM) or NaOH (0.1 M) showed that the C4-AP
precursor (1) is not incised by the enzyme. In contrast,
the amount of cleavage by NaOH or endonuclease IV
measured after irradiation (3a) was within experimental
error of one and other. This suggests that if 2a is
converted into a product(s) other than C4-AP (3a), the
undesired product is also a substrate for endonuclease
IV, whereas the photochemical precursor is not. A simpler
explanation, which is consistent with all observations,
is that the precursor that is converted to NaOH labile
material is transformed into C4-AP exclusively.
a (a) i. Dimethoxy N,N-diisopropylphosphoramidite, tetrazole,
CH3CN; ii. t-BuOOH, decane. (b) NaI, acetone
Epimerization of the C4′-Oxidized Abasic Lesion.
The original report describing the photochemical syn-
thesis of oligonucleotides containing C4-AP advocated
using mixtures of stereoisomers. It was assumed that the
configurational composition of the lesion in DNA would
rapidly equilibrate to the appropriate thermodynamic
mixture dictated by the duplex. This assumption was
based upon the half-lives for pyranose mutarotation in
water, which are approximately 30 min.26,27 Moreover,
the respective half-life for the epimerization of the
furanose form of ribose is considerably faster.28 Despite
the indication that C4-AP should readily adopt its
thermodynamically preferred distribution of isomers in
DNA, we sought evidence to support this supposition.
Individual diastereomers of bis-phosphate diester 4a,b
were synthesized from the previously reported diols
(5a,b, Scheme 2).9 The individual phosphate triesters
were prepared by phosphitylation and in situ oxi-
dation. The purified phosphate triesters (6a,b) were
deprotected using sodium iodide, and the desired sub-
strates were purified by reverse-phase HPLC as their
triethylammonium salts, followed by exchange with
FIGURE 1. 1H NMR analysis following photolysis of indi-
vidual diastereomers of 4. (A) 4a. (B) 4b.
sodium using Dowex resin. Diastereomers 4a and 4b
were readily distinguished by a significant upshield shift
of the C1-proton in the latter, which is occluded by the
residual water peak.29 Solutions (D2O) of individual
diastereomers of 4 (0.1 mM) were photolyzed in a Rayonet
photoreactor (20 min) and immediately analyzed by
1H NMR (with water suppression). Dilute solutions were
irradiated, despite the fact that obtaining NMR data was
more difficult, to mimic conditions in which oligonucleo-
tides are photolyzed and to minimize bimolecular inter-
actions. Although the spectra were obtained with differ-
ing single-to-noise ratios, analysis of the region where
the C1-protons resonate (5.30-5.75 ppm) indicated that
each diastereomer of 4 was converted into the same
mixture of 7. This was evident by examining the ratio of
peak intensities in the region where the C1-proton of 7
was expected to resonate (Figure 1). This experiment
supports the supposition that stereoisomer distribution
(26) Moriyasu, M.; Kato, A.; Okada, M.; Hashimoto, Y. Anal. Lett.
1984, 17, 1533-1538.
(27) Panov, M. Y.; Sokolova, O. B. Russ. J. Gen. Chem. 2004, 74,
1451-1453.
(28) Ryu, K.-S.; Kim, C.; Park, C.; Choi, B.-S. J. Am. Chem. Soc.
2004, 126, 9180-9181.
(29) See Supporting Information.
8124 J. Org. Chem., Vol. 70, No. 20, 2005

C4′-Oxidized Abasic Site Containing Oligonucleotides
SCHEME 3. Strategy for the Synthesis of
Oligonucleotides Containing 4S-AP
SCHEME 4. Synthesis of the Phosphoramidite
Required for Preparing Oligonucleotides
Containing 4S-APa
in a C4-AP precursor (e.g., 2) is unimportant, because
the lesion will readily adopt the distribution of isomers
dictated by its environment in duplex DNA provided the
neighboring nucleotides do not impose a kinetic barrier.
Synthesis of Oligonucleotides Containing Ana-
logues of C4-AP. The configurational lability of the
C4′-oxidized abasic site, particularly at the C4-position,
raises the question of whether the lesion’s extraordinary
effect on replication in E. coli is attributable to the
4S-configuration and/or the presence of a hydroxyl group
at this position. We are using configurationally stable
analogues of C4-AP to investigate these issues. The
4S-epimer of an AP site (4S-AP) lesion was designed as
a configurationally stable probe to investigate the effect
of an altered backbone, which could be introduced into
DNA by the C4′-oxidized abasic lesion independent of the
hydrogen bonding hydroxyl group. We previously re-
ported that the 4S-AP did not give rise to three nucleotide
deletions upon replication in E. coli, but did not describe
the synthesis of oligonucleotides containing this useful
probe.13
a (a) Aqueous acetic acid (75%). (b) p-Anisaldehyde dimethyl
acetal, camphorsulfonic acid, CH2Cl2. (c) i. Diethyl azo dicarboxy-
late, triphenylphosphine, p-nitrobenzoic acid, THF; ii. LiOH,
H2O/THF. (d) OsO4, NMO, t-BuOH, H2O. (e) tert-Butyldimethysilyl
triflate, 2,6-lutidine, CH2Cl2. (f) Pd(OH)2/C, H2, EtOH. (g) Dimeth-
oxytriyl chloride, pyridine. (h) N,N-Diisopropyl 2-cyanoethyl phos-
phoramidic chloride, diisopropylethylamine, CH2Cl2.
Oligonucleotides containing 4S-AP were prepared us-
ing the strategy employed by Johnson and Kim in their
syntheses of DNA containing AP and C2′-oxidized AP
sites, respectively.30,31 In these syntheses, the alkali labile
lesions were introduced following purification of an
oligonucleotide containing an appropriate vicinal diol (9)
using NaIO4. Applying this approach to synthesizing
oligonucleotides containing 4S-AP required that we
prepare 8 (Scheme 3). Phosphoramidite 8 was prepared
starting from the product obtained from Lewis acid
mediated allyl addition to D-glyceraldehyde (Scheme 4).32
The acetonide was converted into the 1,3-protected triol
(11), which was previously prepared by a different
method.33 Preparation of the benzylidene acetal allowed
us to invert the stereochemistry at the carbon that
ultimately becomes C4. Following Mitsunobu inversion,
12 was osmylated and the crude diastereomeric mixture
of triols was persilylated. Phosphoramidite 8 was ob-
tained following hydrogenolysis via dimethoxytritylation
of the primary alcohol and phosphitylation.
The triol containing DNA (9) was obtained using
standard solid-phase oligonucleotide synthesis methods
and commercially available fast deprotecting phosphor-
amidites, with the exception of coupling 8. Extending the
reaction time for the protected triol to 5 min provided
>75% coupling. Following deprotection and cleavage from
the resin using concentrated aqueous ammonia (2 h,
55 °C), the silyl protecting groups were removed using
Et3N‚3HF in N-methylpyrrolidine (3 h, 65 °C).34 The triol
containing oligonucleotides (9a-c) were purified by
denaturing PAGE and characterized by ESI-MS. The
4S-AP lesion was introduced on an as needed basis
(nanomole scale) by treating a 0.1 M sodium acetate
solution (pH 6.0) for 30 min with sodium periodate
(30) Shishkina, I. G.; Johnson, F. Chem. Res. Toxicol. 2000, 13, 907-
912.
(31) Kim, J.; Weledji, Y.; Greenberg, M. M. J. Org. Chem. 2004, 69,
6100-6104.
(32) Almendros, P.; Thomas, E. J. J. Chem. Soc., Perkin Trans. 1
1997, 2561-2568.
J. Org. Chem, Vol. 70, No. 20, 2005 8125

Kim et al.
TABLE 3. UV Melting Temperatures of Duplexes
(5 mM). Following desalting, no further purification of
10a-c was necessary, as evidenced by ESI-MS analysis.
Containing the Stereoisomers of C4-MeOAPa
The dimethoxy abasic site (C4-MeOAP) was envisioned
to act as a configurationally stable analogue of the
C4′-oxidized abasic site, which retains the oxidation state
of C4-AP. Oligonucleotides containing the methoxy ana-
logue of an AP site (MeOAP) have been prepared using
standard solid-phase oligonucleotide synthesis condi-
tions.35 However, C4-MeOAP is unstable to the acidic
detritylation conditions. Consequently, we employed the
strategy used for synthesizing oligonucleotides containing
C4-AP, which takes advantage of the 5′-silyloxy protect-
ing groups to prepare the biopolymer without exposing
it to acid following incorporation of the labile compo-
nent.9,36 Implementation of this approach required that
we synthesize phosphoramidites 16a,b, which was ac-
complished in a manner similar to that used for synthe-
sizing oligonucleotides containing C4-AP (Scheme 5).
a [Duplex] ) 2.2 µM.
major isomers of 19 was silylated, and 20a and 20b were
separated from one another as well as from bis-silylated
materials.
Stereochemical assignments were made based upon
ROSEY analysis of the mixture of the two major isomers
1
of 19 and comparison of the H NMR spectra of 20a,b.
The chemical shifts of the C3 and C5 protons in 20a,b
are very similar to one another and suggest that the
stereochemistry at C3 and C4 is the same. The 1H NMR
spectra of 20a and 20b are most readily distinguished
from one another in the regions where the C1 and C2
protons resonate. The chemical shifts of anomeric protons
are often very sensitive to structural variation. In 20a,b,
the chemical shifts of the diastereotopic C2 protons are
very similar to one another in the major isomer (20a)
but are separated by ∼0.5 ppm in the minor stereoisomer.
The large difference in C2-proton chemical shifts in 20b
is ascribed to the cis relationship of the pro-R proton to
the C1 and C3 oxygen substitutents, whereas each
C2-proton in 20a is cis to one of the oxygen containing
substituents and the protons exhibit similar chemical
shifts. The individual isomers of 20 were phosphitylated
and purified. Care must be taken during chromatogra-
phy, as the phosphoramidites (16a,b) are relatively
nonpolar and are not detectable using UV light.
SCHEME 5. Synthesis of the Phosphoramidite
Required for Preparing Oligonucleotides
Containing C4-MeOAPa
a (a) NBS, MeOH, CH3CN. (b) TBAF‚hydrate, THF. (c) Cyclo-
dodecyloxy bis-trimethylsilyloxy chloride, pyridine, DMAP, CH2Cl2.
(d) N,N-Diisopropyl methyl phosphoramidic chloride, diisopro-
pylethylamine, CH2Cl2.
Oligonucleotide synthesis (21a,b) was carried out using
typical dimethoxytrityl protected phosphoramidites until
C4-MeOAP is incorporated, because the coupling ef-
ficiency of 16a and 16b as well as the corresponding
silyloxy protected phosphoramidites of the native nucleo-
tides cannot be monitored. Automated synthesis was
paused prior to introduction of 16, at which time the
reagents were switched, and the remainder of 21a,b was
synthesized using the method previously described for
oligonucleotides containing C4-AP.9 Deprotection was
also carried out as described. The UV melting tempera-
tures of the respective duplexes were measured at
2.2 µM (Table 3). The TM’s of the two diastereomers were
within experimental error of one another. Furthermore,
Cyclization of in situ deprotected 17 in the presence of
methanol produced silylated C4-MeOAP (18) as a mix-
ture of isomers.9 Separation of the two major stereo-
isomers (∼83% of the mixture) from the minor isomers
was achieved upon desilylation. The mixture of the two
(33) Furstner, A.; Schlede, M. Adv. Synth. Catal. 2002, 344, 657-
665.
(34) Wincott, F.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz, D.;
Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S.; Usman, N.
Nucleic Acids Res. 1995, 23, 2677-2684.
(35) McCullough, A. K.; Sanchez, A.; Dodson, M. L.; Marapaka, P.;
Taylor, J.-S.; Lloyd, R. S. Biochemistry 2001, 40, 561-568.
(36) Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. J. Am. Chem.
Soc. 1998, 120, 11820-11821.
8126 J. Org. Chem., Vol. 70, No. 20, 2005

C4′-Oxidized Abasic Site Containing Oligonucleotides
purified by chromatography (1-5% MeOH in CH2Cl2) to give
the triol (1.13 g, 55.5%). 1H NMR (acetone-d6 with TMS)
δ 5.99-5.89 (m, 1H), 5.11-4.99 (m, 2H), 3.90 (d, J ) 5.2 Hz,
1H), 3.83-3.47 (m, 6H), 2.48-2.42 (m, 1H), 2.25-2.17 (m, 1H);
13C NMR (acetone-d6 with TMS) δ 136.7, 116.7, 74.9, 73.0, 64.5,
38.6.
they were very similar to those reported for C4-AP and
the tetrahydrofuran analogue of an abasic site in other-
wise identical duplexes, but distinct from the 4S-AP
containing DNA.13 The latter indicates that the config-
uration at C4 of C4-MeOAP was R in each instance, as
suggested above based upon analysis of the NMR data.
To a solution of the triol (1.10 g, 8.32 mmol) in CH2Cl2
(30 mL) was added camphorsulfonic acid (0.20 g, 0.86 mmol)
and p-anisaldehyde dimethyl acetal (3.10 g, 17.0 mmol). After
18 h, the reaction mixture was neutralized by addition of
triethylamine, concentrated, and purified by flash chromatog-
raphy (10-30% EtOAc in hexanes) to give the benzylidene
alcohol 11 (1.23 g, 59.1%). Rf 0.34 (1:2 EtOAc in hexanes);
1H NMR (CDCl3) δ 7.41 (d, J ) 8.4 Hz, 2H), 6.91 (d, J )
8.8 Hz, 2H), 6.04-5.94 (m, 1H), 5.42 (s, 1H), 5.22-5.11 (m,
2H), 4.20 (dd, J ) 4.0, 10.4 Hz, 1H), 3.80 (s, 3H), 3.61-3.49
(m, 3H), 2.64-2.58 (m, 1H), 2.46-2.39 (m, 1H), 2.26 (brs, 1H);
13C NMR (CDCl3) δ 159.9, 134.2, 130.2, 127.3, 117.4, 113.5,
100.7, 81.0, 70.9, 65.3, 55.2, 36.4; IR (film) 3446, 3076, 2910,
1614, 1519, 1255, 1081, 827 cm-1; HRMS (EI) M - H+ calcd,
for C14H17O4 249.1127; found, 249.1137.
Experimental Methods
Preparation of 4a and 4b. Dimethoxy N,N-diisopropyl-
phosphoramidite (43 mg, 0.22 mmol) was added to 5a9 (22 mg,
41 µmol) in an acetonitrile solution of tetrazole (0.5 mL,
0.45 M). A solution of tert-butyl hydroperoxide (5 M) in decane
(0.1 mL) was added after being stirred for 30 min. After being
stirred for 2 h, the mixture was concentrated under vacuum.
The crude material was taken up in CH2Cl2 (10 mL) and
washed with saturated aqueous NaHCO3, water, and brine.
The organic layer was dried over Na2SO4, filtered, concen-
trated, and purified by column chromatography (2% MeOH
in CH2Cl2) to provide the bis-phosphate triester, 6a (20 mg,
64%). Rf 0.31 (1:15 MeOH:CH2Cl2); 1H NMR (CDCl3) δ 7.62
(s, 1H), 7.56 (s, 1H), 7.39 (s, 1H), 6.95 (s, 1H), 5.38 (dd, J )
4.0, 5.6 Hz, 1H), 5.13 (d, J ) 7.2 Hz, 2H), 5.00-4.89 (m, 3H),
4.28-4.18 (m, 2H), 3.93 (s, 6H), 3.91 (s, 3H), 3.83 (s, 3H), 3.80
(d, J ) 1.6 Hz, 3H), 3.79 (d, J ) 2.0 Hz, 3H), 3.78 (d, J ) 2 Hz,
3H), 3.76 (d, J ) 2.0 Hz, 3H), 2.84-2.77 (m, 1H), 2.45-2.38
(m, 1H); 31P NMR (CDCl3) δ 1.26, 0.77. The bis-phosphate
triester was refluxed in acetone (2 mL) in the presence of
excess NaI (178 mg, 1.19 mmol) for 6 h. The mixture was
cooled to room temperature and evaporated to dryness. The
residue was taken up in water and extracted with EtOAc. The
water layer was concentrated, and the product (4a) was
purified by reverse-phase HPLC (Waters µ-Bondpak, 7.8 ×
25 cm; solvent A: 100 mM TEAA (pH 7.8); solvent B: 50%
CH3CN in 100 mM TEAA (pH 7.8); 0-100% B linearly over
30 min). The triethylammonium ion was then exchanged by
passing over a Dowex-Na+ column to provide 4a (6 mg).
1H NMR (D2O, water suppression) δ 7.45 (s, 1H), 7.44 (s, 1H),
7.08 (s, 1H), 6.75 (s, 1H), 5.66 (dd, J ) 4.4, 7.0 Hz, 1H), 4.89-
4.56 (m, 5H), 4.22-4.08 (m, 2H), 3.84 (s, 6H), 3.81 (s, 3H), 3.71
(s, 3H), 3.61 (d, J ) 10.8 Hz, 3H), 3.56 (d, J ) 10.8 Hz, 3H),
2.74-2.68 (m, 1H), 2.51-2.45 (m, 1H); 31P NMR (D2O) δ 1.25,
0.18. ESI-MS (M - H) calcd for C25H34N2O19P2, 727.48; found,
727.27.
Diastereomeric 4b was prepared from 5b on the same scale
in identical manner. 6b: 1H NMR (CDCl3) δ 7.59 (s, 1H), 7.51
(s, 1H), 7.22 (s, 1H), 6.84 (s, 1H), 5.60 (t, J ) 4.8 Hz, 1H),
5.06-4.92 (m, 5H), 4.26 (dd, J ) 6.4, 10.8 Hz, 1H), 4.21 (dd,
J ) 5.6, 11.2 Hz, 1H), 3.90 (s, 6H), 3.70 (d, J ) 2.0 Hz, 3H),
3.69 (d, J ) 2.0 Hz, 3H), 2.69-2.63 (m, 1H), 2.52-2.46 (m,
1H); 31P NMR (CDCl3) δ 1.2, 0.4. 4b: 1H NMR (D2O, water
suppression) δ 7.72 (s, 1H), 7.55 (s, 1H), 7.05 (s, 1H), 6.99 (s,
1H), 4.98-4.50 (m, 6H), 4.20-4.05 (m, 2H), 3.90 (s, 3H), 3.87
(s, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 3.57 (d, J ) 10.8 Hz, 3H),
3.54 (d, J ) 10.8 Hz, 3H), 2.53-2.48 (m, 1H), 2.23-2.20 (m,
1H); 31P NMR (D2O) δ 1.23, 0.03. ESI-MS (M - H) calcd for
C25H34N2O19P2, 727.48; found, 727.13.
Preparation of 11. To a -78 °C solution of allyl tributyltin
(6.29 g, 19.0 mmol) in CH2Cl2 (30 mL) under an Ar atmosphere
was added a solution of SnBr4 (8.08 g, 18.4 mmol) in CH2Cl2
(20 mL).31 After 30 min, D-glyceraldehyde acetonide (2.00 g,
15.4 mmol) in CH2Cl2 (30 mL) was added via cannular. After
1.5 h, the reaction mixture was quenched with saturated
aqueous NaHCO3, warmed to room temperature, and stirred
for an additional 1 h. The reaction mixture was diluted with
EtOAc. The organic layer was washed with saturated NH4Cl,
saturated NaHCO3, and brine, and then dried over Na2SO4.
The solution was filtered and concentrated on a rotary
evaporator. The residue was purified by chromatography
(10-33% EtOAc in hexanes) to yield the alcohol. The alcohol
was hydrolyzed in 75% aqueous acetic acid at 60 °C for 3 h,
cooled to room temperature, and concentrated. The crude was
Preparation of 12. Diethyl azo dicarboxylate (0.25 g,
1.43 mmol) was added dropwise to a solution of Ph3P (365 mg,
1.39 mmol), p-nitrobenzoic acid (230 mg, 1.38 mmol), and 11
(230 mg, 0.92 mmol) in THF (9 mL) at -45 °C. The mixture
was allowed to warm to room temperature and was stirred
overnight. The reaction mixture was partitioned with ether
and H2O, extracted with ether, washed with brine, and dried
over Na2SO4. The organics were filtered, concentrated, and
purified by column chromatography (2-10% EtOAc in hex-
anes) to afford the inverted p-nitrobenzoate derivative of 11
as a white solid (250 mg, 68.0%). Rf 0.68 (1:4 EtOAc/hexanes);
1
mp 115 °C; H NMR (CDCl3) δ 8.30 (d, J ) 8.8 Hz, 1H), 8.20
(d, J ) 8.8 Hz, 1H), 7.45 (d, J ) 8.4 Hz, 1H), 6.92 (d, J )
8.8 Hz, 1H), 5.96-5.89 (m, 1H), 5.55 (s, 1H), 5.14-5.04 (m,
3H), 4.50 (dd, J ) 5.2, 10.4 Hz, 1H), 4.05-4.00 (m, 1H), 3.81
(s, 3H), 3.76 (t, J ) 10.4 Hz, 1H), 2.54-2.44 (m, 2H); 13C NMR
(CDCl3) δ 163.5, 160.2, 150.7, 134.7, 133.0, 130.8, 129.7, 127.4,
123.6, 117.8, 113.6, 101.2, 78.4, 67.8, 55.3, 36.6; IR (KBr) 2866,
1720, 1616, 1529, 1310, 1274, 1123, 833 cm-1; HRMS (FAB)
+
M
calcd for C21H21NO6, 400.1396; found, 400.1372.
The benzoate (250 mg, 0.63 mmol) was stirred with LiOH
(20 mg, 0.84 mmol) in THF/H2O (10:1, 6.6 mL) overnight. The
reaction was quenched with aqueous NH4Cl, extracted with
ethyl acetate, washed with brine, and dried over Na2SO4. The
filtrate was concentrated and purified by column chromatog-
raphy (10-30% EtOAc in hexanes) to afford 12 (150 mg, 95%).
1
Rf 0.28 (1:2 EtOAc/hexanes); H NMR (CDCl3) δ 7.42 (d, J )
8.4 Hz, 2H), 6.88 (d, J ) 8.8 Hz, 2H), 6.04-5.94 (m, 1H), 5.43
(s, 1H), 5.22-5.11 (m, 2H), 4.22 (dd, J ) 4.4, 10.4 Hz, 1H),
4.19-4.10 (m, 1H), 3.80 (s, 3H), 3.64-3.58 (m, 2H), 3.53
(t, J ) 10.0 Hz, 1H), 2.65-2.58 (m, 1H), 2.47-2.37 (m, 1H),
2.09 (brs, 1H); 13C NMR (CDCl3) δ 159.9, 134.2, 130.2, 127.3,
117.4, 113.5, 100.8, 81.0, 70.9, 65.5, 55.2, 36.5; IR (film) 3439,
2840, 1614, 1518, 1251, 1079, 827 cm-1; HRMS (EI) M + calcd
for C14H18O4, 250.1205; found, 250.1207.
Preparation of 13. A solution of OsO4 (12 mg, 47.2 µmol)
and N-methyl morpholine-N-oxide (141 mg, 1.2 mmol) in
t-BuOH/H2O (1:1, 6 mL) was added to 12 (150 mg, 0.60 mmol)
at 0 °C. The mixture was allowed to warm to room temperature
and was stirred overnight. Sodium bisulfite (1.0 g) was added
and stirred for 30 min. The mixture was diluted with CH2Cl2,
washed with brine, dried over Na2SO4, filtered, concentrated,
and purified by column chromatography (5-10% MeOH in
CH2Cl2) to afford the triol, which was carried on immediately
to 13. tert-Butyldimethylsilyl triflate (544 mg, 2.1 mmol) and
2,6-lutidine (286 mg, 2.7 mmol) were added to the triol in
CH2Cl2 (10 mL) at 0 °C. After 1 h, the solution was poured
into cold aqueous NH4Cl, extracted with CH2Cl2, and washed
with NaHCO3 and brine. The organic layer was dried over
Na2SO4, filtered, concentrated, and purified by column chro-
matography (2-5% EtOAc in hexanes) to afford a mixture of
J. Org. Chem, Vol. 70, No. 20, 2005 8127

Kim et al.
two diastereomers of 13 (220 mg, 58.3% over two steps).
Rf 0.65 (1:9 EtOAc/Hexanes); 1H NMR (CDCl3) δ 7.43-7.38
(m, 2H), 6.91-6.86 (m, 2H), 5.43 (s, 1H), 4.19-4.14 (m, 1H),
4.02-3.95 (m, 1H), 3.81 (s, 3H), 3.76-3.47 (m, 5H), 2.18-1.90
(m, 1H), 1.70-1.62 (m, 1H), 0.92 (s, 9H), 0.91 (s, 9H), 0.90 (s,
9H), 0.11 (s, 3H), 0.09 (s, 6H), 0.07 (s, 3H), 0.06 (s, 3H), 0.06
(s, 3H); 13C NMR (CDCl3) δ 159.8, 130.6, 127.3, 113.5, 113.4,
100.6, 100.5, 79.2, 78.6, 71.9, 70.6, 68.9, 68.3, 68.0, 67.2, 66.8,
55.2, 37.1, 36.9, 26.0, 25.9, 25.8, 25.7, 18.4, 18.1, 18.0, 17.9,
-3.9, -4.1, -4.3, -4.6, -4.7, -4.9, -5.3; IR (film) 2929, 2857,
1518, 1463, 1251, 1107, 1039, 836 cm-1; HRMS (MALDI-TOF)
M + Na+ calcd for C32H62O6NaSi3, 649.3747; found, 649.3734.
Preparation of 14. To a solution of benzylidene 13
(160 mg, 0.26 mmol) in EtOH (6 mL) was added Pd(OH)2/C
(20%, 50 mg) under H2 (50 psi). After 0.5 h, the mixture was
filtered through a Celite pad, washed with EtOH, and con-
centrated. The crude was purified by column chromatography
(0-2% MeOH in CH2Cl2) to afford a mixture of two diastere-
omers of 14 (110 mg, 84.7%). Rf 0.31 (1:4 EtOAc/hexanes);
1H NMR (CDCl3) δ 3.97-3.45 (m, 8H), 2.62-2.51 (m, 1H),
2.07-1.87 (m, 1H), 1.70-1.59 (m, 1H), 0.89 (s, 18H), 0.88 (s,
9H), 0.11-0.05 (m, 18H); 13C NMR (CDCl3) δ 75.2, 75.0, 73.0,
72.7, 72.1, 71.3, 67.5, 66.6, 65.1, 64.8, 37.9, 37.1, 25.9, 25.8,
25.7, 18.3, 18.0, 17.9, -4.2, -4.4, -4.5, -4.8, -5.0, -5.4; IR
(film) 3445, 2956, 2858, 1464, 1256, 1091, 837 cm-1; HRMS
(FAB) M + H+ calcd for C24H57O5Si3, 509.3514; found, 509.3525.
Preparation of 15. A solution of 14 (135 mg, 0.27 mmol)
in dimethoxytrityl chloride (120 mg, 0.35 mmol) was stirred
in pyridine (3 mL) overnight. The solution was quenched with
MeOH (1 mL), concentrated, and purified by column chroma-
tography (10-20% EtOAc in hexanes) to give 15 (200 mg,
93.2%). Rf 0.52 (1:4 EtOAc/Hexanes); 1H NMR (CDCl3) δ 7.46-
7.17 (m, 9H), 6.82-6.80 (m, 4H), 4.00-3.90 (m, 2H), 3.79 (s,
6H), 3.76-3.72 (m, 1H), 3.61-3.40 (m, 2H), 3.21-3.05 (m, 2H),
2.97 (d, J ) 3.2 Hz, 0.5H), 2.75 (d, J ) 3.6 Hz, 0.5H), 1.75-
1.44 (m, 2H), 0.88-0.86 (m, 27H), 0.09-0.03 (m, 18H);
13C NMR (CDCl3) δ 158.4, 144.9, 136.2, 136.1, 130.1, 128.2,
127.7, 126.6, 113.0, 86.3, 75.0, 72.3, 71.1, 70.6, 69.8, 67.5, 65.1,
55.1, 36.4, 35.9, 26.0, 25.9, 18.3, 18.0, -4.3, -4.4, -4.8, -4.9,
-5.4; IR (film) 3518, 2954, 2857, 1608, 1510, 1464, 1252,
1082, 835 cm-1; HRMS (MALDI-TOF) M + Na+ calcd for
C45H74O7NaSi3, 833.4635; found, 833.4625.
ether. The combined organic layers were washed with satu-
rated aqueous NaHCO3, H2O, and brine. The organics were
dried over Na2SO4 and concentrated, and the crude compound
was purified by column chromatography (2-10% EtOAc in
hexanes) to afford 18 (a mixture of 4 isomers, 0.74 g, 91.9%).
Rf 0.51 (1:9 EtOAc/hexanes); 1H NMR (CDCl3) δ 7.83-7.30 (m,
20H), 5.16-4.97 (m, 1H), 4.84-4.53 (m, 1H), 4.10-3.62 (m,
2H), 3.48-2.98 (m, 6H), 2.21-1.80 (m, 2H), 1.15-0.93 (m,
18H); 13C NMR (CDCl3) δ 136.1, 135.9, 135.8, 135.5, 133.6,
133.5, 129.7, 129.5, 127.6, 127.5, 110.1, 106.5, 104.8, 103.0,
74.7, 71.4, 63.7, 63.1, 56.0, 55.7, 50.8, 49.1, 40.2, 39.6, 27.0,
26.9, 26.7, 19.4, 19.2, 19.1; IR (neat) 3049, 2932, 2857, 1589,
1472, 1113, 1027, 823 cm-1; HRMS (FAB) M - OCH3 calcd
+
for C38H47O4Si2, 623.3013; found, 623.3238.
Preparation of 19. TBAF‚hydrate (1.49 g, 4.74 mmol) was
added to a 0 °C solution of 18 (620 mg, 0.95 mmol) in THF
(12 mL). The mixture was allowed to warm to room temper-
ature and was stirred overnight, at which time it was
concentrated on a rotary evaporator. The crude compound was
purified by column chromatography (0-5% MeOH in CH2Cl2)
to afford 20 as two sets of mixtures, each containing two
diastereomers of 19 (minor set, 20 mg, 11.8%; major set,
97 mg, 57.4%). Only the major set of isomers was characterized
and carried on. 19: Rf 0.34 (10% MeOH/CH2Cl2); 1H NMR
(CDCl3) δ 5.25 (dd, J ) 4.4, 5.6 Hz, 1H), 4.35-4.33 (m, 1H),
3.85-3.69 (m, 2H), 3.45 (s, 3H), 3.31 (s, 3H), 3.13 (brs, 1H),
2.29-2.12 (m, 2H); 13C NMR (CDCl3) δ 109.4, 106.7, 104.2,
103.8, 75.8, 72.6, 60.6, 58.9, 56.2, 56.0, 49.7, 48.9, 40.0, 39.0;
IR (neat): 3430, 2937, 1447, 1266, 1100, 1022, 739 cm-1
.
Preparation of 20a and 20b. A solution of cyclododecyloxy
bis-trimethylsilyloxy chloride (188 mg, 0.44 mmol) in CH2Cl2
(3 mL) was added to a 0 °C solution of 19 (45 mg, 0.25 mmol),
pyridine (391 mg, 4.95 mmol), and DMAP (8 mg, 0.07 mmol)
in CH2Cl2 (3 mL) over 10 min. After 5 h, the solution was
warmed to room temperature, quenched with saturated aque-
ous NaHCO3, and diluted with EtOAc (100 mL). The organic
layer was washed with saturated aqueous NaHCO3, H2O, and
brine, and dried over Na2SO4. The solution was then concen-
trated, and the crude compound was purified by column
chromatography (0-10% EtOAc in hexanes containing 0.5%
triethylamine) to afford 20a and 20b (20a 40 mg, 28.2%; 20b
25 mg, 17.6%). 20a: Rf 0.43 (1:4 EtOAc/hexanes); 1H NMR
(CDCl3) δ 5.27 (t, J ) 5.6 Hz, 1H), 4.25-4.22 (m, 1H), 4.04-
4.00 (m, 1H), 3.93 (dd, J ) 11.6, 48 Hz, 2H), 3.73-3.71 (dd,
J ) 1.6, 3.6 Hz, 1H), 3.44 (s, 3H), 3.30 (s, 3H), 2.27-2.13 (m,
2H), 1.69-1.25 (m, 22H), 0.15 (s, 9H), 0.14 (s, 9H); 13C NMR
(CDCl3) δ 110.1, 107.1, 75.5, 71.3, 58.4, 56.1, 48.6, 39.4, 31.9,
24.3, 23.8, 23.4, 23.3, 23.2, 20.9, 20.7, 1.5, 1.4; IR (film) 3463,
2932, 2864, 1469, 1252, 1083, 843 cm-1; HRMS (FAB) M -
OCH3+ calcd for C24H51O7Si3, 535.2943; found, 535.2924. 20b:
Rf 0.35 (1:4 EtOAc/hexanes); 1H NMR (CDCl3) δ 4.97 (dd, J )
4.0, 5.6 Hz, 1H), 4.27 (dd, J ) 8.0, 14.8 Hz, 1H), 4.02-3.97
(m, 1H), 3.72 (dd, J ) 15.2, 53.2 Hz, 2H), 3.43 (s, 3H), 3.42 (s,
3H), 2.75 (d, J ) 7.6 Hz, 1H), 2.48-2.41 (m, 1H), 1.98-1.92
(m, 1H), 1.67-1.25 (m, 22H), 0.13 (s, 18H); 13C NMR (CDCl3)
δ 104.5, 104.4, 72.4, 70.8, 61.5, 55.9, 49.9, 39.4, 32.0, 24.4, 24.0,
23.3, 23.2, 20.8, 1.5; IR (film) 3566, 2932, 2852, 1470,
Preparation of 8. N,N-Diisopropyl 2-cyanoethyl phos-
phoramidic chloride (233 mg, 0.98 mmol) was added to a
solution of 15 (160 mg, 0.20 mmol) and diisopropylethylamine
(257 mg, 2.0 mmol) in CH2Cl2 (1.8 mL). After 7 h at room
temperature, the mixture was diluted with EtOAc (80 mL) and
washed with saturated aqueous NaHCO3 (2×), H2O, and brine.
The organic layer was dried over Na2SO4, filtered, and
concentrated. The crude compound was purified by column
chromatography (oven dried silica gel, 20% EtOAc in hexanes)
to afford a diastereomeric mixture of 8 (150 mg, 75.1%).
Rf 0.48 (1:4 EtOAc/hexanes); 1H NMR (CDCl3) δ 7.46-7.17 (m,
9H), 6.85-6.79 (m, 4H), 4.32-4.03 (m, 2H), 3.93-3.51 (m, 6H),
3.79 (s, 6H), 3.42-3.35 (m, 1H), 3.15-3.03 (m, 2H), 2.59-2.39
(m, 2H), 1.89-1.34 (m, 2H), 1.19-1.03 (m, 12H), 0.91-0.84
(m, 27H), 0.13-0.05 (m, 18H); 13C NMR (CDCl3) δ 158.4, 144.0,
136.3, 136.1, 130.1, 130.0, 128.3, 128.2, 127.6, 126.6, 117.6,
117.5, 113.0, 86.4, 75.6, 74.8, 74.0, 70.5, 70.1, 67.9, 65.4, 65.2,
58.2, 58.0, 57.8, 57.6, 55.1, 43.2, 43.1, 43.0, 42.8, 37.1, 36.1,
35.4, 26.0, 25.9, 25.8, 24.8, 24.7, 24.6, 24.5, 20.2, 20.1, 18.4,
18.3, 18.1, 18.0, -3.7, -3.8, -3.9, -4.0, -4.1, -4.3, -4.4, -4.8,
-4.9, -5.2, -5.3, -5.4; 31P NMR (CDCl3) δ 151.3, 151.0, 147.5,
147.1; IR (film) 2929, 2857, 1608, 1510, 1463, 1252, 1077,
+
1252, 1083, 847 cm-1; HRMS (FAB) M - OCH3 calcd for
C24H51O7Si3, 535.2943; found, 535.3060.
Preparation of 16a and 16b. N,N-Diisopropyl methyl
phosphoramidic chloride (35 mg, 0.18 mmol) was added to a
0 °C solution of 20a (40 mg, 70 µmol) and diisopropylethy-
lamine (45 mg, 0.35 mmol) in CH2Cl2 (1 mL). The solution was
allowed to warm to room temperature and was stirred over-
night. The reaction mixture was diluted with EtOAc and
washed with saturated aqueous NaHCO3, H2O, and brine. The
layers were separated, and the aqueous phase was extracted
with CH2Cl2. The combined organic layers were dried over
Na2SO4 and concentrated, and the crude compound was
purified by column chromatography (0-5% EtOAc in hexanes
containing 0.5% triethylamine) to afford a mixture of two
diastereomers of 16a (25 mg, 49%). 16a: Rf 0.67 (1:4 EtOAc/
834 cm-1
; HRMS (MALDI-TOF) M +
Na+ calcd for
C54H91N2O8NaSi3P, 1033.5713; found, 1033.5744.
Preparation of 18. N-Bromosuccinimide (1.09 g,
6.15 mmol) was added to a -10 °C solution of 179 (0.86 g,
1.23 mmol) in a mixture of CH3CN/MeOH (30:1, 15.5 mL).
After 1 h, the solution was warmed to room temperature and
quenched with saturated aqueous NaS2O3 (5 mL). The layers
were separated, and the aqueous phase was extracted with
8128 J. Org. Chem., Vol. 70, No. 20, 2005

C4′-Oxidized Abasic Site Containing Oligonucleotides
hexanes); 1H NMR (CDCl3) δ 5.23-5.18 (m, 1H), 4.40-4.30
(m, 1H), 4.01-3.78 (m, 3H), 3.63-3.54 (m, 2H), 3.45-3.32 (m,
9H), 2.43-2.28 (m, 1H), 2.23-2.15 (m, 1H), 1.64-1.25 (m,
22H), 1.19-1.15 (m, 12H), 0.13-1.12 (m, 18H); 31P NMR
(CDCl3) δ 149.4, 148.7; IR (film) 2933, 2867, 1470, 1364, 1252,
formamide loading buffer. Lesion integrity was determined by
reacting both the precursor (2a-d) and the lesion (3a-d)
containing duplexes (5 µL) with 5 µL of a 2× enzyme solution
containing 50 mM HEPES-KOH (pH 7.5), 50 mM KCl, 10%
glycerol, 1 mM dithiothreitol, 100 µg/mL BSA, and 100 nM
endonuclease IV. The reactions were incubated at 37 °C for
45 min and then quenched by the addition of 10 µL of
formamide. Background cleavage was determined by treating
aliquots (5 µL) with the 2× enzyme buffer (5 µL) described
above without enzyme and quenching with 10 µL of formamide
to determine background cleavage. All samples were analyzed
on a 12% denaturing polyacrylamide gel.
UV Melting Experiments. Duplexes (5 µM) were prepared
by heating 21a or 21b and the appropriate complement in a
one-to-one ratio in 10 mM PIPES (pH 7.0), 10 mM MgCl2, and
100 mM NaCl at 90 °C for 5 min. The duplexes were cooled to
room temperature over 2.5 h, followed by an additional period
of 2.5 h at 4 °C. Solutions of 2.2 µM were prepared by diluting
the hybridized duplex in PIPES buffer. Melting experiments
were carried out in 1-cm path length quartz cells containing
150 µL of duplex solution covered by 100 µL of mineral oil.
UV absorbance was recorded as the temperature was ramped
at 0.5 °C/min from 15 to 85 °C.
+
1185, 1090, 1060, 976, 846 cm-1; HRMS (FAB) M - OCH3
calcd for C31H67NO8PSi3, 696.3912; found, 696.4069.
Phosphitylation was carried out on 20b (25 mg, 43.8 µmol)
under the same conditions to provide 16b (20 mg, 63%). 16b:
Rf 0.62 (1:4 EtOAc/hexanes); 1H NMR (CDCl3) δ 4.97-4.94 (m,
1H), 4.57-4.43 (m, 1H), 4.00-3.80 (m, 2H), 3.70-3.56 (m, 3H),
3.45-3.36 (m, 9H), 2.57-2.41 (m, 1H), 2.09-2.01 (m, 1H),
1.63-1.28 (m, 22H), 1.20-1.16 (m, 12H), 0.14-1.13 (m, 18H);
31P NMR (CDCl3) δ 149.7, 149.1; IR (film) 2932, 1469, 1379,
1261, 1184, 1086, 847 cm-1; HRMS (FAB) M + H+ calcd for
C32H71NO9PSi3, 728.4174; found, 728.3860.
Preparation of Duplexes Containing C4-AP. Duplexes
containing 1 (2) were prepared by hybridizing the 32P-labeled
lesion containing oligonucleotide with 1.5 equiv of the ap-
propriate complement in a solution containing 100 mM KCl
and 10 mM MgCl2. Hybridization was carried out at 90 °C for
5 min, followed by cooling to room temperature over 5 h.
Duplexes 2a-d (120 nM) were photolyzed at 350 nm for
30 min in 50 mM KCl and 10 mM MgCl2 immediately prior to
use.
Photoconversion of 1 to C4-AP and Analysis Using
Endo IV. Duplexes containing the bases A, T, G, and C (2a-
d) opposite 1 were prepared as described above. The strand
containing 1 was 5′-labeled prior to hybridization. Duplexes
2a-d (20 nM) were photolyzed at 350 nm for 30 min in
100 mM KCl and 10 mM MgCl2. Portions of 2a-d (20 nM) in
100 mM KCl and 10 mM MgCl2 were also prepared and stored
in the dark. The photoconversion of 1 in each solution was
determined by incubating 5-µL aliquots with 0.2 M NaOH
(5 µL) for 20 min at 37 °C. The reactions were quenched by
adding 1 M HCl (1 µL) and diluting with 9 µL of 95%
Acknowledgment. We are grateful for support of
this research from the National Institute of General
Medical Sciences (GM-063028). We thank Aaron C.
Jacobs for assistance with the UV melting tempera-
tures.
Supporting Information Available: General experimen-
tal methods. NMR spectral data of 4, 6, 8, 12-16, 18-20.
ESI-MS of oligonucleotides (9, 10, 21). This material is
available free of charge via the Internet at http://pubs.acs.org.
JO0512249
J. Org. Chem, Vol. 70, No. 20, 2005 8129