Synthesis and analysis of oligonucleotides containing abasic site analogues

Article abstract of DOI:10.1021/jo702614p

(Figure Presented) DNA damage results in the formation of abasic sites from the formal hydrolysis of the glycosidic bond (AP) and several oxidized abasic lesions. Previous studies on AP sites revealed that DNA polymerases preferentially incorporated dA op

Full text of DOI:10.1021/jo702614p

Synthesis and Analysis of Oligonucleotides Containing Abasic Site
Analogues
Haidong Huang and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins UniVersity, 3400 N. Charles St., Baltimore, Maryland 21218
mgreenberg@jhu.edu
ReceiVed December 6, 2007
DNA damage results in the formation of abasic sites from the formal hydrolysis of the glycosidic bond
(AP) and several oxidized abasic lesions. Previous studies on AP sites revealed that DNA polymerases
preferentially incorporated dA opposite them in ∼80% of the replication events in Escherichia coli.
These results were consistent with the hypothesis that the AP sites are noninstructive lesions due to the
absence of a Watson-Crick base whose bypass adheres to the “A-rule.” Recent replication studies of the
oxidized abasic lesion, 2-deoxyribonolactone (L), revealed that DNA polymerase(s) does not apply the
A-rule when bypassing it and incorporates large amounts of dG opposite L. These studies suggested that
abasic sites such as L do direct polymerases to selectively incorporate nucleotides opposite them. However,
it was not possible to determine the structural basis for this molecular recognition from these experiments.
A group of oligonucleotides containing analogues of the AP and L lesions were synthesized and
characterized as probes to gain insight into the structural basis for the distinct effect of 2-deoxyribonolactone
on replication. These molecules will be useful tools for studying replication in cells and in vitro.
Introduction
hydrogen atom to diffusible species such as hydroxyl radical,
L is formed in 3-times greater yield than AP sites.6,7 Further-
more, C4-AP results from abstraction of the C4′-hydrogen atom
that rests on the outer edge of the minor groove and forms a
carbon hydrogen bond that is only marginally stronger than that
between the C1′-carbon and hydrogen.6 Nonetheless, the γ-ra-
diolysis-induced yield of C4-AP is only ∼1/10 that of 2-deoxy-
ribonolactone (L).8 One explanation for the higher than expected
yield of L involves its formation as the 5′-component of tandem
lesions that result from initial radical addition to the pyrimidine
base (Scheme 1).9,10
Abasic sites are a family of DNA lesions that lack the
heterocycles involved in Watson-Crick base pair formation in
duplex DNA. The unoxidized abasic site, AP, results from
spontaneous cleavage of the glycosidic bond but is also formed
when DNA is exposed to oxidative stress.1 A variety of oxidized
abasic sites (e.g., L, C4-AP) are produced when DNA is exposed
to oxidative stress. 2-Deoxyribonolactone (L) is produced by a
(3) Xue, L.; Greenberg, M. M. Angew. Chem., Int. Ed. 2007, 46, 561-
564.
(4) Roginskaya, M.; Razskazovskiy, Y.; Bernhard, W. A. Angew. Chem.,
Int. Ed. 2005, 44, 6210-6213.
variety of anti-tumor agents that bind in the minor groove of
DNA and is the major abasic lesion produced when DNA is
exposed to γ-radiolysis or Fe‚EDTA, both of which produce
hydroxyl radical.2-5 Despite the inaccessibility of the C1′-
(5) Roginskaya, M.; Bernhard, W. A.; Marion, R. T.; Razskazovskiy,
Y. Radiat. Res. 2005, 163, 85-89.
(6) Miaskiewicz, K.; Osman, R. J. Am. Chem. Soc. 1994, 116, 232-
238.
(7) Balasubramanian, B.; Pogozelski, W. K.; Tullius, T. D. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9738-9743.
(8) Dhar, S.; Kodama, T.; Greenberg, M. M. J. Am. Chem. Soc. 2007,
129, 8702-8703.
(1) Lindahl, T. Nature 1993, 362, 709-715.
(2) Xi, Z.; Goldberg, I. H. In ComprehensiVe Natural Products Chemistry;
Kool, E. T., Ed.; Elsevier: Amsterdam, 1999; Vol. 7, pp 553-592.
10.1021/jo702614p CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/07/2008
J. Org. Chem. 2008, 73, 2695-2703
2695

Huang and Greenberg
SCHEME 1. 2-Deoxyribonolactone Formed via Internucleotidyl Hydrogen Atom Abstraction
Nucleobase radicals are believed to account for as much as
90% of the reactive intermediates resulting from hydroxyl
radical addition to the π-bonds of the Watson-Crick bases, and
tandem lesions containing L account for more than 10% of the
chemistry attributable to at least one of these.9,11 The respective
nucleobase peroxyl radicals overcome the inaccessibility of the
C1′-hydrogen atom faced by a highly reactive diffusible species
(e.g., hydroxyl radical) by taking advantage of the helical twist
of the duplex, which brings the C1′-hydrogen atom of the 5′-
adjacent nucleotide into close proximity. The biochemical
significance of 2-deoxyribonolactone’s formation is exemplified
by its ability to form cross-links with DNA repair enzymes, as
well as its effects on replication.12,13 The distinct effect that L
has on replication in Escherichia coli provided the impetus for
the synthesis and characterization of the oligonucleotide probes
presented in this study.14
affects the conformation of the five-membered ring (Figure 1).
In order to probe the structural source for the differences in
polymerase bypass of AP and L, we synthesized a group of
oligonucleotides containing analogues of these lesions.
Results and Discussion
Probe Design. When evaluating the structural differences
between the AP and L lesions we focused primarily on the
hydrogen-bonding groups at C1, which are the most obvious.
The effect of the cyclic oxygen that is in conjugation with the
carbonyl in L on the conformation of the ring was also
considered. The structures of the hydroxylated monomeric
components were calculated at the B3LYP/6-31G(d.p) level
(Figures 1 and 2). These calculations were used as a guide
despite the possibility that incorporation into oligonucleotides
could alter their structures. Three molecules were considered
as candidates to probe whether the carbonyl group is directly
responsible for the differences in replication of AP and L. The
lactam (Lm) and ketone (K) retain the hydrogen bond acceptor.
These molecules also allow us to probe the importance of the
cyclic oxygen and the conformation of the 5-membered ring.
The five membered rings of the lactam, 2-deoxyribonolactone
(L), and cyclopentanone (K) adopt different conformations. The
ketone (K) also does not require the cyclic atom at position
five to maintain coplanarity with the carbonyl group. In addition,
by retaining the C1-carbonyl group but varying the nature of
the ring the ketone (K) and Lm provide tests for whether the
cyclic oxygen plays a specific role in the bypass of 2-deoxyri-
bonolactone (L) as a hydrogen bond acceptor. In contrast, the
methylene cyclopentane (MCP) maintains the planarity of the
carbonyl group in L while eliminating the contribution of
hydrogen bonding.
Three other analogues were conceived to test the importance
of the hydrogen bond acceptor at the C1 position of an abasic
site (F, COH), as well as the importance of the conformation
of the 5-membered ring (CPE, CPA). The configurationally
stable, cyclonucleoside analogue of an AP site (COH) has been
studied previously in DNA.19 This molecule probes whether the
cyclic oxygen has any role in polymerase bypass. The tetrahy-
drofuran analogue (F) is commonly used to mimic AP sites
because of its chemical stability.18 The hydrocarbon analogues
(CPA, CPE) probe whether ring conformation significantly
influences polymerase activity. The cyclopentane analogue
As members of the family of abasic lesions, L was expected
to adhere to the “A-rule” and not instruct a DNA polymerase
to incorporate a particular nucleotide opposite it.15,16 2′-
Deoxyadenosine is typically incorporated opposite “noninstruc-
tive” lesions, such as AP and its tetrahydrofuran analogue (F)
in ∼80% of replication events.17,18 Surprisingly, transfection of
E. coli with a single stranded genome containing L resulted in
levels of dG incorporation opposite the lesion that were
comparable to those of dA.14 This led to the proposal that L
instructed the bypass polymerase, DNA polymerase V to
incorporate higher levels of dG than expected for a noninstruc-
tive lesion. The most obvious structural source for the differ-
ences in AP and L bypass was the presence of a hydrogen bond
acceptor in the latter compared to a hydroxyl group at the C1-
position of AP. The presence of the carbonyl group in L also
(9) Hong, I. S.; Carter, K. N.; Sato, K.; Greenberg, M. M. J. Am. Chem.
Soc. 2007, 129, 4089-4098.
(10) Carter, K. N.; Greenberg, M. M. J. Am. Chem. Soc. 2003, 125,
13376-13378.
(11) von Sonntag, C. Free-Radical-Induced DNA Damage and Its Repair;
Springer-Verlag: Berlin, 2006.
(12) Kroeger, K. M.; Hashimoto, M.; Kow, Y. W.; Greenberg, M. M.
Biochemistry 2003, 42, 2449-2455.
(13) Hashimoto, M.; Greenberg, M. M.; Kow, Y. W.; Hwang, J.-T.;
Cunningham, R. P. J. Am. Chem. Soc. 2001, 123, 3161-3162.
(14) Kroeger, K. M.; Jiang, Y. L.; Kow, Y. W.; Goodman, M. F.;
Greenberg, M. M. Biochemistry 2004, 43, 6723-6733.
(15) Strauss, B. S. DNA Repair 2005, 4, 951-957.
(16) Taylor, J.-S. Mutation Res. 2002, 510, 55-70.
(17) Lawrence, C. W.; Borden, A.; Banerjee, S. K.; LeClerc, J. E. Nucleic
Acids Res. 1990, 18, 2153-2157.
(18) Kroeger, K. M.; Goodman, M. F.; Greenberg, M. M. Nucleic Acids
Res. 2004, 32, 5480-5485.
(19) de los Santos, C.; El-khateeb, M.; Rege, P.; Tian, K.; Johnson, F.
Biochemistry 2004, 43, 15349-15357.
2696 J. Org. Chem., Vol. 73, No. 7, 2008

Oligonucleotides Containing Abasic Site Analogues
FIGURE 1. Computational analysis of monomeric 2-deoxyribonolactone (L) and analogues. (A) Energy minimized structures. (B) Electron density
maps viewed from the bottom faces of the molecules. Electron density correlates with color. Blue-red, minimum-maximum.
FIGURE 2. Computational analysis of monomeric abasic site (AP) and analogues. (A) Energy minimized structures. (B) Electron density maps
viewed from the bottom faces of the molecules. Electron density correlates with color. Blue-red, minimum-maximum.
SCHEME 2. Generation of Alkali-Labile Molecules in
Oligonucleotides
(CPA) has been used previously as a probe for methylase
enzyme activity.20
General Synthetic Strategy. Automated solid-phase syn-
thesis was the desired method for preparing oligonucleotides
containing the analogues. Syntheses of oligonucleotides contain-
ing AP, L, F, COH, and CPA have already been reported. We
anticipated that solid-phase oligonucleotide synthesis would be
routine for incorporating CPE and MCP following preparation
of the respective phosphoramidites because each of these
molecules was expected to be stable to the synthesis and alkaline
deprotection conditions. The only modification introduced was
to substitute tert-butyl hydroperoxide for I2 during the solid-
phase synthesis of oligonucleotides containing MCP and CPE
to guard against possible complications due to addition-
elimination reactions.
other alkali-labile lesions (e.g., C4-AP).26-28 In contrast, we
anticipated using mild periodate oxidation of a vicinal diol (1)
to generate K in oligonucleotides (Scheme 2). This method has
Incorporation of Lm and K in oligonucleotides is potentially
complicated by their alkali-lability, just as 2-deoxyribonolactone
(L) is. The instability of L is circumvented by incorporating it
in a more stable disguised form (e.g., 1, Scheme 2), which is
also been used for generating labile molecules in oligonucle-
otides.27,29,30 Although we expected that the lactam (Lm) would
photochemically transformed into the lesion following oligo-
nucleotide purification on an as-needed basis.21-25 Photochem-
(21) Kotera, M.; Bourdat, A.-G.; Defrancq, E.; Lhomme, J. J. Am. Chem.
istry has proven useful for preparing oligonucleotides containing
Soc. 1998, 120, 11810-11811.
(22) Kotera, M.; Roupioz, Y.; Defrancq, E.; Bourdat, A.-G.; Garcia, J.;
(20) Marquez, V. E.; Wang, P.; Nicklaus, M. C.; Maier, M.; Manoharan,
M.; Chrisman, J. K.; Banavali, N. K.; Mackerell, A. D. J. Nucleosides,
Nucleotides Nucleic Acids 2001, 20, 451-459.
Coulombeau, C.; Lhomme, J. Chem. Eur. J. 2000, 6, 4163-4169.
(23) Hwang, J.-T.; Tallman, K. A.; Greenberg, M. M. Nucleic Acids Res.
1999, 27, 3805-3810.
J. Org. Chem, Vol. 73, No. 7, 2008 2697

Huang and Greenberg
SCHEME 5. Ketone (K) Phosphoramidite Synthesisa
SCHEME 3. Lactam (Lm) Phosphoramidite Precursor
Synthesisa
a Reagents and conditions: (a) 9-BBN, THF; (b) Dess-Martin perio-
dinane, CH2Cl2; (c) Nysted’s reagent, THF; (d) OsO4, NMO, t-BuOH/H2O;
(e) TBSOTf, 2,6-lutidine, CH2Cl2; (f) Pd/C, H2, MeOH/CH2Cl2; (g) DMTCl,
Et3N, DMAP, CH2Cl2; (h) N,N′-diisopropyl 2-cyanoethyl phosphoramidic
chloride, N,N′-diisopropylethylamine, CH2Cl2.
a Reagents and conditions: (a) allyltributyltin, BF3‚Et2O; (b) camphor-
sulfonic acid, acetone; (c) O3, NaOH, MeOH/CH2Cl2; (d) 1:1 TFA/CH2Cl2;
(e) 3% TFA, MeOH; (f) K2CO3, MeOH; (g) DMTCl, pyridine.
alkaline conditions.35 In order to avoid competitive cyclization
by the primary alcohol the Boc and acetonide were deprotected
sequentially and the crude trifluoracetate salt was then depro-
tonated, resulting in spontaneous cyclization to 9.
SCHEME 4. Lactam (Lm) Phosphoramidite Synthesisa
Initially, phosphitylation was attempted directly on the
dimethoxytritylated lactam (10, Scheme 4). However, this led
to a mixture of N-phosphitylated product and material in which
the nitrogen and oxygen were functionalized. Consequently, the
lactam nitrogen was selectively acetylated (11) by transiently
protecting the hydroxyl group prior to preparing the phosphora-
midite (12). The oligonucleotides were synthesized using fast
deprotecting phosphoramidites. The oxidized abasic site was
introduced using a 15 min coupling time, while the native
nucleotides were coupled using standard coupling times.
Although some cleavage occurred at the site of the modification,
modest yields of 25a,b were obtained by deprotecting the
oligonucleotides in concentrated aqueous ammonia hydroxide
at room temperature for 3 h and purifying them by denaturing
polyacrylamide gel electrophoresis in a cold room.
a Reagents and conditions: (a) TMSCl, Et3N, THF; (b) AcBr, Et3N, THF;
(c) Et3N‚3HF, MeOH; (d) N,N′-diisopropyl 2-cyanoethyl phosphoramidic
chloride, N,N′-diisopropylethylamine, CH2Cl2.
be somewhat labile to alkaline conditions, we predicted that
the higher pKa of a lactam compared to the lactone would enable
us to deprotect oligonucleotides containing Lm under mild
alkaline conditions.31
Synthesis of Oligonucleotides Containing the Cyclopen-
tanone (K). The preparation of the requisite phosphoramidite
(17) proceeded through the dibenzyl protected cyclopentene (13,
Scheme 5).36 Benzyl protection of the alcohols was desirable
because of the ether’s orthogonality to the silyl protecting group
in 17. Cyclopentene 13 was obtained via either of two methods,
one of which was previously reported starting from cyclopen-
tadienide.36,37 The other proceeded through the diol (18, Scheme
6). We opted to transform 13 into the alcohol (14) via
regioselective hydroboration instead of the reported multistep
phenylselenylation, followed by hydrolysis and radical mediated
deselenylation.37,38 The desired regioisomer was observed as a
mixture of stereoisomers (85:15) to the exclusion of that
resulting from borane addition to the more hindered carbon. A
variety of oxidation methods were explored due to the propensity
for 15 to undergo â-elimination.39 Although PCC and Swern
oxidation conditions provided modest yields of the ketone, 15
was obtained in almost quantitative yield using the Dess-Martin
Synthesis of Oligonucleotides Containing the Lactam (Lm)
Analogue. The stereoselective preparation of an appropriate
phosphoramidite (12, Scheme 4) started with 5, which is derived
from the methyl ester of serine (Scheme 3).32 The aldehyde (5)
was allylated by allyltributyltin to yield an inseparable 6:1
mixture of diastereomers of 6. Comparison to the literature
indicated that the major allyl alcohol contained the desired
S-configuration.33 Separation of the diastereomers was achieved
following rearrangement of 6 to the acetonide (7) and only the
desired S isomer was carried forward.34 The ultimate C1-carbon
was introduced as the methyl ester (8) via ozonolysis under
(24) Tronche, C.; Goodman, B. K.; Greenberg, M. M. Chem. Biol. 1998,
5, 263-271.
(25) Goodman, B. K.; Greenberg, M. M. J. Org. Chem. 1996, 61, 2-3.
(26) Kim, J.; Gil, J. M.; Greenberg, M. M. Angew. Chem., Int. Ed. 2003,
42, 5882-5885.
(27) Kodama, T.; Greenberg, M. M. J. Org. Chem. 2005, 70, 9916-
9924.
(28) Aso, M.; Usui, K.; Fukuda, M.; Kakihara, Y.; Goromaru, T.;
Suemune, H. Org. Lett. 2006, 15.
(29) Shishkina, I. G.; Johnson, F. Chem. Res. Toxicol. 2000, 13, 907-
912.
(35) Marshall, J. A.; Garofalo, A. W. J. Org. Chem. 1993, 58, 3675-
3680.
(36) Ezzitouni, A.; Russ, P.; Marquez, V. E. J. Org. Chem. 1997, 62,
4870-4873.
(37) Ludek, O. R.; Meier, C. Synthesis 2003, 2101-2109.
(38) Marquez, V. E.; Russ, P.; Alonso, R.; Siddiqui, M. A.; Hernandez,
S.; George, C.; Nicklaus, M. C.; Dai, F.; Ford, H. J. HelV. Chim. Acta 1999,
82, 2119-2129.
(39) Bisacchi, G. S.; Chao, S. T.; Bachard, C.; Daris, J. P.; Innaimo, S.;
Jacobs, G. A.; Kocy, O.; Lapointe, P.; Martel, A.; Merchant, Z.; Slusarchyk,
W. A.; Sundeen, J. E.; Young, M. G.; Colonno, R.; Zahler, R. Bioorg. Med.
Chem. Lett. 1997, 7, 127-132.
(30) Kim, J.; Weledji, Y. N.; Greenberg, M. M. J. Org. Chem. 2004,
69, 6100-6104.
(31) Bordwell, F. G.; Fried, H. E. J. Org. Chem. 1991, 56, 4218-4223.
(32) Qi, X.; Wang, X.; Wang, L.; Wang, Q.; Chen, S.; Suo, J.; Chang,
J. Eur. J. Med. Chem. 2005, 40, 805-810.
(33) Marshall, J. A.; Seletsky, B. M.; Coan, P. S. J. Org. Chem. 1994,
59, 5139-5140.
(34) Hafner, A.; Duthaler, R. O.; Marti, R.; Rihs, G.; Rothe-Streit, P.;
Schwarzenbach, F. J. Am. Chem. Soc. 1992, 114, 2321-2336.
2698 J. Org. Chem., Vol. 73, No. 7, 2008

Oligonucleotides Containing Abasic Site Analogues
SCHEME 6. Methylenecyclopentane (MCP)
Phosphoramidite Synthesisa
standard solid-phase synthesis conditions, with the exceptions
that oxidation was carried out using t-BuOOH and that the
coupling time for 22 or 23 was extended to 15 min. Oligo-
nucleotides were purified by denaturing PAGE and characterized
by ESI-MS, following deprotection using concentrated aqueous
ammonia hydroxide.
a Reagents and conditions: (a) iPrO-Si(CH3)2CH2MgCl, CuCN, THF;
(b) KF, K2CO3, H2O2, MeOH/THF; (c) TBSOTf, 2,6-lutidine, THF; (d)
9-BBN, THF; (e) Dess-Martin periodinane; (f) Nysted’s reagent, THF; (g)
TBAF, THF; (h) DMTCl, Et3N, DMAP, CH2Cl2; (i) N,N′-diisopropyl
2-cyanoethyl phosphoramidic chloride, N,N′-diisopropylethylamine, CH2Cl2.
periodinane reagent. The tendency for 15 to undergo elimination
also presented a challenge for carrying out the subsequent
methylenation reaction. Although conventional Wittig olefina-
tion was unsuccessful, the more acidic Nysted’s reagent provided
a 55% yield of the desired alkene.39 Osmylation produced an
inseparable 60:40 mixture of diastereomers, which were carried
on together as their bis-TBDMS ethers (16). After cleaving the
benzyl protecting groups in 16 by hydrogenolysis. The new diol
was carried on to 17 via standard phosphoramidite preparative
procedures.
Comparison of the Stability of Oligonucleotides Contain-
ing 2-Deoxyribonolactone (L) and Alkali-Labile Analogues
(K, Lm). The sensitivity of the oligonucleotides to alkali
conditions is important for determining whether they will be
amenable to conditions required for their incorporation into
larger nucleic acid constructs (e.g., plasmid DNA) and form
determining the thermal stability of duplexes containing the
abasic sites. The sensitivity of the 32P-labeled oligonucleotides
to various buffer conditions was determined by measuring their
fragmentation by gel electrophoresis. Previous studies have
documented the susceptibility of L to alkaline treatment.23,41,42
Hence, it was not surprising that more than 90% of 24a was
cleaved upon mild NaOH treatment (0.1 M, 37 °C, 20 min)
(Table 1). The lactam (Lm, 25b) was more stable to these
conditions, which is consistent with our ability to deprotect
oligonucleotides containing Lm with concentrated aqueous
ammonia. The ketone (K, 26a) was considerably more suscep-
tible to NaOH treatment and was almost completely cleaved
even in formamide loading buffer without any alkaline treatment
(Table 1). Treatment of 26a with hydroxylamine to produce
the oxime stabilized the oligonucleotide toward cleavage,
supporting the proposal that the loading buffer induced cleavage.
The ketone also proved to be unstable to some conditions
commonly used for carrying out enzymatic transformations (e.g.,
Tris pH 7.5) required for incorporating the respective oligo-
nucleotides in single stranded genomes43 or UV-melting experi-
ments. For instance, more than 80% of 26a was cleaved in
PIPES (10 mM, pH 7.0) at 75 °C in 1 h. In contrast, no cleavage
of the Lm (25a) was observed and <15% of an oligonucleotide
containing 2-deoxyribonolactone (24a) was cleaved under these
conditions. Furthermore, although K (26b) underwent significant
cleavage in Tris (pH 7.5, 100 mM), less than 20% of the
oligonucleotide was cleaved at pH 7.0 after 3 h at 25 °C (Table
1).
Oligonucleotides (33a,b) containing the vicinal diol precursor
(3) were prepared using the same procedures described above
for introducing the lactam. The vicinal diol (3) was revealed
using Et3N‚3HF, following aqueous ammonia deprotection of
the other protecting groups and cleavage of the oligonucleotides
from the solid-phase support. The vicinal diol containing oligo-
nucleotides (33a,b) were purified by denaturing PAGE, and due
to its lability (see below) the ketone (26a,b) was generated on
an as needed basis using NaIO4 (5 mM) in sodium acetate buffer
(pH 6.0) (Scheme 2). ESI-MS analysis of 26a,b following
desalting indicated that the oxidation was complete after 60 min.
Synthesis of Oligonucleotides Containing the Methyl-
enecyclopentane (MCP). Postsynthetic modification was un-
necessary for preparing oligonucleotides containing MCP or
CPE because the molecules are stable to the alkaline deprotec-
tion conditions. Successful synthesis of 17 suggested that the
preparation of the necessary phosphoramidite for MCP incor-
poration (22) could proceed via a similar route. However,
selective debenzylation proved difficult in the presence of the
exocyclic alkene. Consequently, the bis-TBDMS cyclopentene
(19) was prepared from 1R,4S-1-acetoxy-4-hydroxycyclopent-
2-ene (Scheme 6).40 The silyl protected cyclopentene (19) was
hydroborated, oxidized (20), and methylenated (21) using the
same reagents as the dibenzyl derivative (13) described above
in 67% overall yield. Following desilylation of 21, the diol was
carried on to the phosphoramidite (22).
UV-Melting Measurements of Duplexes Containing Abasic
Sites. Abasic sites typically significantly depress the thermal
(40) Matsuumi, M.; Ito, M.; Kobayashi, Y. Synlett 2002, 1508-1510.
The synthesis of the phosphoramidite (23) needed for
preparing oligonucleotides containing these abasic site analogues
was straightforward and started from the diol (18) obtained from
1R,4S-1-acetoxy-4-hydroxycyclopent-2-ene (Scheme 6). Oli-
gonucleotides containing MCP or CPE were prepared using
(41) Roupioz, Y.; Lhomme, J.; Kotera, M. J. Am. Chem. Soc. 2002, 124,
9129-9135.
(42) Zheng, Y.; Sheppard, T. L. Chem. Res. Toxicol. 2004, 17, 197-
207.
(43) Delaney, J. C.; Essigmann, J. M. Methods Enzymol. 2006, 408,
1-15.
J. Org. Chem, Vol. 73, No. 7, 2008 2699

Huang and Greenberg
% cleavage
TABLE 1. Chemical Stability of Oligonucleotides Containing L, Lm, and K
molecule
conditions
temp (°C)
time (min)
1 (32a)
NaOH (0.1 M)′
NaOH (0.1 M)
NaOH (0.1 M)′
NaOH (0.1 M)′
NaOH (0.1 M)′
formamide loading buffer
PIPES (10 mM, pH 7.5)
PIPES (10 mM, pH 7.5)
PIPES (10 mM, pH 7.5)
PIPES (10 mM, pH 7.5)
PIPES (10 mM, pH 7.5)
PIPES (10 mM, pH 7.5)
Tris (100 mM, pH 7.5)
Tris (100 mM, pH 7.5) then
Tris (100 mM, pH 7.5)
Bis-Tris-propane‚HCl (10 mM, pH 7.0)
Bis-Tris-propane‚HCl (10 mM, pH 7.0) then
Bis-Tris-propane‚HCl (10 mM, pH 7.0)
37
37
37
37
37
25
75
75
75
75
75
75
0
20
20
20
20
20
5
60
180
60
180
60
180
60
60
180
60
60
0
93
61
0
>99
>95
14
92
0
L (24a)
Lm (25a)
3 (33a)
K (26a)
K (26a)
L (24a)
L (24a)
Lm (25a)
Lm (25a)
K (26a)
K (26a)
K (26a)
K (26a)
0
80
96
50
0
16
0
0
16
92
∼1
K (26a)
K (26a)
180
18
TABLE 2. UV-Melting Temperatures (°C) of Duplexes Containing
all of these molecules are sufficiently stable, even the cyclo-
pentanone (K), so as to permit their utilization as probes of
replication in cells.43 These molecules should prove useful for
elucidating why L induces high levels of dG incorporation
opposite it in E. coli, whereas AP sites adhere to the A-rule.14,18
Abasic Sitesa
N:N′ ) T:A
N:N′ ) C:G
X
P ) A
P ) G
P ) A
P ) G
Experimental Methods
T
F
68.0 ( 1.2b
58.1 ( 0.2
57.7 ( 0.3
58.0 ( 0.6
57.0 ( 0.5
56.2 ( 0.2
57.8 ( 0.6
58.1 ( 0.5
70.2 ( 0.6b
59.9 ( 0.3
59.1 ( 0.5
59.6 ( 0.2
58.9 ( 0.3
59.4 ( 0.4
58.9 ( 0.3
60.1 ( 0.4
Compound 7. Compound 529 (3.5 g, 15.3 mmol), allyltributyltin
(6.1 mL, 19.9 mmol), and boron trifluoride etherate (2.91 mL, 23
mmol) were stirred at -78 °C in methylene chloride (50 mL) for
3 h. Saturated sodium bicarbonate (30 mL) was added, the solution
was warmed to room temperature, and stirring was continued for
30 min. The mixture was extracted with ether (3 × 50 mL). The
organic layers were combined and washed with brine. The organic
layer was dried over magnesium sulfate and filtered through Celite.
The filtrate was concentrated to 30 mL and filtered through Celite
again. The solvent was evaporated and the crude product was
purified by column chromatography (2:1 hexanes/ethyl acetate) to
yield 6 as a mixture of diastereomers (2.6 g, 9.6 mmol, 63%).
A solution of compound 6 (2.6 g, 9.6 mmol) and 10-camphor-
sulfonic acid (222 mg, 0.96 mmol) was stirred at room temperature
in acetone (45 mL) for 48 h, until equilibrium was reached. Acetone
was evaporated, and the residue was dissolved in ether (50 mL)
and washed successively with saturated sodium bicarbonate solution
and brine. The organic layer was dried over sodium sulfate, and
the solvent was evaporated. The crude product was purified by
column chromatography (5:1 hexanes/ethyl acetate) to yield 7 (1.8
57.1 ( 0.5
56.6 ( 0.2
57.0 ( 0.7
56.6 ( 0.5
56.7 ( 0.3
57.4 ( 0.3
59.1 ( 0.3
60.3 ( 0.6
60.4 ( 0.3
60.5 ( 0.5
60.1 ( 0.5
58.3 ( 0.2
59.6 ( 0.6
61.5 ( 0.5
COH
CPA
CPE
L
Lm
MCP
a Tm’s are the average of 3 measurements ( SD. b Data taken from ref
39.
stability of duplex DNA. UV-melting temperatures of duplexes
containing the various abasic sites were measured, with the
exception of K, which was too unstable to heating. Melting
studies were carried out in two sequence contexts, with either
dA or dG opposite the abasic sites (Table 2). Purines were
chosen as the opposing nucleotides because they are most
frequently incorporated opposite L, AP, and F by polymerases
in E. coli.14,18 All of the abasic site analogues decreased the
Tm’s of duplexes containing the native nucleotides by ∼10 °C.44
The Tm’s of the duplexes containing dA opposite F were within
experimental error of those previously reported, supporting the
verity of the data in Table 2.44 Overall, duplexes containing a
flanking dC melted slightly higher than those in which the abasic
site was flanked by a 5′-dT. This was to be expected based
upon the greater stability of a G‚C base pair compared with an
A‚T base pair. In addition, with the exception of the duplex
containing L, the melting temperature was greater when the
abasic site was opposed by dG than dA in the same sequence
context. However, overall there were no significant differences
in thermal stability of duplexes containing the various abasic
sites.
1
g, 6.6 mmol, 69%). H NMR (CDCl3) δ 5.79 (m, 1H), 5.02 (m,
2H), 4.60 (s, br, 1H), 3.82 (br, 1H), 3.49 (br, 3H), 2.48 (br, 1H),
2.20 (m, 1H), 1.35 (m, 15H). 13C NMR (CDCl3) δ 155.1, 134.2,
116.8, 98.7, 79.5, 63.2, 49.0, 36.9, 28.2, 19.8. IR (KBr) 3334, 2987,
1722, 1533, 1388, 1369, 1241, 1201, 1044 cm-1. FAB-HRMS (M
+ H+) calcd for C14H26O4N 272.1862, found 272.1848.
Compound 8. A methanolic NaOH solution (2.5 M, 5 mL) was
added to a solution of 7 (780 mg, 2.9 mmol) in methylene chloride
(20 mL). Ozone was bubbled through at -78 °C while a yellow
precipitate formed. The solution turned deep blue after 45 min.
The reaction was extracted with ether (3 × 20 mL). The organic
layers were combined and dried over magnesium sulfate. The
solvent was checked for the presence of peroxides (KI) prior to
concentration in vacuo, and the crude product was purified by
column chromatography (2:1 hexanes/ethyl acetate) to yield 8 (475
mg, 1.6 mmol, 54%). 1H NMR (CDCl3) δ 4.42 (br, 1H), 4.06 (m,
1H), 3.68 (s, 3H), 3.52 (m, 2H), 3.64 (m, 1H), 2.50 (m, 1H), 1.45
Conclusions
The abasic site analogues described above are useful for
determining the structural features of abasic sites (AP) and
2-deoxyribonolactone (L) that are responsible for their distinct
biochemical effects. Under the appropriate buffer conditions,
(44) Kroeger, K. M.; Kim, J.; Goodman, M. F.; Greenberg, M. M.
Biochemistry 2004, 43, 13621-13627.
2700 J. Org. Chem., Vol. 73, No. 7, 2008

Oligonucleotides Containing Abasic Site Analogues
(m, 15H). 13C NMR (CDCl3) δ 155.1, 134.1, 116.8, 98.7, 79.5,
72.3, 63.2, 49.0, 36.9, 28.2, 28.0, 19.8. IR (KBr) 3369, 2994, 2947,
2872, 1747, 1719, 1688, 1528, 1459, 1438, 1390, 1367, 1299, 1201,
1167, 1080, 1043, 833 cm-1. FAB-HRMS (M + H+) calcd for
C14H26O6N 304.1760, found 304.1743.
(3:1 hexanes/ethyl acetate) to yield 15 (2.33 g, 7.53 mmol, 98%).
1H NMR (CDCl3) δ 7.38 (m, 10H), 4.58 (m, 4H), 4.20 (m, 1H),
3.61 (dd, 1H, J) 9, 5 Hz), 3.53 (dd, 1H, J ) 9, 3 Hz), 2.63 (m,
3H), 2.35 (dd, 1H, J ) 5, 1 Hz), 2.20 (dd, 1H, J ) 5, 1 Hz). 13C
NMR (CDCl3) δ 215.4 137.7(9), 137.7(5), 128.1(0), 128.0(8),
127.3(9), 127.3(4), 127.3(0), 127.2, 77.7, 72.8, 70.9, 70.1, 44.0,
42.0, 39.6. IR (KBr) 3064, 3030, 2857, 1740, 1497, 1454, 1365,
1199, 1168, 1146, 1121, 1028 cm-1. FAB-HRMS (M + H+) calcd
for C20H23O3 311.1647, found 311.1636.
Compound 16. TiCl4 (7.53 mmol, 7.53 mL, 1 M in methylene
chloride) was added dropwise to a suspension of Nysted’s reagent
in THF (17.17 g, 7.53 mmol, 20 wt % in THF) at 0 °C. Additional
THF (20 mL) was added to facilitate stirring. The suspension was
kept at 0 °C for 10 min before neat 15 (2.33 g, 7.53 mmol) was
added. The reaction was warmed to room temperature and stirred
overnight. Saturated sodium bicarbonate solution (5 mL) was added
to quench the reaction. The suspension was diluted with ethyl
acetate (50 mL) and filtered. Water (20 mL) was added to the
filtrate. The organic layer was separated, washed with brine, and
dried over magnesium sulfate. The solvent was removed in vacuo
and the crude product was purified by column chromatography (10:1
hexanes/ethyl acetate) to yield the olefination product of 15 (1.28
g, 4.15 mmol, 55%). 1H NMR (CDCl3) δ 7.35 (m, 10H), 4.92 (m,
2H), 4.54 (m, 4H), 3.90 (dd, 1H, J ) 12, 6 Hz), 3.35 (dd, 1H, J )
10, 1.5 Hz), 3.43 (dd, J ) 10, 1.5 Hz), 2.68 (m, 2H), 2.45 (m, 2H),
2.20 (dd, 1H, J ) 16, 6 Hz). 13C NMR (CDCl3) δ 148.2, 138.7,
138.5, 128.2(8), 128.2(6), 127.5(9), 127.4(6), 127.4(3), 127.4(0),
107.1, 80.9, 73.0, 71.4, 71.1, 45.2, 38.6, 34.0. IR (KBr) 3064, 3028,
2983, 2940, 2854, 1658, 1496, 1454, 1364, 1205, 1099, 1028, 878,
751, 697 cm-1. FAB-HRMS (MH+) calcd for C25H21O2 309.1855,
found 309.1851.
Compound 9. Compound 8 (400 mg, 1.3 mmol) was treated
with TFA/CH2Cl2 (1:1, 10 mL) at room temperature for 30 min.
The volatile fraction was removed in vacuo. A 3% solution of TFA
in methanol (10 mL) was added, and the reaction was stirred at
room temperature overnight. The solvent was removed in vacuo.
The residue was dissolved in methanol (10 mL). Potassium
carbonate (538 mg, 3.9 mmol) was added, and the reaction was
stirred at room temperature for 3 h. The reaction was diluted with
methanol (10 mL) and centrifuged. The supernatant was saved, and
the solid was washed with CH2Cl2/MeOH (1:1, 2 × 5 mL). The
combined supernatant was concentrated and purified by column
chromatography (1:3 methanol/CH2Cl2) to yield 9 (167 mg, 1.27
1
mmol, 97%). H NMR (d4-MeOH) δ 4.32 (m, 1H), 3.59 (m, 2H),
3.51 (m, 1H), 2.74 (dd, 2H, J ) 17, 7 Hz), 2.18 (dd, 2H, J) 17,
3 Hz). 13C NMR (d4-MeOH) δ 177.6, 68.6, 65.3, 62.1, 39.6. IR
(KBr) 3300 (br), 2928, 1696, 1445, 1270, 1084, 713 cm-1. FAB-
HRMS (M + H+) calcd for C5H10O3N 132.0661, found 132.0661.
Compound 10. Pyridine (5 mL) was added to a mixture of 9
(150 mg, 1.15 mmol) and dimethoxytrityl chloride (507 mg, 1.50
mmol). The reaction was stirred at room temperature for 12 h before
quenching with methanol (1 mL). The solvent was removed and
the crude product was purified by column chromatography (1:20
methanol/ethyl acetate) to yield 10 (500 mg, 1.15 mmol, 100%).
1H NMR (CDCl3) δ 7.42 (d, 2H, J ) 8 Hz), 7.19-7.31 (m, 7H),
6.80 (m, 5H), 4.19 (s, br 1H), 3.94 (b, 1H), 3.75 (m, 6H), 3.69 (b,
1H), 3.24 (m, 1H), 3.09 (m, 1H), 2.70 (dd, 1H, J ) 17, 7 Hz), 2.28
(dd, J ) 17, 3 Hz). 13C NMR (CDCl3) δ 176.68, 158.57, 158.54,
144.49, 135.70, 135.54, 130.06, 129.95, 128.06, 127.96, 126.95,
113.27, 86.50, 69.85, 64.44, 63.53, 55.20, 40.10. IR (KBr) 3398
(br), 2957, 2834, 1696, 1607, 1577, 1508, 1449, 1301, 1250, 1178,
1089, 1034, 910, 831, 731, 700 cm-1. ESI-HRMS (M + Na+) calcd
for C26H27NO5Na 456.1787, found 456.1789.
A solution of N-methylmorphine N-oxide (305 mg, 2.6 mmol)
and osmium tetraoxide (17 mg, 0.065 mmol) in a mixture of
t-BuOH/H2O (1:1, 12 mL) was added to the above olefination
product (400 mg, 1.3 mmol) at 0 °C. The reaction was warmed to
room temperature and stirred for 24 h. Sodium bisulfite (1.5 g)
was added, and the suspension was stirred for 30 min. The mixture
was diluted with methylene chloride (30 mL) and water (10 mL).
The aqueous layer was extracted with methylene chloride (3 × 30
mL). The organic layers were combined and dried over magnesium
sulfate. The solvent was evaporated, and the crude product was
purified by column chromatography (1:4 hexanes/ethyl acetate) to
give the dihydroxylation product (380 mg, 1.11 mmol, 85%) as a
mixture of stereoisomers. 1H NMR (CDCl3) δ 7.32 (m, 10H), 4.52
(m, 4H), 4.12 and 3.98 (1H), 3.52-3.31 (m, 5H), 2.70 and 2.36
(2H), 2.19-2.02 (m, 2H), 1.83 and 1.75 (1H), 1.61 and 1.46 (1H).
13C NMR (CDCl3) δ 138.4, 138.2, 138.0, 137.6, 128.4(0), 128.2-
(9), 128.2(6), 127.7(5), 127.6(7), 127.5(8), 127.5(6), 127.5(3),
127.4(9), 127.4(7), 127.4(0), 82.7, 82.3, 81.6, 80.3, 73.3, 72.9, 72.1,
Compound 11. TMSCl (1.32 mmol, 145 mg, 169 µL) was added
to a solution of 10 (1 mmol, 440 mg) and triethylamine (2.64 mmol,
167 mg, 154 µL) in THF (10 mL) at 0 °C. The reaction was warmed
to room temperature and stirred for 2 h. Triethylamine (20 mmol,
2.02 g, 2.8 mL) was added, followed by acetyl bromide (10 mmol,
12.30 g, 0.75 mL). The reaction was continued at room temperature
for 2 h. Triethylamine trihydrofluoride (1 mmol, 161 mg, 16 µL)
and methanol (5 mL) were added, and the reaction was stirred at
room temperature for 15 min. The mixture was diluted with
methylene chloride (100 mL) and washed with saturated sodium
bicarbonate solution, followed by brine. The organic layer was dried
over magnesium sulfate. The solvent was removed, and the crude
product was purified by column chromatography (1:2 hexanes/ethyl
acetate) to give 11 (255 mg, 0.52 mmol, 52%). 1H NMR (CDCl3)
δ 7.22 (m, 10H), 6.82 (m, 4H), 4.30 (m, 2H), 3.78 (s, 6H), 3.49
(dd, 1H, J) 10, 4 Hz), 3.32 (dd, 1H, J ) 10, 2 Hz), 3.17 (dd, 1H,
J ) 18, 6 Hz), 2.52 (m, 3H), 2.49 (m, 1H), 1.81 (br, 1H). 13C
NMR (CDCl3) δ 174.3, 171.0, 158.6, 144.3, 135.6, 135.4, 129.9-
(1), 129.8(7), 127.9(6), 127.9(3), 127.0, 113.3, 86.8, 77.2, 67.3,
65.9, 61.9, 55.2, 42.9, 29.7, 25.2. IR (KBr), 3503(br), 2959, 2929,
2837, 1745, 1700, 1608, 1509, 1465, 1444, 1370, 1302, 1251, 1208,
1078, 1036, 834 cm-1. ESI-HRMS (M + Na+) calcd for C28H29-
NO6Na 498.1893, found 498.1895.
Compound 15. A solution of 1437 (2.4 g, 7.7 mmol) and Dess-
Martin periodane (3.27 g, 7.7 mmol) was stirred at room temper-
ature in methylene chloride (70 mL) for 1 h. The reaction was
diluted with diethyl ether (150 mL) and washed with a mixture of
10% sodium thiosulfate (50 mL), followed by saturated sodium
bicarbonate (50 mL). The aqueous layer was back-washed with
diethyl ether (100 mL). The organic layers were combined, washed
with brine, and dried over magnesium sulfate. The solvent was
evaporated, and the crude was purified with column chromatography
71.7, 71.3, 70.7, 69.3, 68.7, 44.6, 44.4, 43.4, 41.4, 38.7, 38.3 cm-1
.
IR (KBr) 3400 (br), 3005, 2932, 2852, 1474, 1397, 1113, 1098,
909, 734, 697 cm-1. FAB-HRMS (M + H+) calcd for C21H27O4
343.1909, found 343.1900.
TBSOTf (1.53 mmol, 404 mg, 351 µL) was added dropwise to
a solution of the above dihydroxylation product (0.51 mmol, 175
mg) and 2,6-lutidine (2.04 mmol, 219 mg, 238 µL) in methylene
chloride (10 mL) at 0 °C. The reaction was warmed to room
temperature, and stirring was continued for 6 h. The mixture was
diluted with ethyl acetate (100 mL) and washed with ammonium
chloride (1 M), followed successively with saturated sodium
bicarbonate and brine. The organic layer was dried over magnesium
sulfate. The solvent was removed in vacuo, and the crude product
was purified by column chromatography (20:1 hexanes/ethyl
acetate) to yield 16 (0.44 mmol, 249 mg, 81%) as a mixture of
1
diastereomers. H NMR (CDCl3) δ 7.34 (m, 10H), 4.58 (m, 4H),
3.98 and 3.72 (1H), 3.62-3.41 (m, 4H), 2.59-2.24 (m, 2H), 2.00
to 1.66 (m, 3H), 0.95 (m, 18H), 0.11 (m, 12H). 13C NMR (CDCl3)
δ 138.9(2), 138.8(4), 138.7(6), 138.6(8), 128.2(6), 128.2(4), 128.2-
(2), 128.2(0), 127.6(3), 127.6(0), 127.5(3), 127.4(6), 127.3(8),
J. Org. Chem, Vol. 73, No. 7, 2008 2701

Huang and Greenberg
127.3(2), 83.0, 81.9, 81.4, 80.8, 72.9, 72.8, 71.6(0), 71.5(8), 71.4,
69.3, 69.2, 45.0, 44.9, 43.4, 42.9, 38.9, 38.8, 26.0, 25.9, 25.8(4),
25.8(0), 18.5, 18.3, 18.0(7), 18.0(6), -2.4(0), -2.4(3), -2.5, -2.6,
-5.4, -5.5. IR (KBr) 3030, 2958, 2927, 2883, 2855, 1497, 1463,
1388, 1259, 1112, 832, 773, 732, 697 cm-1. ESI-HRMS (M + Na+)
calcd for C33H54O4NaSi2 593.3458, found 593.3464.
39.3, 25.8, 25.7, 18.2, 18.0, -4.7, -4.9, -5.5, -5.7, IR (KBr),
2961, 2936, 2894, 2855, 1750, 1469, 1400, 1361, 1254, 1195, 1107,
1002, 912, 839, 776, 670 cm-1. ESI-HRMS (M + H+) calcd for
C18H39O3Si2 359.2438, found 359.2438.
Compound 21. The Nysted olefination of 20 was performed at
a 2.60 mmol scale (930 mg of 20, 5.91 g Nysted’s reagent (20 wt
% in THF)) using the same procedure for the preparation of 16.
The crude product was purified by column chromatography (50:1
Hydrogenolysis of 16. The preparation of 16 was repeated to
obtain enough material for the next reaction. Pd/C (84 mg) was
added to a solution of 16 (420 mg, 0.74 mmol) in a mixture of
MeOH/CH2Cl2 (1:2, 10 mL). The suspension was stirred under H2
pressure (50 psi) for 2 h. The solution was filtered through Celite
and washed with ethyl acetate (50 mL). The filtrate was concen-
trated in vacuo, and the crude was purified by column chromatog-
raphy (1:10 hexanes/ethyl acetate) to give the diol (263 mg, 0.67
mmol, 91%). 1H NMR (CDCl3) δ 4.19 and 3.94 (m, 1H), 3.72 (m,
1H), 3.61-3.39 (m, 3H), 3.20 (br, 2H), 2.30-2.02 (m, 4H), 1.46
and 1.30 (1H), 0.85 (m, 18H), 0.08 (m, 12H). 13C NMR (CDCl3)
δ 83.5, 82.4, 76.8, 76.1, 69.5, 68.7, 66.0, 65.6, 49.2, 48.9, 46.1(4),
46.0(7), 38.6, 37.9, 25.9(2), 25.8(8), 25.7(5), 25.7(2), 13.4, 18.3,
17.9(6), 17.9(5), -2.4(2), -2.4(6), -2.4(9), -5.51. IR (KBr) 3405
(br), 2958, 2937, 2839, 2854, 1465, 1429, 1390, 1348, 1248, 1158,
1099, 1056, 999, 830, 767 cm-1. ESI-HRMS (M + NH4+) calcd
for C7H16NO2 146.1181, found 146.1178.
1
hexanes/ethyl acetate) to yield 21 (0.76 g, 2.12 mmol, 82%). H
NMR (CDCl3) δ 4.86 (m, 2H), 4.08 (dd, J ) 10, 6 Hz), 3.60 (m,
2H), 2.63 (dd, 1H, J ) 16, 6 Hz), 2.50 (dd, 1H, J ) 16, 8 Hz),
2.27 (dd, J ) 16, 6 Hz), 2.17 (m, 1H), 2.06 (m, 1H), 0.87 (m,
18H), 0.08 (m, 12H). 13C NMR (CDCl3) δ 148.6, 106.5, 73.5, 62.9,
50.1, 42.1, 33.0, 26.0, 25.9, 18.3, 18.1, -4.5, -4.8, -5.4, -5.5.
IR (KBr) 3074, 2957, 2934, 2894, 2855, 1659, 1469, 1390, 1365,
1115, 1057, 1010, 878, 840, 775, 671 cm-1. ESI-HRMS (M + H+)
calcd for C19H41O2Si2 357.2645, found 357.2657.
Desilylation of 21. A solution of compound 21 (0.76 g, 2.1
mmol) and tetrabutylammonium fluoride monohydrate (2.35 g, 8.4
mmol) in THF (15 mL) was stirred at room temperature for 18 h.
The solvent was evaporated, and the crude was purified by column
chromatography (ethyl acetate) to give the desilylation product of
21 (296 mg, 1.62 mmol, 77%). 1H NMR (CDCl3) δ 4.87 (m, 2H),
4.05 (dd, 1H, J ) 14, 7 Hz), 3.72 (dd, 1H, J ) 10, 5 Hz), 3.56 (dd,
1H, J ) 10, 7 Hz), 3.70-3.30 (br, 2H), 2.71 (dd, 1H, J ) 7, 1
Compound 18. A suspension of the dimethylisopropoxysilyl
ether of 1837 (1.4 g, 6.3 mmol), potassium fluoride (1.86 g, 32
mmol), potassium bicarbonate (1.9 g, 19 mmol) and hydrogen
peroxide (6 mL, 30% in H2O) in methanol/THF (1:1, 40 mL) was
heated at reflux for 15 h. Sodium thiosulfate (500 mg) was added.
The solution was diluted with ethyl acetate (400 mL) and
magnesium sulfate (2 g) was added. The mixture was filtered
through Celite, and the filtrate was concentrated in vacuo. The crude
product was purified by column chromatography (ethyl acetate) to
Hz), 2.67 (m, 1H), 2.33 (m, 1H), 2.12 (m, 1H), 0.95 (m, 1H). 13
NMR (CDCl3) δ 146.9, 107.5, 75.8, 65.1, 48.8, 41.4, 33.1. IR (KBr)
C
3284 (br), 2945, 1658, 1430, 1346, 1156, 1076, 1021, 881 cm-1
.
ESI-HRMS (M + NH4+) calcd for C7H16NO2 146.1181, found
146.1178.
Oligonucleotide Synthesis. The 16-nucleotide long oligonucle-
otides were synthesized on an Applied Biosystems Incorporated
394 DNA synthesizer using standard reagents (Glen Research). Bz-
dA-CE, iBu-dG-CE, Ac-dC-CE and T were used as the phosphora-
midites for oligonucleotides containing F, L, CPA, CPE and MCP.
Fast deprotecting phosphoramidites (Pac-dA-CE, iPac-dG-CE, and
Ac-dC-CE) and T were used for synthesizing oligonucleotides
containing Lm, COH and K. Pivaloyl anhydride/2,6-Lutidine/THF
(1:1:8) was used as the capping reagent when fast deprotecting
phosphoramidites were used.45 t-BuOOH in toluene (1 M) was used
as oxidation reagent for CPA, CPE, and MCP. Deprotection was
carried out in concentrated aqueous ammonia at room temperature
for COH (12 h), K (12 h), and Lm (3 h), and at 55 °C for all other
oligonucleotides (12 h). The oligonucleotides were purified by 20%
denaturing PAGE and desalted by C-18 Sep-Pak cartridge. The
oligonucleotides were characterized by ESI-MS or MALDI-MS
(Lm) after ammonium acetate precipitation.
UV Melting Temperatures. Oligonucleotides and their comple-
mentary stands (2.2 µM for each strand) were hybridized in 10
mM PIPES (pH 7.0), 10 mM MgCl2 and 100 mM NaCl in a total
volume of 200 µL. The DNA was denatured at 90 °C (70 °C for
Lm), slowly cooled to room temperature, and kept at 0 °C overnight.
During the melting experiment the temperature was increased from
25 to 75 °C at 0.5 °C/min. Readings were taken every 0.2 °C. The
melting temperatures were derived from the first derivative of the
melting curves.
1
yield 18 (578 mg, 5 mmol, 79%). H NMR (CDCl3) δ 5.78 (m,
1H), 5.54 (m, 1H), 4.34 (m, 1H), 3.73 (dd, J ) 10, 5 Hz), 2.75 (m,
2H), 2.50 (br, 1H), 2.30 (m, 2H). 13C NMR (CDCl3) δ 130.3, 129.0,
74.8, 63.9, 57.5, 41.4. IR (KBr) 3296 (br), 2959, 2922, 2853, 1467,
1351, 1054, 1017 cm-1. ESI-HRMS (M + NH4+) calcd for C6H14-
NO2 132.1025, found 132.1019.
Compound 19. The reaction was performed at a 4.79 mmol scale
(546 mg of 18, 14.37 mmol TBSOTf, 19.16 mmol 2,6-lutidine)
using the same silylation procedure employed for the preparation
of 16. The crude product was purified by column chromatography
(50:1 hexanes/ethyl acetate) to yield 19 (1.53 g, 4.47 mmol, 93%).
1H NMR (CDCl3) δ 5.67 (m, 1H), 5.61 (m, 1H), 4.24 (m, 1H),
3.49 (m, 1H), 2.70-2.56 (m, 2H), 2.21 (m, 1H), 0.87 (s, 18H),
0.51 (m, 12H). 13C NMR (CDCl3) δ 130.5, 129.3, 74.4, 57.9, 42.1,
25.9(4), 25.9(1), 18.4, 18.1, -4.5(6), -4.6(5), -5.3(8), -5.4(0).
IR (KBr), 3058, 2959, 2894, 2934, 2894, 2856, 1468, 1364, 1254,
1175, 1103, 1060, 1001, 840, 775 cm-1. ESI-HRMS (M + H+)
calcd for C18H39O2Si2 343.2489, found 343.2501.
Compound 20. 9-BBN (0.5 M solution in THF, 17 mL, 8.5
mmol) was added to a solution of 18 (2.85 g, 4.27 mmol) in THF
(10 mL) at 0 °C. The reaction was warmed to room temperature
and continued for 24 h. The reaction was cooled to 0 °C before
methanol (4 mL), NaOH (3 N, 10 mL) and hydrogen peroxide
(30%, 10 mL) were added successively. The mixture was stirred
at room temperature for 12 h and extracted with diethyl ether (4 ×
50 mL). The organic layers were combined and dried over
magnesium sulfate. The solvent was removed, and the crude product
was partially purified by column chromatography (1:1 hexanes/
ethyl acetate). The Dess-Martin oxidation (3.38 mmol Dess-
Martin periodinane) of the crude material was performed using the
same procedure employed for the preparation of 15. The crude
product was purified by column chromatography (hexanes/ethyl
Base Treatment of Oligonucleotides Containing L (24a), Lm
(25a), and K (26a). The 5′-OH of oligonucleotides 24a, 25a, and
26a were radiolabeled using a standard protocol.46 An aliquot (∼
0.25 pmol, 5 µL) of the 7-nitroindole precursor of 24a was irradiated
at 350 nm for 1 h using a Rayonet Photochemical Reactor equipped
with 16 λmax ) 350 nm lamps. An aliquot (∼ 0.40 pmol, 8 µL) of
the vicinal diol precursor of 26a was treated with NaIO4 (5 mM)
in NaOAc buffer (10 mM, pH 6.0) at room temperature for 1 h.
1
acetate, 10:1) to yield 20 (1.03 g, 2.87 mmol, 67% from 18). H
NMR (CDCl3) δ 4.38 (dd, 1H, J ) 12, 5 Hz), 3.75 (dd, 1H, J )
10, 4 Hz), 3.60 (dd, J ) 12, 5 Hz), 2.59 (dd, 1H, J ) 6, 1 Hz),
2.54 (dd, J ) 6, 1 Hz), 2.31 (m, 1H), 2.18 (m, 2H), 0.90 (m, 18H),
0.08 (m, 12H). 13C NMR (CDCl3) δ 216.5, 71.0, 62.1, 47.7, 47.4,
(45) Zhu, Q.; Delaney, M. O.; Greenberg, M. M. Bioorg. Med. Chem.
Lett. 2001, 11, 1105-1108.
(46) Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular Cloning; Cold
Spring Harbor Laboratory: Cold Spring Harbor, NY, 1982.
2702 J. Org. Chem., Vol. 73, No. 7, 2008

Oligonucleotides Containing Abasic Site Analogues
An aliquot of the precursor of 24a (1.8 µL) was treated with NaOH
(0.2 µL, 1 M) and incubated at 37 °C before being quenched with
HCl (1 equiv). DNA 24a, 25a, and 26a were treated with NaOH
by the same method. An aliquot of 26a (1.8 µL) was treated with
hydroxylamine (0.2 µL, 1 M, pH 4.0) and incubated at room
temperature for 30 min. The samples were mixed with 2 µL loading
buffer (95% formamide, 10 mM EDTA) and subjected to 20%
denaturing PAGE.
Thermal Stability of Oligonucleotides Containing L, K and
Lm. The radiolabeled oligonucleotides (∼0.25 pmol, 25 µL) 24a,
25a and 26a were heated to 75 °C in PIPES buffer (10 mM, pH
7.0). Ketone 26a was precipitated in 3 volumes of EtOH (-78 °C,
20 min) and resuspended in cold water prior to heating. An aliquot
(4.5 µL) was transferred to a new tube in an ice bath after 1, 2,
and 3 h. Ketone 26a was treated with hydroxylamine (0.5 µL, 1
M) and incubated at room temperature for 30 min. The samples
were mixed with 2 µL loading buffer (95% formamide, 10 mM
EDTA) and subjected to 20% denaturing PAGE.
Stability of 26a in Various Buffer Conditions. Ketone 26a (1.8
µL) was incubated at 0 °C for 1 h and then 16 °C for 3 h in TRIS
buffer (100 mM, pH 7.5) or buffer 1 (NEB, 20 mM bis-tris-propane,
10 mM MgCl2, pH 7.0). The samples were treated with hydroxy-
lamine (0.2 µL, 1 M), incubated at room temperature for 30 min,
mixed with 2 µL loading buffer (95% formamide, 10 mM EDTA),
and subjected to 20% denaturing PAGE.
Acknowledgment. We are grateful for support of this
research from the National Institute of General Medical Sciences
(GM-063028).
Supporting Information Available: General experimental
methods and procedures for the synthesis of small molecules and
oligonucleotides. NMR spectral data. ESI-MS and MALDI-TOF-
MS of oligonucleotides. This material is available free of charge
via the Internet at http://pubs.acs.org.
JO702614P
J. Org. Chem, Vol. 73, No. 7, 2008 2703