Preparation and analysis of oligonucleotides containing lesions resulting from C5′-oxidation

Article abstract of DOI:10.1021/jo051666k

Hydrogen atom abstraction from the C5′-position of nucleotides in DNA results in direct strand scission. The newly formed 5′-termini of the cleaved DNA consists of alkali-labile fragments of the oxidized nucleotide. One terminus contains a 5′-aldehyde as

Full text of DOI:10.1021/jo051666k

Preparation and Analysis of Oligonucleotides Containing Lesions
Resulting from C5′-Oxidation
Tetsuya Kodama and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218
mgreenberg@jhu.edu
Received August 8, 2005
Hydrogen atom abstraction from the C5′-position of nucleotides in DNA results in direct strand
scission. The newly formed 5′-termini of the cleaved DNA consists of alkali-labile fragments of the
oxidized nucleotide. One terminus contains a 5′-aldehyde as part of an otherwise undamaged
nucleotide (T-al). A second more structurally distinct product that is produced in lower yields results
from cleavage of the C4′-C5′ carbon-carbon bond. The 1,4-dioxo-2-phosphorylbutane (DOB) is a
precursor of the alkylating agent but-2-ene-1,4-dial. To facilitate studies on these lesions, methods
for synthesizing oligodeoxynucleotides containing DOB or T-al at their 5′-termini were developed.
The effects of these lesions on the UV-melting temperatures of duplex DNA, and their cleavage
labilities were determined. T-al cleaves very slowly (t1/2 ) 100.7 h), whereas DOB has a half-life
at 37 °C (pH 7.2) of less than 11 h. In addition, DOB forms a stable adduct very efficiently with
Tris, which protects the abasic site against cleavage.
Introduction
the mechanisms of their formation, and their biological
effects are well characterized.6-10 These types of studies
are facilitated by independent synthesis of oligonucle-
otides containing individual lesions.11,12 The products
resulting from C5′-oxidation are less well understood, in
part because methods for preparing oligonucleotides
containing them have not been reported. We describe
herein the synthesis and characterization of oligonucle-
otides containing the fragments produced upon C5′-
oxidation.
C5′-Hydrogen atom abstraction gives rise to direct
strand breaks containing two major families of products
at the newly formed termini (Scheme 1). Some reagents
produce fragments containing 3′-phosphate and 5′-
termini containing a C5′-oxidized but otherwise intact
DNA strand breaks resulting from oxidative processes
require hydrogen atom abstraction from the carbohydrate
backbone. Pathways for direct strand scission emanating
from C3′-, C4′-, or C5′-hydrogen atom abstraction are
precedented.1,2 The latter two positions are abstracted
by several minor groove binding molecules. The position-
ing of the C4′- and C5′-hydrogen atoms on the edge of
the minor groove also makes them accessible to diffusible
species.3,4 Some strand scission processes produce termini
that retain nucleotide fragments, which must be removed
as part of the repair process.5 Hence, consideration of the
interactions of these terminal fragments with biomol-
ecules could have important biological consequences. The
products resulting from C4′-hydrogen atom abstraction,
(6) Rabow, L. E.; Stubbe, J.; Kozarich, J. W. J. Am. Chem. Soc. 1990,
112, 3196-3203.
* To whom correspondence should be addressed. Phone: 410-516-
8095. Fax: 410-516-7044.
(7) Rabow, L. E.; McGall, G. H.; Stubbe, J.; Kozarich, J. W. J. Am.
Chem. Soc. 1990, 112, 3203-3208.
(1) Pratviel, G.; Bernadou, J.; Meunier, B. Angew. Chem., Int. Ed.
Engl. 1995, 34, 746-769.
(8) McGall, G. H.; Rabow, L. E.; Ashley, G. W.; Wu, S. H.; Kozarich,
J. W.; Stubbe, J. J. Am. Chem Soc. 1992, 114, 4958-67.
(9) Plastaras, J. P.; Dedon, P. C.; Marnett, L. J. Biochemistry 2002,
41, 5033-5042.
(2) Pogozelski, W. K.; Tullius, T. D. Chem. Rev. 1998, 98, 1089-
1107.
(3) Miaskiewicz, K.; Osman, R. J. Am. Chem. Soc. 1994, 116, 232-
238.
(4) Balasubramanian, B.; Pogozelski, W. K.; Tullius, T. D. Proc. Nat.
Acad. Sci. U.S.A. 1998, 95, 9738-9743.
(5) Demple, B.; Harrison, L. Annu. Rev. Biochem. 1994, 63, 915-
948.
(10) Kroeger, K. M.; Kim, J.; Goodman, M. F.; Greenberg, M. M.
Biochemistry 2004, 43, 13621-13627.
(11) Kim, J.; Gil, J. M.; Greenberg, M. M. Angew. Chem., Int. Ed.
2003, 42, 5882-5885.
(12) Chen, J.; Stubbe, J. Biochemistry 2004, 43, 5278-5286.
10.1021/jo051666k CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/14/2005
9916
J. Org. Chem. 2005, 70, 9916-9924

DNA Lesions from C5′-Oxidation
SCHEME 1. DNA Fragmentation Products
following C5′-Hydrogen Atom Abstraction
SCHEME 2. General Approach for Synthesizing
Oligonucleotides Containing T-al
SCHEME 3. Synthesis of a Phosphoramidite
Precursor (1) to T-ala
nucleotide (T-al).13-15 T-al is susceptible to cleavage upon
treatment with heat and/or alkali, and it forms adducts
with nucleophiles.15,16 Alternatively, the initially formed
C5′-radical ultimately gives rise to products resulting
from C4′-C5′ carbon-carbon bond fragmentation. The
newly formed 3′-terminus contains a phosphate group,
formed via hydrolysis of an activated formate.17 An
unusual oxidized abasic lesion, 1,4-dioxo-2-phosphorylbu-
tane (DOB), a 2-deoxytetrodialdose, is formed concomi-
tantly at the 5′-terminus of the other DNA fragment.18,19
The elimination product, 1,4-dioxo-but-2-ene alkylate
nucleosides, suggest that DOB may be toxic.20,21
a Key: (a) AD mix-â, tBuOH/H2O; (b) DMTCl, pyridine; (c) Ac2O,
Et3N, DMAP, CH3CN; (d) TBAF, THF; (e) 2-cyanoethyl diisopro-
pylchlorophosphoramidite, DIPEA, CH2Cl2.
Although phosphoramidites similar to 1 were previ-
ously reported, we synthesized the regioisomer via a
slightly different route (Scheme 3).24 Instead of utilizing
ring opening of an epoxide, the previously reported alkene
(3) was converted to the diol (4).24,25 Ligand-free OsO4
produced 4 as a 1.5:1 ratio of isomers in good yield (88%).
The alkene (3) was osmylated using AD mix-â to produce
a 4-5:1 mixture of diastereomers of 4 in comparable yield
(85%). Interestingly, AD mix-R produced the same dias-
tereoselectivity but the reaction was very slow and only
∼50% of 3 was converted after 24 h. Although it was not
necessary to control the stereoselectivity of the dihy-
droxylation reaction, AD mix-â was used in the synthesis
of 1 on a preparative scale because it facilitated purifica-
tion and characterization of the products. The diol was
then carried on to the phosphoramidite (1) via standard
protecting group transformations (Scheme 3).
During solid-phase synthesis, phosphoramidite 1 was
activated using tetrazole and coupled in >99% yield to
the solid-phase supported oligodeoxynucleotide for 15 min
prior to standard I2 oxidation. Following detritylation,
which enabled us to determine the coupling yield of 1,
and concentrated aqueous ammonia deprotection, the
vicinal diol (2, Scheme 2) containing oligonucleotide (7)
was purified by denaturing polyacrylamide gel electro-
phoresis and characterized by ESI-MS.26 The final step
in the synthesis of DNA containing T-al (8) was carried
out on nanomole quantities of 7 using NaIO4 in sodium
acetate buffer (pH 6.0) at 37 °C for 1 h. The crude
reactions were desalted, precipitated from ammonium
acetate and characterized by ESI-MS (Figure 1A). Two
species were observed in addition to the expected ion for
8. One ion corresponded to the anticipated elimination
product of T-al (m/z ) 4320.0). The third ion possessed
Results and Discussion
Synthesis and Characterization of DNA Contain-
ing a 5′-Aldehyde Nucleoside (T-al) at Its 5′-Termi-
nus. Oligonucleotides containing 5′-oxidized nucleosides
at their 5′-termini are labile to the alkaline conditions
typically used to deprotect oligonucleotides synthesized
on solid-phase supports and to heat.15,16 Consequently,
we introduced T-al into the oligonucleotide postsyntheti-
cally through phosphoramidite 1 (Scheme 2). The alde-
hyde was produced via NaIO4 treatment of the depro-
tected oligonucleotide containing the vicinal diol (2), a
method used previously to introduce alkali-labile alde-
hydes in DNA.22,23
(13) Kappen, L. S.; Goldberg, I. H.; Liesch, J. M. Proc. Nat. Acad.
Sci. U.S.A. 1982, 79, 744-748.
(14) Sugiyama, H.; Fujiwara, T.; Kawabata, H.; Yoda, N.; Hirayama,
N.; Saito, I. J. Am. Chem Soc. 1992, 114, 5573-8.
(15) Angeloff, A.; Dubey, I.; Pratviel, G.; Bernadou, J.; Meunier, B.
Chem. Res Toxicol. 2001, 14, 1413-1420.
(16) Pratviel, G.; Pitie´, M.; Bernadou, J.; Meunier, B. Angew. Chem.,
Int. Ed. 1991, 30, 702-704.
(17) Chin, D. H.; Kappen, L. S.; Goldberg, I. H. Proc. Nat. Acad.
Sci. U.S.A. 1987, 84, 7070-7074.
(18) Kawabata, H.; Takeshita, H.; Fujiwara, T.; Sugiyama, H.;
Matsuura, T.; Saito, I. Tetrahedron Lett. 1989, 30, 4263-4266.
(19) Chen, B.; Bohnert, T.; Zhou, X.; Dedon, P. C. Chem. Res. Toxicol.
2004, 17, 1406-1413.
(20) Gingipalli, L.; Dedon, P. C. J. Am. Chem Soc. 2001, 123, 2664-
2665.
(23) Kim, J.; Weledji, Y. N.; Greenberg, M. M. J. Org. Chem. 2004,
(21) Bohnert, T.; Gingipalli, L.; Dedon, P. C. Biochem. Biophys. Res.
Commun. 2004, 323, 838-844.
69, 6100-6104.
(24) Wang, G.; Middleton, P. J. Tetrahedron Lett. 1996, 37, 2739-
(22) Shishkina, I. G.; Johnson, F. Chem. Res. Toxicol. 2000, 13, 907-
912.
42.
(25) Sorensen, A. M.; Nielsen, P. Org. Lett. 2000, 2, 4217-4219.
J. Org. Chem, Vol. 70, No. 24, 2005 9917

Kodama and Greenberg
FIGURE 1. ESI-MS analysis of 8 prior to (A) or after (B) treatment with NaBH4. R ) CCG TAA TGC AGT CT-3′. / ) Na+
adduct.
m/z that could be unreacted 7 or the methanol adduct of
8 (m/z ) 4573.7), which is present in the matrix solution
used for sample analysis. The latter ion was absent
following treatment of the crude oxidized mixture with
NaBH4, suggesting that it was not unreacted 7 (Figure
1B). This indicates that conversion of 7 was complete.
In addition, analysis of the reduced material reveals that
most of the elimination product is an artifact of the ESI-
MS (Figure 1B).
Further evidence that T-al formation was complete and
FIGURE 2. Denaturing PAGE analysis of formation of T-al
did not undergo elimination to a significant extent was
in 8 from 2 by treatment with NaIO4. / ) slower migrating
obtained using freshly prepared 3′-32P-8. Formation and
product attributable to ARP adduct of T-al. R ) CCG TAA
reaction of T-al was examined using denaturing poly-
acrylamide gel electrophoresis (PAGE) (Figure 2). Reac-
tion of 3′-32P-7 (4 pmol, 80 nM) with NaIO4 (25 mM) for
1 h quantitatively converted the diol to T-al (3′-32P-8).
Less than 6% of elimination product was evident on the
gel using independently synthesized 5′-phosphate con-
taining fragmentation product as a migration marker.
The aldehyde did not react efficiently to form an oxime
(3.2%) with aldehyde reactive probe (ARP) at pH 7.2 but
was efficiently reduced (g89%) by NaBH4 and cleaved
using NaOH (0.1 M, 37 °C, 30 min).27 T-al was previously
shown to form an oxime but at pH 9.15
TGC AGT CT-3′-32P-ddA.
by the carbonyl group. Photochemistry and the o-ni-
trobenzyl photoredox reaction in particular are useful for
synthesizing oligonucleotides containing oxidized abasic
sites (L, C4-AP).11,28-30 On the basis of our reported
preparation of C4-AP, we pursued the synthesis of
phosphoramidite 9 as a precursor for DOB (Scheme 4).
The bis(o-nitrobenzyl)-protected abasic site would be
stable to the alkaline oligonucleotide deprotection condi-
tions and would enable us to unmask DOB on an as
Synthesis and Characterization of Oligonucle-
otides Containing 1,4-Dioxo-2-phosphorylbutane
(DOB) at Their 5′-Termini. Abasic sites (e.g. AP, L,
C4-AP) are typically very labile to alkaline conditions
due to the increased acidity of the R-protons imparted
(27) Ide, H.; Akamatsu, K.; Kimura, Y.; Michiue, K.; Makino, K.;
Asaeda, A.; Takamori, Y.; Kubo, K. Biochemistry 1993, 32, 8276-8283.
(28) Hwang, J.-T.; Tallman, K. A.; Greenberg, M. M. Nucleic Acids
Res. 1999, 27, 3805-3810.
(29) Crey-Desbiolles, C.; Lhomme, J.; Dumy, P.; Kotera, M. J. Am.
Chem. Soc. 2004, 126, 9532-9533.
(30) Zheng, Y.; Sheppard, T. L. Chem. Res. Toxiccol. 2004, 17, 197-
207.
(26) Supporting Information.
9918 J. Org. Chem., Vol. 70, No. 24, 2005

DNA Lesions from C5′-Oxidation
SCHEME 4. General Approach for Synthesizing Oligonucleotides Containing DOB
SCHEME 5. Attempted Synthesis of a
Phosphoramidite Precursor to DOBa
dimethoxytrityl ether of R-glycidol in place of the trieth-
ylsilyl group did not improve the overall yield (data not
shown). In situ deprotection of the dithiane and cycliza-
tion provided 18 as a mixture of 4 stereoisomers, along
with incompletely cyclized material. Complete separation
of diastereomers from undesired compounds was achieved
by silica gel chromatography of the desilylated products
(19). Although it was impractical and unnecessary for
preparative purposes, it was possible to separate 19 into
pairs of diastereomers by flash chromatography. After
doing so the 1S,4R-diastereomer of 19 was crystallized
from ethyl acetate and characterized by X-ray diffrac-
tion.26 In practice, the 4 isomers of 19 were carried on
together to produce a mixture of 8 diastereomers of 9.
a Key: (a) DMTCl, pyridine; (b) TBDPSCl, imidazole, DMF; (c)
DIBAL-H, toluene; (d) MsCl, Et3N, CH2Cl2; (e) I2, PPh3 imidazole,
toluene; (f) Li salt of dithiane.
SCHEME 6. Synthesis of a Phosphoramidite
Precursor (9) to DOBa
Oligonucleotides containing protected DOB (10, Scheme
4) were prepared using commercially available fast
deprotecting phosphoramidites and deprotected in con-
centrated aqueous ammonia at 25 °C for 12 h. Phos-
phoramidite 9 was coupled for an extended period (15
min). It was not possible to determine the coupling
efficiency of 9 in real time due to the lack of a dimethoxy-
trityl (DMT) group. However, examination of the puri-
fication gel and comparison of the intensity of the product
band with that of the single nucleotide deletion indicated
that the phosphoramidite coupled as well as those of the
native nucleotides (∼98%). Oligonucleotides containing
10 (20-22) were characterized by ESI-MS.26 Oligonucle-
otides containing 10 were irradiated (λmax ) 350 nm) for
20 min inside the cavity of a Rayonet photoreactor. ESI-
MS analysis revealed a similar distribution of types of
products detected during examination of T-al. No start-
ing material was detected by ESI-MS, indicating that the
precursor was completely converted. Analysis of 24 in a
MeOH/H2O (1:1 by volume) mixture indicated the pres-
ence of DOB, eliminated product, and a third product
exhibiting m/z 32 amu greater than that calculated for
the DOB containing oligonucleotide (Figure 3A). As was
the case above when analyzing T-al (8) by ESI-MS, the
higher molecular weight ion was believed to be a MeOH
adduct of DOB (or mixture of such adducts). Unlike the
C4-AP lesion, the cyclized form of DOB is either not
detected in the ESI-MS or present as a minor compo-
nent.11 The source of the ion (m/z ) 4333.8) corresponding
to eliminated material was of greater concern because
of its possible ramifications with respect to the photo-
chemical process. To distinguish between elimination
occurring as a result of photolysis or mass spectral
analysis, the crude photolyzate was treated with NaBH4
and then analyzed by ESI-MS (Figure 3B). Neither
a Key: (a) TBDMSCl, imidazole, DMF; (b) AcOH, THF, H2O (3:
2:1); (c) Dess-Martin periodinane, K2CO3, CH2Cl2; (d) 4,5-
dimethoxy-2-nitrobenzyl alcohol, NBS, CH3CN, CH2Cl2; (e) TBAF,
THF; (f) 2-cyanoethyl diisopropylchlorophosphoramidite, DIPEA,
CH2Cl2.
needed basis using a high yielding, reliable photochemi-
cal reaction.
Using the synthesis of the respective C4-AP phos-
phoramidite as a guide, we viewed 11 (Scheme 6) as a
key synthetic intermediate.11 Stereocontrolled synthesis
of 11 was initially pursued by attempted nucleophilic
substitution of the protected glycerol derivatives (14, 15)
(Scheme 5). The stereochemistry was set using the
asymmetric dihydroxylation of the p-methoxybenzoate of
allyl alcohol (12).31 However, dithiane anion displacement
of the mesylate (14) or iodide (15) was unsuccessful. The
dithiane protected dialdehyde (11) was prepared from 16,
which was synthesized using the previously reported
dithiane anion induced ring opening of the triethylsilyl
ether of R-glycidol (Scheme 6).32 Migration of the trieth-
ylsilyl group decreased the overall yield of 17. Using the
(31) Corey, E. J.; Guzman-Perez, A.; Noe, M. C. J. Am. Chem Soc.
1995, 117, 10805-10816.
(32) Mori, Y.; Asai, M.; Okumura, A.; Furukawa, H. Tetrahedron
1995, 51, 5299-5314.
J. Org. Chem, Vol. 70, No. 24, 2005 9919

Kodama and Greenberg
FIGURE 3. ESI-MS analysis of DOB in 24 (A) in MeOH:H2O and (B) after NaBH4 reduction. R ) TCG TAA TGC AGT CT-3′.
/ ) Na+ adduct.
FIGURE 5. Denaturing PAGE analysis of DOB (23) with or
without incubation with Tris (pH 7.2, 37 °C, 24 h). R ) CCG
TAA TGC AGT CT-3′-pddA.
FIGURE 4. Denaturing PAGE analysis of DOB (23) produced
upon photolysis of 20. R ) CCG TAA TGC AGT CT-3′-32P-
ddA.
converted it completely to an even faster moving product
that comigrated with independently synthesized mate-
rial. Similarly, treatment with NaBH4 produced a single
product that migrated more rapidly than 3′-32P-23 but
slightly more slowly than independently prepared DNA
containing a 5′-hydroxyl group. Overall, denaturing
PAGE and ESI-MS reveal that DOB is produced cleanly
and efficiently from the o-nitrobenzyl precursor within
DNA (10).
elimination product nor MeOH adduct was observed
under these conditions. The sole products observed
corresponded to the diol and its sodium ion adduct.
These experiments and all others described below were
carried out in nonnucleophilic buffer solutions due to
reaction of DOB with tris(hydroxymethyl)aminomethane
(Tris). Reaction of DOB with Tris was discerned by ESI-
MS on unlabeled material and via denaturing PAGE
using 3′-32P-23. Incubation of 3′-32P-23 (37 °C, 24 h) in
phosphate buffer pH 7.2 (20 mM) results in extensive
cleavage of DOB to product containing 5′-phosphate
(Figure 5). Treatment of the incubated material with
NaBH4 or NaOH results in reduction or elimination of
the remaining DOB lesion, respectively. In contrast, less
than 2% of 3′-32P-23 is cleaved following incubation in
Tris buffer (20 mM, pH 7.2) for 24 h (37 °C). Furthermore,
the oligonucleotide is not modified by treatment with
NaBH4 or NaOH. The change in reactivity suggests that
Additional evidence that supports the proposal that the
phosphate elimination product was an artifact of the ESI-
MS analysis was gleaned from denaturing PAGE analysis
of 3′-32P-20 (Figure 4). Photolysis produced a single
product (DOB, 3′-32P-23) that migrated more rapidly
than the photocaged material. The absence of any
photochemical precursor indicated that the photochemi-
cal conversion was complete. Mild treatment of the
photolyzed material with NaOH (0.1 M, 37 °C, 30 min)
9920 J. Org. Chem., Vol. 70, No. 24, 2005

DNA Lesions from C5′-Oxidation
confirmed that decomposition occurred via â-elimination
(Figure 2 and data not shown). T-al was very stable
under these conditions, exhibiting a 100.7 h half-life (k
) 1.84 × 10-6 s-1) for elimination (Figure 7A).
The dioxobutane abasic lesion (DOB) was much more
labile. Elimination from DOB was also monitored by
denaturing PAGE. However, the remaining lesion in
aliquots removed for analysis was stabilized by reducing
with NaBH4.34 Again, the independently synthesized 5′-
phosphate product produced upon elimination confirmed
the nature of the product produced from DOB (Figure 4
and data not shown) The half-life for the disappearance
of DOB in the respective ternary complex (Table 1) was
10.7 h (k ) 1.86 × 10-5 s-1). The DOB lesion is
considerably less stable than AP, L, or C4-AP are in
duplex DNA.12,30
UV-Melting Temperatures of DNA Containing
T-al and DOB. When T-al and DOB are formed in DNA,
they are adjacent to a 5′-fragment containing a 3′-
phosphate. In the case of T-al, stabilization due to
π-stacking is still possible. Melting temperatures were
determined using a 30 nucleotide long template in the
presence or absence of the corresponding 5′-fragment. It
is not possible to determine whether the melting of the
ternary complexes involves an equal and simultaneous
dissociation of all three strands. However, comparison
of the Tm’s measured for the duplex and ternary systems
indicate that there is some interaction between the
fragmented strands (Table 1). The Tm’s of the respective
ternary complexes are higher than the duplexes when
T-al or thymidine is present at the 5′-terminus of the
fragment. In addition, all of the Tm’s are higher than the
duplex containing the 5′-fragment employed in the
ternary complex hybridized to the template. It is also
interesting to note that DNA containing T-al melts at
higher temperatures than those containing thymidine.
It will be interesting to determine whether there is any
correlation between the high Tm of DNA containing T-al
and its recognition by DNA repair enzymes.
FIGURE 6. ESI-MS analysis of the reaction of DOB (23) with
Tris. R ) CCG TAA TGC AGT CT-3′. / ) Na+ adduct.
TABLE 1. UV-Melting Temperatures of Duplexes and
Ternary Complexesa,b
In contrast, ternary complexes containing the tetrahy-
drofuran analogue of an abasic lesion (F), DOB, or the
phosphate elimination product melted at lower temper-
atures than the respective duplexes. Furthermore, the
Tm’s of duplexes containing thymidine, DOB, F, or
phosphate were very similar to one another. Although
one might expect the duplexes that contain the same
charge but lack a 5′-terminus containing a nucleobase
to melt at similar temperatures (e.g. DOB, F, phosphate),
the similarity to the duplex containing thymidine might
seem surprising at first. However, the results are pre-
dicted using the HyTher program in which the dA
opposite thymidine is treated as a dangling end group
in duplexes containing the phosphate or either abasic site
(Table 2).35
a Oligonucleotides, 2 µM, sodium phosphate (pH 7.2), 10 mM,
NaCl, 100 mM. b p ) phosphate terminus.
the DOB lesion is modified by reaction with Tris. ESI-
MS analysis of the reaction between 23 (2.5 µM) and Tris
(1 mM, pH 7.2) revealed that the major product is a
monoadduct whose molecular weight corresponds to the
tricyclic monoadduct (Figure 6). A small amount of a bis-
adduct is also evident. The efficient trapping of DOB is
consistent with that of other 1,4-dial compounds and
appears to be more efficient than the previously reported
reaction between T-al and Tris at this pH.15,33
Chemical Stability of T-al and DOB Lesions. The
respective rate constants for â-elimination by the lesions
as part of ternary complexes (see Table 1) were deter-
mined in phosphate buffer (10 mM, pH 7.2) at 37 °C using
denaturing PAGE. Comparison of the migratory ability
of independently synthesized 5′-phosphate product with
that of the product observed from reaction of T-al
(33) Maiereanu, C.; Darabantu, M.; Ple´, G.; Berghian, C.; Con-
damine, E.; Ramondenc, Y.; Silaghi-Dumitrescu, I.; Mager, S. Tetra-
hedron 2002, 58, 2681-2693.
(34) Bailly, V.; Verly, W. G. Biochem. J. 1989, 259, 761-768.
(35) HyTher is available at: http://ozone2.chem.wayne.edu/Hyther/
hytherm1main.html.
J. Org. Chem, Vol. 70, No. 24, 2005 9921

Kodama and Greenberg
FIGURE 7. Decomposition within ternary complexes in sodium phosphate (50 mM, pH 7.2) of (A) T-al from 8 and (B) DOB from
23. See Table 1 for ternary complex structures.
TABLE 2. Predicted UV-Melting Temperatures of
Duplexesa,b
72.3, 71.4, 71.1, 64.8, 63.6, 40.1. 39.9, 25.70, 25.67, 17.9, 17.8,
12.5, 12.4, -4.5, -4.7, -4.9; IR (film) 3396, 3062, 2930, 2857,
1698, 1472, 1362, 1276, 1253, 1198, 1105, 1065, 843, 778 cm-1
;
FAB-HRMS (M+ + H+) calcd for C17H31N2O6Si 387.1951, found
387.1958.
Preparation of 5. A mixture of compound 4 (193 mg, 0.50
mmol) and DMTCl (339 mg, 1.0 mmol) in pyridine (5.0 mL)
was stirred at room temperature for 12 h. The resulting
mixture was diluted with ethyl acetate, washed with H2O,
saturated NaHCO3, and brine. After concentration of the dried
(Na2SO4) mixture, the crude mixture was purified by column
chromatography (25-33% ethyl acetate in hexanes) to give a
diastereomeric mixture (ca. 6:1) of 5 (344 mg, 0.50 mmol,
100%) as a foam: 1H NMR (CDCl3) δ 8.65 and 8.39 (each br s,
total 1 H), 7.71-7.16 (m, 32 H), 6.25 (t, 0.86 H, J ) 7.0 Hz),
6.18 (dd, 0.14 H, J ) 7.3 and 6.8 Hz), 4.49 (ddd, 0.15H, J )
5.9, 3.3, 3.0 Hz), 4.39 (m, 0.85 H), 4.09 (m, 1 H), 3.79 and 3.78
(each s, total 6H), 3.38 (t, 0.14 H, J ) 8.6 Hz), 3.24 (dd, 1 H,
J ) 9.4, 3.7 Hz), 3.15 (t, 0.86, J ) 9.2 Hz), 2.85 (br s, 0.86 H),
2.81 (br s, 0.14 H), 2.14 (dd, 1 H, J ) 7.0, 4.0 Hz), 1.92 and
1.91 (each d, total 3 H, each J ) 1.1 Hz), 0.89, 081 (each s,
a Predictions made using HyTher.35 Oligonucleotides, 2 µM,
Na+, 100 mM.
Conclusions
Methods for the synthesis of oligonucleotides contain-
ing the previously observed products resulting from C5′-
oxidation of thymidine in DNA have been developed. Both
T-al and DOB are alkali-labile, but the former is
significantly less so. T-al does not destabilize duplex
DNA compared to thymidine. DOB affects the thermal
stability of a duplex to a similar extent as does the
tetrahydrofuran model (F) of an abasic site. The chemical
properties and physical effects of T-al and DOB suggest
that their interactions with DNA repair enzymes warrant
examination. The synthetic method reported herein will
facilitate examination of the lesions’ biochemistry.
total 9 H), 0.07, 0.04, -0.04 and -0.12 (each s, total 6 H); 13
C
NMR (CDCl3) δ 171.2, 170.0, 169.6, 163.9, 163.8, 158.5, 150.3,
150.2, 144.4, 135.6, 135.5, 135.0, 134.6, 129.91, 129.85, 127.9,
127.8, 126.8, 113.1, 110.9, 110.7, 86.3, 86.2, 85.9, 85.8, 85.3,
84.7, 72.4, 72.3, 72.2, 71.9, 64.3, 62.6, 62.3, 60.3, 55.1, 53.8,
41.0, 40.4, 30.5, 29.2, 25.6, 25.4, 21.1, 21.0, 20.94, 20.91, 19.0,
17.8, 14.1, 13.6, 12.6, 12.5, -4.7, -4.8, -5.0; IR (film) 3448,
3189, 3061, 2929, 2856, 1694, 1608, 1510, 1470, 1251, 1177,
1058, 1035, 833, 778 cm-1; FAB-HRMS (M+) calcd for
C38H48N2O8Si 688.3180, found 688.3120.
Preparation of 6. A mixture of 5 (344 mg, 0.50 mmol),
Et3N (110 µL, 0.79 mmol, 80 mg), acetic anhydride (71 µL, 0.75
mmol, 77 mg), and DMAP (6 mg, 0.05 mmol) in acetonitrile
(5.0 mL) was stirred at room temperature for 2 h. The resulting
mixture was diluted with ethyl acetate, washed with H2O,
saturated NaHCO3, and brine. The dried (Na2SO4) mixture
was concentrated and purified by column chromatography
(33-50% ethyl acetate in hexanes) to give a diastereomeric
mixture (ca. 6:1) of 6 (359 mg, 0.49 mmol, 98%) as a yellow
foam: 1H NMR (CDCl3) δ 8.13 (br s, 1 H), 7.41-7.17 (m, DMTr,
CHCl3), 6.95 (d, 1H, J ) 1.2 Hz), 6.83-6.80 (m, 4 H), 6.25 (dd,
0.16 H, J ) 7.6, 5.6 Hz), 6.17 (dd, 0.84 H, J ) 8.8 and 5.2 Hz),
5.31 (m, 0.16 H), 5.25 (ddd, 0.84 H, J ) 6.0, 4.4 Hz), 4.33 (ddd,
0.84 H, J ) 5.6, 2.0, 1.8 Hz), 4.21 (m, 0.16 H), 4.08 (dd, 0.84
H, J ) 6.0, 2.0 Hz), 3.78 (s, 6 H), 3.40 (dd, 0.16 H, J ) 10.0,
7.6 Hz), 3.31-3.20 (m, 0.16 H), 3.27 (dd, 0.84 H, J ) 10.2, 6.2
Hz), 3.25 (dd, 0.84 H, J ) 10.2, 4.6 Hz), 2.26-2.14 (m, total 4
H), 1.92-1.81 (m, 4 H), 0.87, 0.86 (each s, total 9 H), 0.04,
0.03 and -0.01 (each s, total 6 H); 13C NMR (CDCl3) δ 171.2,
170.0, 169.6, 163.9, 163.8, 158.5, 150.3, 150.2, 144.4, 135.6,
135.5, 135.0, 134.6, 129.91, 129.85, 127.9, 127.8, 126.8, 113.1,
Experimental Methods
Preparation of 4. A mixture of AD-mix â (1.4 g) in tBuOH-
H2O (1:1, 10 mL) was cooled to 0 °C. A solution of compound
3 (350 mg, 1.0 mmol) was added to the suspension, and stirred
vigorously at 4 °C for 24 h. Solid sodium bisulfite (1 g) was
added to the reaction mixture, followed by extraction with CH2-
Cl2. The organic layers were combined, dried over Na2SO4, and
concentrated to dryness. The crude mixture was purified by
column chromatography (25-80% ethyl acetate in hexanes)
to give a diastereomeric mixture (ca. 5:1) of 4 (340 mg, 0.88
mmol, 88%) as a colorless amorphous solid: 1H NMR (CDCl3)
δ 9.53, 9.48 (each br s, total 1 H), 7.50 (d, 0.15 H, J ) 1.2 Hz),
7.40 (d, 0.85 H, J ) 1.2 Hz), 6.12 (t, 0.15 H, J ) 6.8 Hz) and
6.08 (dd, 0.85 H, J ) 7.6, 6.3 Hz), 4.59 (ddd, 0.85 H, J ) 5.9,
2.9, 2.9 Hz), 4.55 (ddd, 0.15 H, J ) 6.2, 3.1 Hz), 3.98-3.62 (m,
5 H), 3.06 and 2.95 (each br s, total 1 H), 2.40 (m, 1 H), 2.15
(ddd, 1 H, J ) 13.3, 6.2, 2.9 Hz), 1.89 and 1.88 (each d, total
3 H, each J ) 1.2 Hz), 0.88 and 0.88 (each s, total 9 H), 0.08,
0.08 and 0.08 (each s, total 6 H); 13C NMR (CDCl3) δ 164.10,
164.06, 150.6, 137.5, 111.12, 111.07, 88.0, 87.9, 87.4, 87.3, 72.8,
9922 J. Org. Chem., Vol. 70, No. 24, 2005

DNA Lesions from C5′-Oxidation
110.9, 110.8, 86.3, 86.2, 85.9. 85.8, 85.3, 84.7, 72.4, 72.3, 72.2,
71.9, 64.3, 62.6, 62.3, 60.3, 55.1, 53.8, 41.0, 40.4, 30.5, 29.2,
25.6, 25.4, 21.1, 21.0, 20.94, 20.91, 19.0, 17.8, 14.1, 13.6, 12.6,
12.5, -4.7, -4.8, -5.0; IR (film) 3183, 3056, 2954, 2931, 2857,
1745, 1694, 1608, 1510, 1464, 1371, 1251, 1177, 1059, 1035,
833, 779, 702, 596, 584 cm-1; FAB-HRMS (M+) calcd for
C40H50N2O9Si 730.3286, found 730.3301.
solid: 1H NMR (CDCl3) δ 4.05 (m, 2 H), 3.61 (m, 1 H), 3.49
(ddd, 1 H, J ) 12.0, 8.0, 4.0 Hz), 2.92-2.80 (m, 4 H), 2.10 (m,
1 H), 1.94-1.84 (m, 4 H), 0.91 (s, 9 H), 0.14 (s, 3 H), 0.11 (s 3
H); 13C NMR (CDCl3) δ 69.3, 66.3, 43.7, 39.4, 30.4, 30.1, 25.9,
25.8, 18.1, -4.57, -4.62; IR (film) 3284, 3186, 2926, 2895, 2853,
1248, 1126, 1110, 1071, 1039, 934, 836, 782, 744 cm-1; FAB-
HRMS (M+ + H) calcd for C13H29O2S2Si 309.1378, found
309.1347.
Preparation of Desilylated 6. A solution of TBAF (1.0 M
in THF, 900 µL) was added to a solution of 6 (435 mg, 0.60
mmol) in THF (5.0 mL) at room temperature, and the mixture
was stirred at the same temperature for 12 h. The resulting
mixture was concentrated, and the crude mixture was purified
by column chromatography (75-100% ethyl acetate in hex-
anes) to give the desilylated product as a mixture of diaster-
eomers (336 mg, 0.55 mmol, 92%) as a white foam: 1H NMR
(CDCl3) δ 8.50 (br s, 1 H), 7.42-7.21 (m, 9.12 H), 7.06 (d, 0.88
H, J ) 0.9 Hz), 6.85-6.83 (m, 4.12 H), 6.20 (t, 1 H, J ) 6.6
Hz), 5.29 (dd, 1 H, J ) 7.5 and 3.8 Hz), 4.68 (m, 1 H), 4.24 (m,
0.12), 4.10-3.97 (m, 1 H), 3.79-3.73 (m, 6.48 H), 3.52-3.47
(m, 1.88 H), 3.39 (dd, 0.12 H, J ) 10.2, 6.1 Hz), 3.31 (dd, 0.12
H, J ) 10.2, 4.5 Hz), 3.21 (dd, 0.88 H, J ) 10.8, 4.0 Hz), 2.45-
2.35 (m, total 1 H), 2.18 (s, 3 H), 1.91 (s, 3 H); 13C NMR (CDCl3)
δ 170.2. 170.0, 163.9, 163.8, 158.6, 158.54, 158.50, 150.39,
150.35, 144.3, 144.1, 135.5, 135.4, 135.3, 135.1, 134.8, 129.9,
129.8, 127.9, 127.8, 127.0, 126.9, 113.2, 113.2, 111.1, 86.8, 86.5,
85.9, 84.7, 84.4, 72.0, 71.3, 70.3, 62.6, 55.1, 40.0, 21.0, 12.6,
12.5; IR (film) 3417, 3194, 3055, 2933, 2837, 1694, 1608, 1509,
1465, 1371, 1250, 1177, 1035, 829 cm-1; FAB-HRMS (Na+)
calcd for C34H36N2O9 616.2421, found 616.2426.
Preparation of 11. A mixture of 17 (926 mg, 3.0 mmol),
Dess-Martin periodinane (1.91 g, 4.5 mmol), and anhydrous
K2CO3 (1.24 g, 9.0 mmol) in CH2Cl2 (90 mL) was stirred at 25
°C for 30 min. A mixture of saturated Na2S2O3 and saturated
NaHCO3 (1:4, 100 mL) was added to the resulting mixture and
stirred vigorously for 5 min. The organic layer was washed
with saturated NaHCO3 and brine and dried over Na2SO4. The
mixture was concentrated to give 11 (925 mg, 3.0 mmol, 100%)
as an amorphous solid: 1H NMR (CDCl3) δ 9.66 (d, 1 H, J )
1.2 Hz), 4.24 (ddd, 1H, J ) 6.8, 5.2, 0.8 Hz), 4.07 (t, 1 H, J )
7.4 Hz), 2.89 (ddd, 1 H, J ) 14, 6.8, 2.8 Hz), 2.82-2.69 (m, 3
H), 2.20-2.16 (m, 2 H), 1.97-1.88 (m, 1 H), 0.93 (s, 9 H), 0.11
(s, 6 H); 13C NMR (CDCl3) δ 203.3, 74.7, 41.2, 38.7, 28.7, 28.5,
25.7, 25.6, 18.1, -4.6, -4.9; IR (film) 2953, 2930, 2897, 2856,
1735, 1472, 1424, 1361, 1253, 1120, 938, 838, 779 cm-1; FAB-
HRMS (M+ + H) calcd for C13H27O2S2Si 307.1222, found
307.1247.
Preparation of 19. To a mixture of compound 11 (340 mg,
1.1 mmol) and 4,5-dimethoxy-2-nitrobenzyl alcohol (1.17 g, 5.5
mmol) in MeCN-CH2Cl2 (2:1, 30 mL) was added NBS (1.17
g, 6.6 mmol) at -10 °C. The resulting mixture was stirred for
1 h at this temperature and poured into an ice-cooled mixture
of saturated Na2S2O3 and saturated NaHCO3 (1:4). The
mixture was extracted with CH2Cl2, and the organic layer was
washed with saturated NaHCO3 brine, followed by drying over
Na2SO4 prior to concentration. The residue was purified by
column chromatography (20-25% ethyl acetate in hexanes)
to give an amorphous solid (319 mg) containing 18 and one or
more other products that are derivatized with 4,5-dimethoxy-
2-nitrobenzyl alcohol. A solution of the yellow amorphous solid
(281 mg) in THF (5.0 mL) was added to a solution of TBAF
(1.0 M in THF, 500 µL, 0.5 mmol) at 0 °C and stirred for 2 h
at this temperature. The mixture was evaporated, and the
residue was purified by column chromatography (a small
amount of CH2Cl2 was used as a loading solution and the
mixture was eluted using 60-70% ethyl acetate in hexanes)
to give a diastereomeric mixture of 19 (199 mg, 0.39 mmol,
40% from 11) as a yellow solid. One diastereomer was
crystallized from a mixture of two isomers using ethyl acetate
after further purification by column chromatography (65%
ethyl acetate in hexanes with 0.5% Et3N). Data for the
crystallized diastereomer (1S,4R-19): 1H NMR (CDCl3) δ 7.65,
7.64 (each s, each 1 H), 7.16, 7.08 (each s, each 1 H), 5.30 (dd,
1 H, J ) 5.6, 4.0 Hz), 5.15 (d, 1 H, J ) 4.4 Hz), 5.11 (s, 2 H),
5.03 (d, 1 H, J ) 14.6 Hz), 4.94 (d, 1 H, J ) 14.6 Hz), 4.34 (m,
1 H), 3.94, 3.92, 3.89, 3.88 (each s, each 3 H), 2.58 (ddd, 1 H,
J ) 13.2, 7.6, 5.6 Hz), 2.52 (d, 1 H, J ) 14.0 Hz), 2.09 (ddd, 1
H, J ) 13.2, 7.2, 4.0 Hz); 13C NMR (CDCl3) δ 153.4, 147.84,
147.80, 139.63, 139.57, 129.3, 129.1, 110.4, 110.3, 108.0, 103.4,
101.6, 71.4, 67.2, 67.0, 56.31, 56.27, 39.5. Data for a mixture
of 4 diastereomers: 1H NMR (CDCl3) δ 7.61, 7.60 (each s, each
1 H), 7.09, 7.02 (each s, each 1 H), 5.57 (dd, 1 H, J ) 5.6, 4.0
Hz), 5.16 (s, 1 H), 5.03-4.91 (m, 4 H), 4.50 (dd, 1 H, J ) 5.6,
2.8 Hz), 3.91, 3.91, 3.86, 3.83 (each s, each 3 H), 2.70 (br s, 1
H), 2.38 (ddd, 1 H, J ) 14.0, 5.6, 4.0 Hz), 2.25 (ddd, 1 H, J )
14.0, 5.6, 2.8 Hz); 13C NMR (CDCl3) δ 153.47, 153.45, 153.1,
153.33, 153.30, 153.23, 153.19, 147.9, 147.8, 147.61, 147.59,
147.55, 139.6, 139.5, 139.44, 139.41, 139.3, 139.2, 129.6,
129.40, 129.36, 129.30, 129.21, 129.15, 129.0, 110.6, 110.3,
110.2, 109.9, 109.7, 109.2, 108.8, 108.0, 107.94, 107.92, 107.85,
107.80, 107.7, 105.5, 105.0, 103.3, 102.5, 101.5, 101.3, 75.6,
74.4, 71.3, 70.7, 67.4, 67.2, 67.1, 66.8, 66.5, 66.2, 56.34, 56.25,
56.19, 56.17, 56.12, 56.07, 39.9, 38.8, 38.59, 38.57; IR (film)
Preparation of Phosphoramidite 1. To a solution of the
above alcohol (185 mg, 0.3 mmol) and DIPEA (105 µL, 0.60
mmol, 78 mg) in CH2Cl2 (3 mL) was added 2-cyanoethyl
diisopropylchlorophosphoramidite (100 µL, 0.45 mmol, 107 mg)
at 0 °C, and the resulting solution was stirred at room
temperature for 4 h. The reaction mixture was diluted with
ethyl acetate and washed with saturated NaHCO3, H2O, and
brine, After concentration of the dried (Na2SO4) mixture,
residue was purified by column chromatography (25-35%
ethyl acetate in hexanes with 0.5% Et3N) to give 1 (195 mg,
0.24 mmol, 80%) as a pale yellow amorphous solid: 1H NMR
(CDCl3) δ 7.42-7.18 (m, 9.14 H), 6.99-6.96 (m, 0.86 H), 6.84-
6.79 (m, 4 H), 6.29 (m, 0.14 H), 6.19 (dd, 0.86 H, J ) 8.4, 5.7
Hz), 5.40-5.23 (m, 1 H), 4.54 (m, 0.86 H), 4.44 (m, 0.14 H),
4.22 (m, 1 H), 3.89 (t, 0.4 H, J ) 6.2 Hz), 3.85-3.48 (m, 11 H),
3.42-3.23 (m, 2 H), 2.63-2.58 (m, 1.4 H), 2.54-2.40 (m, 2 H),
2.15, 2.14, 2.13, 2.12 (each s, total 3 H), 2.06-1.93 (m, 2.3 H),
1.89 (m, 0.4 H), 1.86 (m, 2.6 H), 1.19-1.13 (m, total 12 H); 13
C
NMR (CDCl3) δ 169.9, 163.83, 163.78, 158.4, 158.3, 150.3,
150.2, 144.4, 135.5, 135.4, 134.52, 134.46, 129.8, 127.9, 127.7,
127.6, 126.72, 126.66, 117.5, 117.4, 113.0, 112.9, 111.0, 86.3,
86.2, 84.9, 84.8, 84.6, 84.5, 84.3, 84.2, 73.6, 73.5, 72.8, 72.6,
72.2, 62.2, 62.8, 58.28, 58.27, 58.0, 57.8, 55.0, 43.2, 43.2, 43.1,
43.0, 38.9, 24.5, 24.4, 24.3, 24.2, 21.00, 20.96, 20.2, 20.1, 20.0,
19.9, 12.5, 12.3; 31P NMR (CDCl3) δ 149.36, 149.11, 149.03,
149.01; IR (film) 3189, 3056, 2967, 2933, 2837, 1743, 1693,
1608, 1583, 1509, 1465, 1366, 1250, 1179, 1036, 979, 830, 736
cm-1; FAB-HRMS ([M + Na]+) calcd for C43H53N4O10NaP
839.3397, found 839.3387.
Preparation of 17. Imidazole (450 mg, 6.6 mmol) and
TBDMSCl (500 mg, 3.3 mmol) were added to a solution of 16
(925 mg, 3.0 mmol) in DMF (30 mL) and stirred at room
temperature for 1 h. The reaction mixture was diluted with
diethyl ether, washed with H2O and brine, dried (Na2SO4), and
evaporated to give a yellow oil. A solution of AcOH-H2O (3:1,
24 mL) was added to the crude material in THF (12 mL) at 0
°C, and the mixture was stirred at this temperature for 2 h.
The resulting mixture was poured into ice-cooled saturated
NaHCO3, and the contents were extracted with CH2Cl2. The
combined organic layers were washed with saturated NaHCO3
and brine, dried with Na2SO4, and concentrated. The residue
was purified by column chromatography (5% acetone in
hexanes) to give 17 (400 mg, 1.3 mmol, 43%) as a colorless
J. Org. Chem, Vol. 70, No. 24, 2005 9923

Kodama and Greenberg
1581, 1520, 1325, 1275, 1220, 1066, 986, 873, 796, 756 cm-1
;
a G-25 Sephadex column that was equilibrated with H2O,
followed by washing with 50 µL of H2O. The pH of the
combined fractions was adjusted with 500 mM (5 µL) sodium
phosphate (pH 7.2) and concentrated to 50 µL. Larger scale
preparations of unlabeled 8 (e.g. 4 nmol/1 mL) were carried
out for 1 h at 37 °C. Samples were diluted with NH4OAc (10
mM, 4 mL) and desalted using a C18-Sep pak cartridge, which
was washed with NH4OAc (50 mM).
FAB-HRMS (M+) calcd for C22H26N2O12 510.1486, found
510.1480.
Preparation of Phosphoramidite 9. To a solution of 19
(255 mg, 0.5 mmol) in CH2Cl2 (5 mL) was added DIPEA (175
µL, 1.00 mmol, 130 mg) and 2-cyanoethyl diisopropylchloro-
phosphoramidite (170 µL, 0.76 mmol, 180 mg) at 0 °C. The
resulting solution was warmed to room temperature. After
being stirred for 1 h, the reaction mixture was diluted with
ethyl acetate, washed with saturated NaHCO3, H2O, and brine,
dried (Na2SO4), and concentrated. The residue was purified
by column chromatography (40-50% ethyl acetate in hexanes
with 0.5% Et3N) to give 9 (271 mg, 0.38 mmol, 76%) as a yellow
amorphous solid: 1H NMR (CDCl3) δ 7.74-7.00 (m, 4 H), 5.55-
4.85 (m, 6 H), 4.69-4.50 (m, 0.43 H), 4.40-4.22 (m, 0.57 H),
4.10-3.69 (m, 14 H), 3.67-3.51 (m, 2 H), 2.70-2.20 (m, 4 H),
1.38-1.01 (m, 12 H); 13C NMR (CDCl3) δ 153.6, 153.54, 153.47,
153.29, 153.27, 153.25, 153.23, 147.70, 147.68, 147.63, 147.59,
147.56, 147.52, 147.48, 139.5, 139.42, 139.39, 139.37, 139.35,
139.33, 139.25, 139.1, 138.8, 130.6, 130.3, 130.2, 129.8, 129.6,
129.50, 129.49, 129.42, 129.39, 129.35, 129.30, 117.55 117.50,
110.5, 110.4, 110.3, 110.24, 110.21, 110.15, 110.11, 110.08,
110.05, 110.02, 109.99, 109.85, 109.77, 108.11, 108.05, 108.00,
107.94, 107.91, 107.8, 107.7, 107.6, 105.7, 105.2, 105.1, 102.8,
102.6, 102.1, 100.6, 100.4, 67.3, 67.2, 67.14, 67.05, 66.8, 66.7,
66.7, 66.3, 66.1, 65.9, 58.5, 58.42, 58.36, 58.33, 58.24, 58.17,
58.06, 56.42, 56.36, 56.31, 56.26, 56.25, 56.20, 56.18, 56.17,
56.13, 56.11, 43.22, 43.18. 43.09, 43.05, 39.4, 39.3, 39.10, 39.05,
38.2, 37.1, 24.54, 24.47, 24.42, 24.35, 24.29, 24.27, 24.23, 20.34,
20.32, 20.30, 20.27, 20.23; 31P NMR (CDCl3) δ 148.79, 148.76,
148.70, 148.66, 148.55, 148.32; IR (film) 2967, 1581, 1520,
1413, 1363, 1327, 1276, 1221, 1067, 981, 876, 796 cm-1; FAB-
HRMS (M+) calcd for C31H43N4O13P 710.2564, found 710.2567.
Preparation of DOB-Containing Oligonucleotides.
Following radiolabeling using standard methods (terminal
deoxynucleotidyl transferase, R-32P-ddATP), the oligonucle-
otide (10 pmol) was separated from unincorporated R-32P-
ddATP using a C18-Sep pak cartridge. Photolysis (λmax ) 350
nm) was carried out in sodium phosphate (50 µL, 50 mM, pH
7.2) at room temperature for 20 min. When DOB was to be
used in duplex or ternary complexes, hybridization was carried
out prior to photolysis to avoid elimination of the lesion.
Nanomole scale preparations were carried out in TEAA buffer
(10 mM, pH 7.2). Following irradiation (20 min), TEAA was
removed using a C18-Sep pak cartridge and washing with NH4-
OAc (5 mM).
Reaction of DOB with Tris-HCl. 3′-32P-23 (10 pmol, 45
µL) in 10 mM sodium phosphate (pH 7.2) was mixed with 5
µL of 200 mM Tris-HCl buffer pH 7.2 and incubated at 37 °C
for 24 h. Each solution was divided into 3 tubes and mixed
with 1.8 µL of (I) H2O, (II) 500 mM NaBH4 in H2O, or (III) 1.0
M NaOH. The mixtures were incubated for 30 min at room
temperature (I, II) or at 37 °C (III). The NaOH reaction was
neutralized with 1.0 M HCl. Control experiments were carried
out in 10 mM sodium phosphate buffer (pH 7.2) without the
addition of Tris-HCl. After incubation, each solution was mixed
with 95% formamide loading buffer and analyzed by 20%
denaturing PAGE.
Decomposition Kinetics of DOB and T-al. Ternary
complexes of 3′-32P-7 (T-al) or 3′-32P-20 were prepared in 50
mM sodium phosphate (pH 7.2) and 100 mM NaCl. Complex
containing 10 was irradiated at room temperature for 20 min,
and the DNA was used without further purification. Complex
containing 7 was incubated in 25 mM NaIO4 and 100 mM
NaOAc (pH 6.0) at room temperature for 1 h and then passed
through a G-25 Sephadex column (50 mM sodium phosphate
(pH 7.2), 100 mM NaCl). The ternary complexes were incu-
bated at 37 °C for 24 h (DOB) or 96 h (T-al). Aliquots (4.5 µL)
were mixed with 0.5 µL of 0.5 M NaBH4, incubated at room
temperature for 30 min, and stored at -20 °C while awaiting
denaturing PAGE separation.
Thermal Denaturation Study of Duplexes. Oligonucle-
otides 8 and 23 were prepared as described above. Samples
were 2 µM in each oligonucleotide. Melts were carried out in
10 mM sodium phosphate (pH 7.2) and 100 mM NaCl, and
the temperature was increased at 0.5 °C/min. Readings were
taken every 0.2 °C.
Acknowledgment. We are grateful for support of
this research from the National Institute of General
Medical Sciences (Grant GM-063028). T.K. is grateful
for a Japan Society for the Promotion of Science Post-
doctoral Fellowship for Research Abroad.
Preparation of T-al-Containing Oligonucleotides. Fol-
lowing radiolabeling using standard methods (terminal deoxy-
nucleotidyl transferase, R-32P-ddATP), the oligonucleotide (10
pmol) was separated from unincorporated R-32P-ddATP using
a C18-Bakerbond spe cartridge (100 mg). The 3′-32P-labeled
oligonucleotide was reacted in 100 mM NaOAc (pH 6.0) with
25 mM NaIO4 at room temperature for 60 min (total volume:
50 µL). The oligonucleotide was desalted by passing through
Supporting Information Available: General experimen-
tal methods and procedures for the synthesis of 15 and 16,
NMR spectral data, and ESI-MS of oligonucleotides. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO051666K
9924 J. Org. Chem., Vol. 70, No. 24, 2005