DNA damage and interstrand cross-link formation upon irradiation of aryl iodide C-nucleotide analogues

Article abstract of DOI:10.1021/jo902071y

(Chemical Equation Presented) The 5-halopyrimidine nucleotides damageDNAupon UV-irradiation or exposure to γ-radiolysis via the formation of the 2′-deoxyuridin-5-yl σ-radical. The bromo and iodo derivatives of these molecules are useful tools for probing DNA structure and as therapeutically useful radiosensitizing agents. A series of aryl iodide C-nucleotides were incorporated into synthetic oligonucleotides and exposed to UV-irradiation and γ-radiolysis. The strand damage produced upon irradiation of DNA containing these molecules is consistent with the generation of highly reactive σ-radicals. Direct stand breaks and alkali-labile lesions are formed at the nucleotide analogue and flanking nucleotides. The distribution of lesion type and location varies depending upon the position of the aryl ring that is iodinated. Unlike 5-halopyrimidine nucleotides, the aryl iodides produce interstrand cross-links in duplex regions of DNA when exposed to γ-radiolysis or UV-irradiation. Quenching studies suggest that cross-links are produced by γ-radiolysis via capture of a solvated electron, and subsequent fragmentation to the σ-radical. These observations suggest that aryl iodide C-nucleotide analogues may be useful as probes for excess electron transfer and radiosensitizing agents.

Full text of DOI:10.1021/jo902071y

pubs.acs.org/joc
DNA Damage and Interstrand Cross-Link Formation upon Irradiation of
Aryl Iodide C-Nucleotide Analogues
Hui Ding and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 N. Charles Street, Baltimore, Maryland 21218
mgreenberg@jhu.edu
Received September 25, 2009
The 5-halopyrimidine nucleotides damage DNA upon UV-irradiation or exposure to γ-radiolysis via
the formation of the 20-deoxyuridin-5-yl σ-radical. The bromo and iodo derivatives of these
molecules are useful tools for probing DNA structure and as therapeutically useful radiosensitizing
agents. A series of aryl iodide C-nucleotides were incorporated into synthetic oligonucleotides and
exposed to UV-irradiation and γ-radiolysis. The strand damage produced upon irradiation of DNA
containing these molecules is consistent with the generation of highly reactive σ-radicals. Direct stand
breaks and alkali-labile lesions are formed at the nucleotide analogue and flanking nucleotides. The
distribution of lesion type and location varies depending upon the position of the aryl ring that is
iodinated. Unlike 5-halopyrimidine nucleotides, the aryl iodides produce interstrand cross-links in
duplex regions of DNA when exposed to γ-radiolysis or UV-irradiation. Quenching studies suggest
that cross-links are produced by γ-radiolysis via capture of a solvated electron, and subsequent
fragmentation to the σ-radical. These observations suggest that aryl iodide C-nucleotide analogues
may be useful as probes for excess electron transfer and radiosensitizing agents.
Introduction
(5YL, Scheme 1).5-12 The σ-radical is also produced upon
γ-irradiation of BrdU and IdU, possibly by reaction of the
5-halopyrimidines with solvated electrons, followed by
cleavage of the radical anion. The σ-radical (5YL) produces
alkali-labile lesions by abstracting hydrogen atoms from
50-adjacent nucleotides. The significantly higher reactivity
of 5YL compared to π-carbon radicals is the source for the
formation of alkali-labile lesions without the involvement of
O2, which is often required for “fixing” damage involving
DNA radicals. The ability to produce DNA damage under
O2-deficient conditions has resulted in the use of BrdU and
IdU as radiosensitizing agents in hypoxic tumors.13-15 Re-
cently, BrdU was shown to produce interstrand cross-links
The 5-halopyrimidines, most notably 5-iodo-20-deoxyuri-
dine (IdU) and 5-bromo-20-deoxyuridine (BrdU), are useful
probes for studying excess electron transfer in duplex DNA
and nucleic acid structure.1-4 The weak carbon-halogen
bond and their structural similarity to thymidine provide the
foundation for their utility in these applications. UV-irradia-
tion of BrdU and IdU produces a σ-radical, 20-deoxyuridin-5-yl
*To whom correspondence should be addressed. Phone: 410-516-8095
Fax: 410-516-7044.
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(3) Xu, Y.; Tashiro, R.; Sugiyama, H. Nat. Protoc. 2007, 2, 78–87.
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DOI: 10.1021/jo902071y
r
Published on Web 01/12/2010
J. Org. Chem. 2010, 75, 535–544 535
2010 American Chemical Society

Ding and Greenberg
JOCFeatured Article
SCHEME 1
SCHEME 2
SCHEME 3a
upon γ-irradiation in unpaired (“bubble”) regions of duplex
DNA, albeit not in hybridized regions.16-18 Interstrand cross-
links are an important family of DNA lesions because they are
absolute blocks to replication and transcription.19-21 The
biological significance of one particular ICL was recently
increased by the observation that it gives rise to even more
toxic double-strand breaks upon nucleotide excision repair.22
Molecules that produce ICLs in hybridized regions of DNA
upon γ-radiolysis and/or UV-irradiation might be useful as
new radiosensitizing agents, as well as mechanistic probes of
nucleic acid chemistry. We wish to report on aryl iodide
nucleotide analogues that exhibit these desirable properties.
aKey: (a) NaI, CuI, 5, pentan-1-ol; (b) DMTrCl, pyridine; (c) 2-Cyano-
ethyl-N,N0-diisopropyl chlorophosphoramidite, CH2Cl2.
DNA structure, replication, and nucleic acid-protein inter-
actions.28-32 In particular, IT has been used to explore the
flexibility of protein binding sites.33-36 This molecule provided
the starting point for our investigation, and led to the examina-
tion of the three monoiodinated nucleotide analogues (I2, I3,
and I4). It was assumed that irradiation of IT (Scheme 2), and
the monoiodides would produce the respective aryl radicals.
Synthesis of Iodo-Substituted Aryl Nucleotide Analogues
and Their Incorporation into Oligonucleotides. Oligonucleo-
tides containing IT and I3 were prepared via solid-phase
oligonucleotide synthesis using procedures previously repor-
ted by Kool and Sekine, respectively.37,38 The syntheses of
oligonucleotides containing I2 and I4 were carried out using
a similar strategy. Phosphoramidite 4 was synthesized from
the previously reported bromide (1, Scheme 3).39 Bromide (1)
was displaced by iodide using a mixture of NaI/CuI and
trans-N,N0-dimethyl-1,2-cyclohexanediamine (5) in a pressure
bottle in a manner similar to that previously described by
Kool.38,39 The iodide (2) was carried on to 4 via standard
methods. The phosphoramidite (10) used for synthesizing
Results and Discussion
Our choices of aryl iodides were based upon two disparate
bodies of literature. Aryl iodides are photochemical precur-
sors of aryl radicals.23,24 σ-Radicals are also generated from
their respective radical anions.25-27 The aryl radicals are
highly reactive and readily add to π-bonds and abstract
hydrogen atoms that are bonded to tetrahedral carbons. In
addition, nucleotide analogues containing aryl halides as
substitutes for pyrimidine nucleobases are useful probes of
(28) Lee, H. R.; Helquist, S. S.; Kool, E. T.; Johnson, K. A. J. Biol. Chem.
2008, 283, 14411–14416.
(29) Lee, H. R.; Helquist, S. A.; Kool, E. T.; Johnson, K. A. J. Biol. Chem.
2008, 283, 14402–14410.
(30) Delaney, J. C.; Henderson, P. T.; Helquist, S. A.; Morales, J. C.;
Essigmann, J. M.; Kool, E. T. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4469–
4473.
(16) Dextraze, M.-E.; Cecchini, S.; Begeron, F. O.; Girouard, S.; Turcotte,
K.; Wagner, J. R.; Hunting, D. J. Biochemistry 2009, 48, 2005–2011.
(17) Dextraze, M.-E.; Wagner, J. R.; Hunting, D. J. Biochemistry 2007,
46, 9089–9097.
(18) Cecchini, S.; Girouard, S.; Huels, M. A.; Sanche, L.; Hunting, D. J.
Biochemistry 2005, 44, 1932–1940.
(19) Noll, D. M.; Mason, T. M.; Miller, P. S. Chem. Rev. 2006, 106, 277–
301.
(31) Kool, E. T. Annu. Rev. Biochem. 2002, 71, 191–219.
(32) Pallan, P. S.; Egli, M. J. Am. Chem. Soc. 2009, 131, 12548–12549.
(33) Jarchow-Choy, S. K.; Sjuvarsson, E.; Sintim, H. O.; Eriksson, S.;
Kool, E. T. J. Am. Chem. Soc. 2009, 131, 5488–5494.
(34) Silverman, A. P.; Garforth, S. J.; Prasad, V. R.; Kool, E. T.
Biochemistry 2008, 47, 4800–4807.
€
(20) Scharer, O. D. ChemBioChem 2005, 6, 27–32.
(21) Dronkert, M. L.; Kanaar, R. Mutat. Res. 2001, 486, 217–247.
(22) Sczepanski, J.; Jacobs, A. C.; Van Houten, B.; Greenberg, M. M.
Biochemistry 2009, 7565–7567.
(23) Garden, S. J.; Avila, D. V.; Beckwith, A. L. J.; Bowry, V. W.; Ingold,
K. U.; Lusztyk, J. J. Org. Chem. 1996, 61, 805–809.
(35) Mizukami, S.; Kim, T. W.; Helquist, S. A.; Kool, E. T. Biochemistry
2006, 45, 2772–2778.
(24) Braslau, R.; Anderson, M. O. Tetrahedron Lett. 1998, 39, 4227–4230.
(25) Chami, Z.; Gareil, M.; Pinson, J.; Saveant, J. M.; Thiebault, A.
J. Org. Chem. 1991, 56, 586–95.
(26) Citterio, A.; Minisci, F.; Vismara, E. J. Org. Chem. 1982, 47, 81–8.
ꢀ
(27) M’Halla, F.; Pinson, J.; Saveant, J. M. J. Am. Chem. Soc. 1980, 102,
(36) Kim, T. W.; Delaney, J. C.; Essigmann, J. M.; Kool, E. T. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 15803–15808.
(37) Tawarada, R.; Seio, K.; Sekine, M. J. Org. Chem. 2008, 73, 383–390.
(38) Kim, T. W.; Kool, E. T. Org. Lett. 2004, 6, 3949–3952.
(39) Hwang, G. T.; Romesberg, F. E. Nucleic Acids Res. 2006, 34, 2037–
2045.
4120–4127.
536 J. Org. Chem. Vol. 75, No. 3, 2010

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SCHEME 4a
aKey: (a) (i) 1,4-diiodobenzene, n-BuLi, THF, (ii) Et3SiH, BF3 Et2O,
3
CH2Cl2; (b) Bu4NF, THF; (c) DMTrCl, pyridine; (d) 2-cyanoethyl N,
N0-diisopropylchlorophosphoramidite, CH2Cl2.
oligonucleotides containing I4 was prepared from 1,4-
diiodobenzene and the silylated 2-deoxyribonolactone (6)
employed in the syntheses of related molecules (Scheme 4).40
The phosphoramidites of the iodo nucleotide analogues
were incorporated into oligonucleotides via automated
solid-phase synthesis using an extended (5 min) coupling
time. Oligonucleotides containing Kool’s diiodo molecule
(IT) were prepared using commercially available (Glen
Research) fast-deprotecting phosphoramidites, and t-
BuOOH (1 M) was used as the oxidizing agent in place of
I2. These oligonucleotides were deprotected with concen-
trated NH4OH (9 h, 25 °C). Oligonucleotides containing I2,
I3, or I4 were prepared using standard I2 oxidation and were
deprotected using “AMA” conditions (1:1 aqueous methyl-
amine and concentrated NH4OH).41 The oligonucleotides
were purified by 20% denaturing polyacrylamide gel electro-
phoresis and characterized by ESI-MS following desalting.42
All experiments described herein were carried out using
duplexes (11-18) containing these modified oligonucleo-
tides. With the exception of hydroxyl radical digestion
experiments to determine the site(s) of cross-linking on the
opposing strand, the oligonucleotide containing the nucleo-
tide analogue was radiolabeled at either its 50- or 30-terminus.
FIGURE 1. Direct strand scission and alkali-labile lesion forma-
tion when duplex DNA (11) containing IT is photolyzed: (A)
anaerobic photolysis of 50-32P-11; (B) aerobic photolysis of 50-32P-
11. Key: dsb, direct strand breaks; NaOH, photolysate treated with
0.1 M NaOH at 37 °C; Pip., photolysate treated with 1.0 M
piperidine at 90 °C.
tubes using lamps with maximum emission at 300 nm. When
50-32P-11 (50 nM) containing IT was photolyzed under
anaerobic conditions the nucleotide analogue (X11) and 50-
(T10) and 30- (T12) flanking nucleotides were the major sites
of strand damage (Figure 1A). Direct strand breaks (dsb)
and smaller amounts of NaOH-labile cleavage were pro-
duced. Treatment with piperidine resulted in a slight increase
in cleavage at most nucleotide positions. Although detailed
product analysis was not carried out, previous studies on
oxidative DNA damage suggest that the formation of direct
strand breaks and NaOH labile sites are consistent with
hydrogen atom abstraction from the deoxyribose compo-
nent.43 Positions that are only labile to piperidine are often
associated with nucleobase lesions, which in this study could
result from aryl radical addition to adjacent nucleobases.
The data (Figure 1) suggest that all three types of lesions are
produced upon photolysis of IT containing DNA. Lesser
amounts of strand damage are detected at A8 and T9.
Damage at these sites may be attributed to a diffusible
species, particularly under aerobic conditions where the
relative amounts of damage increases. However, we cannot
rule out direct reaction between the aryl radical and these
nucleotides due to disruption of the duplex structure by the
diiodo precursor. Duplex melting temperatures are reduced
considerably when IT is substituted for thymidine opposite
dA.44 In addition, reactions of a DNA radical two or more
Strand Damage Produced upon UV-Irradiation of Duplex
DNA Containing the 2,4-Diiodo-5-methylbenzene Nucleotide
Analogue (IT). Photolyses (30 min) were carried out in Pyrex
(40) Wichai, U.; Woski, S. A. Org. Lett. 1999, 1, 1173–1175.
(41) Reddy, M. P.; Hanna, N. B.; Faroqui, F. Tetrahedron Lett. 1994, 35,
4311–4314.
(43) Hong, I. S.; Carter, K. N.; Sato, K.; Greenberg, M. M. J. Am. Chem.
Soc. 2007, 129, 4089–4098.
(44) Kim, T. W.; Kool, E. T. J. Org. Chem. 2005, 70, 2048–2053.
(42) See the Supporting Information.
J. Org. Chem. Vol. 75, No. 3, 2010 537

Ding and Greenberg
JOCFeatured Article
nucleotides away, particularly in single-stranded regions, is
precedented.45 The amount and pattern of strand damage at
T10-T12 produced from 50-32P-11 in the presence of O2
(Figure 1B) is similar to that observed under degassed
conditions. However, as mentioned above, cleavage at A8
and T9 is greater under these conditions and could be due to
the production of reactive oxygen species. The general
cleavage pattern is similar when 30-32P-11 is photolyzed
under the respective aerobic or anaerobic conditions.42
Under most conditions, cleavage at T10 and X11 is slightly
favored over T12. In addition, the cleavage products pro-
duced under all conditions from 30- and 50-32P-11 comigrate
in denaturing PAGE with the products of Maxam-Gilbert
sequencing reactions, indicating that they contain phosphate
termini.42 The similarity in cleavage patterns in 30- and
50-32P-11 is in contrast to what is observed when nucleobase
radicals resulting from radical addition to native pyrimidines
are generated by γ-irradiation. In those instances, the result-
ing peroxyl radicals generate tandem lesions by reacting with
flanking nucleotides in duplex DNA.43,46 The position of the
original nucleobase radical and the other nucleotide in the
tandem lesion are alkali-labile. Consequently, the strand
scission pattern is different depending upon which oligonu-
cleotide terminus is labeled because only the shortest labeled
fragment is observed. In contrast, when radicals are gene-
rated from IT in 11, formation of an alkali-labile lesion at this
site need not accompany damage at adjacent nucleotides.
Another difference between the strand damage derived
from IT and nucleobase radicals resulting from radical
addition is the effect of O2 on the level and pattern of strand
damage. The nucleobase radicals require O2 for strand
damage.47 In contrast, strand damage derived from IT is at
most weakly dependent on O2. Comparing the reactivity of
20-deoxyuridin-5-yl (5YL) produced from the 5-halopyri-
midines to the presumptive radical(s) derived from IT is
perhaps a more fair comparison, as both are σ-radicals. An
O2 effect is observed in the reactions where 5YL is produced
photochemically, but this may be complicated by the in-
volvement of photoinduced electron transfer in their genera-
tion.11 Alternatively, the lack of an O2 effect involving
irradiation of IT (with the exception of a small increase in
damage at T8 and A9 in the presence of O2) may suggest that
this radical trap ineffectively competes with intrastrand
reactions of the aryl radical(s). This is consistent with avail-
able kinetic data on aryl radicals. Assuming that O2 is
present at 0.2 mM, the effective first order rate constant
FIGURE 2. Effect of β-mercaptoethanol on direct strand incision
in UV-irradiated 50-32P-11: (A) aerobic conditions; (B) anaerobic
conditions.
intramolecular reactions in this case.25,26 Overall, the avail-
able rate constants support the observation that O2 does not
compete very effectively with the oligonucleotide for the
putative aryl radical.
Dioxygen is also involved in the steps leading to strand
scission and alkali-labile damage that follow intranucleo-
tidyl or internucleotidyl reaction of the aryl radical. Con-
sequently, in the absence of an exogenous quenching agent,
such as a thiol, traces of O2 could give rise to the observed
products. For instance, if 99.9% of the O2 (0.2 mM) is
removed from a sample, the remaining concentration
(0.2 μM) is approximately an order of magnitude greater than
that of 11. Hence, in the absence of thiol it is possible that the
same products are formed in degassed and aerobic reactions.
Additional information was gleaned from examining the
effects of thiol on strand damage. The ability of β-mercapto-
ethanol (BME) to prevent direct strand breaks (Figure 2) and
alkali-labile lesion formation (Figure 3) was studied under
aerobic and anaerobic conditions. Low (1 mM) BME con-
centration had little if any effect under aerobic conditions on
direct strand scission (Figure 2A) and even less of one on
piperidine labile lesions (Figure 3A). Although bimolecular
for its reaction with the aryl radical (kO2 = 2 ꢀ 109 M-1 s-1
)
is ∼4ꢀ105 s-1. In contrast, an aryl radical abstracts hydro-
gen atoms from tetrahydrofuran, which should be less
reactive than 2-deoxyribose, with a bimolecular rate con-
stant, kTHF = 3.7 ꢀ 106 M-1 s-1 23
. Using this rate constant
rate constants for thiol trapping of aryl radicals (kRSH
)
as a guide, we speculate that oxidation of the DNA backbone
will compete with O2 trapping of the aryl radical if the
effective molarity of the sugar is 0.1 M. Rate constants for
intermolecular aryl radical addition to alkenes (107 - 1010
M-1 s-1) are also rapid enough to effectively compete
with reactions with O2 because the former are effectively
are not available, reaction of an aryl radical with Bu3SnH
(kSnH = 7.8 ꢀ 108 M-1 s-1) has been reported.23 Since thiols
typically react slightly more rapidly than Bu3SnH with alkyl
radicals, the rate constant for an aryl radical reacting with
Bu3SnH could be considered a lower limit for reaction of
BME with the radical(s) generated from IT (Scheme 2).48
Based upon these assumptions, the rate constant for aryl
radical trapping by the thiol should be comparable to that of
(45) Crean, C.; Uvaydov, Y.; Geacintov, N. E.; Shafirovich, V. Nucleic
Acids Res. 2008, 36, 742–755.
(46) Carter, K. N.; Greenberg, M. M. J. Am. Chem. Soc. 2003, 125,
13376–13378.
(47) Greenberg, M. M. Org. Biomol. Chem. 2007, 5, 18–30.
(48) Newcomb, M. Tetrahedron 1993, 49, 1151–1176.
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FIGURE 4. Direct strand scission and alkali-labile lesion forma-
tion when duplex DNA (12) containing I2 is photolyzed. (A)
anaerobic photolysis of 50-32P-12; (B) aerobic photolysis of 50-32P-
12. Key: dsb, direct strand breaks; NaOH, photolysate treated with
0.1 M NaOH at 37 °C; Pip., photolysate treated with 1.0 M
piperidine at 90 °C.
FIGURE 3. Effect of β-mercaptoethanol on alkali-labile lesion
formation (piperidine, 1 M, 90 °C) in UV-irradiated 50-32P-11: (A)
aerobic conditions; (B) anaerobic conditions.
O2 and 1 mM BME will compete with O2 for the aryl
radical(s). Therefore, since O2 (0.2 mM) does not compete
with inter- and intranucleotidyl reactions of the aryl radical-
(s), we do not expect 1 mM BME to either. The competition
between O2 and BME for reaction with alkyl (π) radicals
produced in DNA is expected to be very different from the
reaction(s) of the putative aryl (σ) radicals because the rate
constants with O2 that these radicals are considerably
greater than with BME.48 Consequently, BME (1 mM) will
not compete with O2 (0.2 mM) for any subsequently formed
alkyl radicals. These facts are consistent with the lack of an
effect of 1 mM BME on strand damage from photolysis of
DNA containing IT under aerobic conditions.
In contrast, 1 mM BME significantly reduced the amount
of strand damage under anaerobic conditions (Figures 2B
and 3B). These observations are attributed to reactions
between thiols and alkyl radicals that are produced via
reactions of the initially formed aryl radical(s) with the 20-
deoxyribose rings and/or adjacent nucleobases. β-Fragmen-
tation of sugar radicals produce strand breaks in duplex
DNA with rate constants e2 ꢀ 102 s-1 at pH 7.0.49 These
reactions and alkyl radical trapping by residual O2 are too
slow to compete with quenching (kRSH e 1 ꢀ 107 M-1 s-1) by
1 mM BME.48 At higher BME concentration (100 mM) the
levels of direct strand breaks and alkali-labile lesions at
A8-T12 are reduced to very low levels (<1%). This concen-
tration of BME efficiently competes with O2 for the radicals.
Moreover, using the above rate constants as a guide, it is
reasonable to assume that the 0.1 M thiol competes with the
inter- and intranucleotidyl reactions of the aryl radical(s).
Strand Damage Produced upon UV-Irradiation of Duplex
DNA Containing Monoiodinated Aryl Nucleotide Analogues.
Irradiation of the 2,4-diiodo-5-methylbenzene nucleotide
analogue (IT) could give rise to two different aryl radicals
(Scheme 2), which may react differently from one another
due to conformational constraints in the duplex. Conse-
quently, the photochemistry of duplexes (12, 14) containing
o-iodoaryl (I2) or p-iodoaryl (I4) nucleotide analogues was
examined. Strand damage was produced in lower yields than
from IT (11) when otherwise identical duplexes containing
these compounds were photolyzed (Figures 4 and 5).42 One
explanation for this behavior is that photolysis of IT gene-
rates radicals more efficiently. Although the aryl iodide
nucleotide analogues all absorb weakly at 300 nm, IT
absorbs slightly more strongly than the monoiodoaryl mole-
cules in the region not blocked by the Pyrex glass and has the
longest λmax (242 nm in MeOH).42 However, we cannot
determine from these experiments whether the aryl iodides
exhibit comparable quantum efficiency for photochemical
conversion.
Although UV-irradiation of the o-iodoaryl (12, Figure 4)
and p-iodoaryl (14, Figure 5) nucleotide analogues produced
lower yields of strand damage products than Kool’s com-
pound, these molecules generated distinctive cleavage patterns.
These observations suggest that diffusible species are not res-
ponsible for the majority of strand damage. When the o-iodo-
aryl analogue (I2) is photolyzed (Figure 4), piperidine labile
lesion formation is favored at the 30-adjacent nucleotide (T12)
(49) Giese, B.; Dussy, A.; Meggers, E.; Petretta, M.; Schwitter, U. J. Am.
Chem. Soc. 1997, 119, 11130–11131.
J. Org. Chem. Vol. 75, No. 3, 2010 539

Ding and Greenberg
JOCFeatured Article
FIGURE 6. Molecular modeling (Spartan ’02) of 50-T IT T (top to
bottom in figure) with IT in the (A) anti-conformation or (B) syn-
conformation. The opposing strand is omitted for clarity.
3
3
SCHEME 5
of the 30-adjacent nucleobase (Figure 6A), which could help
explain the formation of piperidine labile lesions at T12
(Figures 1 and 3). Adoption of the “syn” like conformational
isomer (Figure 6B) places the same radical in a better
position to react with the C20-hydrogen atom of its own
FIGURE 5. Direct strand scission and alkali-labile lesion forma-
tion when duplex DNA (14) containing I4 is photolyzed: (A)
Anaerobic photolysis of 50-32P-14; (B) aerobic photolysis of
50-32P-14. Key: dsb, direct strand breaks; NaOH, photolysate
treated with 0.1 M NaOH at 37 °C; Pip., photolysate treated with
1.0 M piperidine at 90 °C.
˚
sugar ring, which is ∼2.6 A away from the pro-radical center
under all conditions.42 Direct strand breaks and NaOH-labile
lesions were produced in <1% yield at T12 regardless
of whether O2 was present or not and independent of
which oligonucleotide terminus was labeled. Intranucleotidyl
cleavage (X11) was the second most prominent cleavage site,
but the majority of lesions at this position in the oligonucleotide
were direct strand breaks and they were produced in low yield.
There was little if any enhancement in cleavage attributable to
NaOH or piperidine treatment. These observations suggest
that the aryl radical produced from I2 preferentially reacts with
the nucleobase of the 30-adjacent nucleotide (T12), less so
with its own 20-deoxyribose ring, and little if at all with the
50-adjacent nucleotide (T10).
and a comparable distance from the C20-hydrogen of the
50-adjacent nucleotide. Although the C10- and C20-hydrogen
0
˚
˚
atoms of the 5 -adjacent thymidine are within 3.3 A and 2.6 A
respectively of the ortho-carbon in IT, there is little evidence
for reaction at these positions in 12 even though there is in the
former (Figure 4). In contrast, the alkene carbons of the 50-
˚
adjacent thymine ring are g4.5 A from the aryl ring carbon
and as expected we do not detect damage (piperidine labile)
consistent with reaction at this site. Assuming that piperidine
labile lesions result from reaction with nucleobases and that
direct strand breaks and NaOH labile lesions involve hydro-
gen atom abstraction from the 20-deoxyribose rings, with the
exception of evidence for direct oxidation of the 50-adjacent
20-deoxyribose ring, these models reflect the observed DNA
damage overall fairly well (Figure 4).
Molecular models also indicate that radicals derived from
the p-iodoaryl molecule (I4) are not well positioned to react
with any of the 20-deoxyribose rings (Figure 6), which is
consistent with the dearth of direct strand breaks or NaOH-
labile lesions from 50-32P-14 (Figure 5). Furthermore, the
helical nature of the duplex provides better overlap between
the incipient aryl radical(s) and the π-bond of the 50-flanking
nucleotide than it does the 30-adjacent nucleobase (Figure 6).
As with the photoreactivity of IT, we cannot rule out the
formation of a diffusible species or the disruption of the
duplex structure as a source for the piperidine-labile lesions
at T9 when 50-32P-14 is photolyzed. Disruption of the duplex
structure by IT or I4 is more likely than by I2. The former
should experience steric clash with the N6-amino of the
opposing dA, whereas an iodide at the ortho-position can
Photolysis of p-iodoaryl containing duplex (50-32P-14)
produces low levels of DNA damage, with modest preference
for the 50-flanking nucleotides (T9 and T10) (Figure 5).
Cleavage is enhanced 2-fold or more when the DNA is
treated with piperidine and there is little if any direct strand
scission. In contrast to the o-iodoaryl molecule, I4 exhibits
little if any specific damage at the 30-flanking nucleotide (T12)
or at its own position (X11).
The damage produced by photolysis of the o-iodoaryl and
p-iodoaryl containing DNA is somewhat reflective of the
proximity of the presumably formed aryl radicals to poten-
tial reactive sites as suggested by simple molecular models of
a trinucleotide containing IT, which take into account only
two conformations of the nucleotide analogue (Scheme 5,
Figure 6). For instance, when IT is in the “anti” like
conformation, the carbon of the incipient radical ortho to
˚
the carbon bonded to the sugar is ∼3.9 A from the C5-carbon
540 J. Org. Chem. Vol. 75, No. 3, 2010

Ding and Greenberg
JOCFeatured Article
FIGURE 8. Molecular modeling (Spartan ’02) of 50-T I3 T (top to
bottom in figure) with IT in the anti-conformation (A) or syn-
conformation (B). The opposing strand is omitted for clarity.
3
3
FIGURE 7. Direct strand scission and alkali-labile lesion forma-
tion when duplex DNA (13) containing I3 is photolyzed: (A)
anaerobic photolysis of 50-32P-13; (B) aerobic photolysis of 50-32P-
13. Key: dsb, direct strand breaks; NaOH, photolysate treated with
0.1 M NaOH at 37 °C; Pip., photolysate treated with 1.0 M
piperidine at 90 °C.
FIGURE 9. Effect of t-BuOH on interstrand cross-link (ICL) yield
in 137Cs irradiated 50-32P-15a-d as a function of nucleotide (Y)
opposite IT.
occupy the minor groove. Overall, the composite reactivity
of o-iodoaryl and p-iodoaryl nucleotide analogues is consis-
tent with much of the strand damage produced by IT. The
most apparent difference is the absence of significant frac-
tions of direct strand breaks and NaOH-labile lesions at T10.
The reactivity of the o- and p-aryl radicals led us to
investigate the third regioisomeric aryl iodide nucleotide
analogue, I3. Strand damage upon photolysis of 50-32P-13
is more efficient than the other monoiodides but less so than
IT (Figure 7).42 Piperidine labile damage is the major form of
lesions produced under all conditions, indicating that little if
any reactions involve hydrogen atom abstraction from the
20-deoxyribose rings. In addition, the cleavage pattern for I3
is different from the other molecules examined. Intranucleo-
tidyl cleavage is minimal under most conditions. The major-
ity of alkali-labile lesions are produced at the 30- and
50-flanking nucleotides, as well as at T9. The reactivity of
the m-aryl iodide molecule is consistent with observations
made in studies on the reactivity of the above aryl iodides.
The m-aryl radical was expected to be inefficient at produ-
cing intranucleotidyl direct strand breaks or NaOH-labile
lesions because of its poor orbital overlap with the hydrogen
atoms of its own deoxyribose. The absence of such lesions at
the 50-adjacent thymidine is a little surprising because the
distances between the C10- and C20-hydrogens at this posi-
tion are not much greater than they are from the ortho-
carbon (Figure 8). However, the lack of reactivity could be
due to the fact that the sp2 like orbital of the incipient radical
is not directed toward the 20-deoxyribose hydrogen atoms. In
contrast, the aryl radical is well positioned to add into the
π-bond of the 50- or 30-adjacent pyrimidine when it is present
in the “syn” or “anti” conformation, respectively. Finally,
the anticipated disruption of base pairing with the opposing
dA by the -iodo substituent may explain why such a signi-
ficant level of damage is observed at T9.
Interstrand Cross-Link Formation upon Irradiation of
Iodinated Nucleotide Analogues. Recently, γ- and UV-
irradiation of 5-bromo-20-deoxyuridine was shown to pro-
duce interstrand cross-links in nonbase paired regions of
duplex DNA.16,17,50 ICL formation from irradiation of
halogenated nucleotides (or analogues) in paired regions
would be even more significant because these regions are
more plentiful in DNA. γ-Radiolysis of 50-32P-15a-d under
anaerobic conditions produced interstrand cross-links with-
out a significant dependence on the identity of the opposing
nucleotide, although the ICL yield was slightly higher when
IT was opposed by a pyrimidine than a purine (Figure 9,
Table 1). Addition of the hydroxyl radical scavenger, t-
BuOH (10 mM) did not affect the cross-link yield
(Figure 9). However, ICL formation was reduced to <1%
in the presence of O2, which is known to scavenge solvated
electrons (data not shown).51 These observations suggest
that cross-linking is induced via solvated electron capture
and subsequent aryl radical formation via fragmentation of
the radical anion.
(50) Cecchini, S.; Masson, C.; La Madeleine, C.; Huels, M. A.; Sanche,
L.; Wagner, J. R.; Hunting, D. J. Biochemistry 2005, 44, 16957–16966.
(51) von Sonntag, C. Free-Radical-Induced DNA Damage and Its Repair;
Springer-Verlag: Berlin, 2006.
J. Org. Chem. Vol. 75, No. 3, 2010 541

Ding and Greenberg
JOCFeatured Article
TABLE 1. Interstrand Cross-Link Yields Following 137Cs Irradiation
TABLE 2. Interstrand Cross-Link Yields Following UV Irradiation of
DNA Containing I4a
(700 Gy)a
aYields are the average ( std dev of three samples.
aYields are the average ( std dev of three samples.
137Cs irradiation of the monoiodide nucleotide analogues
also produced interstrand cross-links (Table 1). The o-
iodoaryl precursor (I2) was the least effective at producing
cross-links, and the p-iodoaryl nucleotide analogue (I4)
produced the highest ICL yields. The cross-link yields
reached almost 16% when I4 (18a-d) was irradiated. Add-
ing t-BuOH generally had little effect on cross-link yield,
although inexplicably when I4 was opposed by dA, the ICL
yield almost doubled.42 The high yields of ICLs produced
from I4 were also observed when 18a was photolyzed (Table
2). However, a control duplex (19) containing a T:A base pair
in place of the nucleotide analogue yielded <2% ICL. Cross-
link yields were higher when the nucleotide analogue was
opposed by pyrimidines than when purines were present at
these positions. This trend is the same as when the duplex was
exposed to γ-radiolysis (Table 1). UV-induced cross-link
yields were modestly reduced when photolyses were carried
out under aerobic conditions. One possible explanation is
that O2 partially quenches the excited state. Alternatively,
assuming that cross-links result from aryl radical addition
into a π-bond of the opposing nucleotide, O2 (kO2 ∼4 ꢀ 105
s-1 when [O2] = 0.2 mM) trapping may compete with this
reaction. If so, the anticipated rate constant for O2 trapping
would indicate that cross-linking by the aryl radical is
considerably faster than that estimated for cross-linking by
the 5-(20-deoxyuridinyl)methyl radical (20), whose rate con-
stant for ICL formation (∼103 s-1) is limited by rotation
about the nucleotide’s glycosidic bond.52,53
strand was cross-linked.42,55 Cross-links produced from
photolysis of I4 in 18a under anaerobic conditions occurred
exclusively with the opposing dA. When I4 was paired
opposite dC (18c), the opposing nucleotide was still the
major position cross-linked. However, a small amount of
cross-linking with T14 was also detected. Similar behavior
was observed when I3 was opposite dT. The majority of
cross-links were formed with the opposing thymidine (T13) in
17d, along with small amounts with T14. Cross-linking in
photolyzed duplex containing I3 opposite dG (17b) was least
specific. Although ICLs were produced with the opposing
purine, considerable cross-linking was observed with T14 and
T15. The less selective cross-linking observed when I3 was
opposite dG may be a consequence of greater disruption of
base pairing in the vicinity of the nucleotide analogue when it
is paired with the larger purine. Assuming that cross-linking
occurs via σ-radical addition to a π-bond, dG may also be
inherently less reactive with the incipient aryl radical because
its major tautomer does not contain a π-bond proximal to
the aryl radical.
Summary. The studies described above demonstrate a new
application of aryl iodide nucleotide analogues. UV-irradia-
tion (300 nm) of duplexes containing these molecules pro-
duces a mixture of direct strand breaks and alkali-labile
lesions. The strand damage produced is consistent with
generation of highly reactive aryl radicals. However, unlike
the 5-halopyrimidines we do not know if the aryl radicals are
formed via direct excitation and/or photoinduced electron
transfer from a neighboring nucleotide. Additional studies
are necessary to more fully characterize the strand products
of these reactions, as well as the mechanisms of their forma-
tion.
The formation of interstrand cross-links upon irradiation
of the iodinated nucleotide analogues in base paired regions
of DNA is distinctive. The 5-(20-deoxyuridinyl)methyl radi-
cal (20) produces ICLs.52-54 However, a synthetic molecule
that generates this reactive species when exposed to γ-radi-
olysis has not been reported.56 5-Bromo-20-deoxyuridine
(BrdU) produces ICLs, but only in nonbase paired regions
of DNA. We speculate that Watson-Crick base pairing
involving BrdU imposes a barrier to rotation about the
glycosidic bond into the syn-conformation required for
cross-linking that prevents this reaction from competing
Although cross-linking by the nucleotide analogues may
be considerably faster than by 20, the latter is more selective.
Cross-links derived from 20 exclusively involve the opposing
nucleotide.54 Hydroxyl radical cleavage experiments were
carried out on a subset of the cross-linked products from the
p- and m-aryl radicals, which gave the highest yields of ICLs
in order to determine which nucleotide(s) on the opposing
(52) Hong, I. S.; Ding, H.; Greenberg, M. M. J. Am. Chem. Soc. 2006,
128, 485–491.
(53) Ding, H.; Majumdar, A.; Tolman, J. R.; Greenberg, M. M. J. Am.
Chem. Soc. 2008, 130, 17981–17987.
(55) Millard, J. T.; Weidner, M. F.; Kirchner, J. J.; Ribeiro, S.; Hopkins,
P. B. Nucleic Acids Res. 1991, 19, 1885–1892.
(54) Hong, I. S.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127, 3692–
3693.
(56) Hong, I. S.; Ding, H.; Greenberg, M. M. J. Am. Chem. Soc. 2006,
128, 2230–2231.
542 J. Org. Chem. Vol. 75, No. 3, 2010

Ding and Greenberg
JOCFeatured Article
with others of the σ-radical. The non-hydrogen-bonding
nucleotide analogues investigated here probably face a lower
barrier to glycosidic bond rotation due to the absence of
hydrogen bonds with the opposing nucleotide. Furthermore,
their destabilization of the duplex may facilitate the con-
formational freedom that facilitates cross-link formation.
Cross-link formation in γ-irradiated samples containing
hydroxyl radical quencher indicates that the aryl iodides
do not react with this species. We hypothesize that the aryl
iodides produce cross-links via reaction with solvated elec-
trons. This is consistent with ICL quenching by O2, which
traps this species. This suggests that these molecules, espe-
cially I3 and I4 could be useful mechanistic tools for probing
excess electron transfer in DNA. One could imagine using
cross-link formation in a PAGE experiment as a simple read
out for excess electron transfer. Furthermore, their ability to
produce cross-links under anaerobic conditions suggests that
the iodinated nucleotide analogues could be useful as radio-
sensitizing agents. Their ultimate utility as radiosensitizing
agents in cells requires that they be incorporated into cellular
DNA. Although it is unlikely that the I3 or I4 would be
incorporated in DNA by existing polymerases, one could
hope that in the future engineered polymerases may accept
such substrates.57 Alternatively, one could utilize advances
made in the design of nucleotides analogues that are accepted
as substrates by DNA polymerases as inspiration for design-
ing new halogenated radiosensitizing agents that produce
interstrand cross-links.58-63
Preparation of 2. A pressure tube was charged with CuI
(57 mg, 0.3 mmol), NaI (1.8 g, 12 mmol), and sealed with a
rubber septum. It was evacuated and backfilled with argon
(repeat three times). A solution of 139 (163 mg, 0.59 mmol)
and trans-N,N0-dimethyl-1, 2-cyclohexanediamine (86 mg,
0.6 mmol) in 1-pentanol (1.6 mL) was added to the pressure
tube via syringe. The reaction mixture was sealed with a Teflon
stopper and stirred at 130 °C for 40 h. The resulting suspension
was cooled to 25 °C, at which time saturated NaHCO3 (20 mL)
was added and the resulting mixture was extracted with diethyl
ether. The combined organic phases were washed with saturated
NaHCO3, water, and brine. After being dried over anhydrous
Na2SO4, the solution was concentrated to give the crude product
which was purified by flash chromatography (30: 1 EtOAc/
MeOH) to yield an oil. 1H NMR showed it is a mixture (1.1:1) of
the desired product and 10, 20-dideoxy-β-10-phenyl-D-ribofura-
nose. The mixture was further purified by HPLC to give 2 (45
mg, 24% yield). HPLC conditions: Waters Delta Pak C18
column (300 ꢀ 7.8 mm); solution A: water; solution B: acetoni-
trile; increase of B from 2% to 50% in 15 min at a flow rate of
4 mL/min. 1H NMR (acetone-d6) δ 7.83 (d, J = 8.0 Hz, 1H), 7.70
(d, J = 7.8 Hz, 1H), 7.39 (dd, J = 7.8, 7.8 Hz, 1H), 7.03 (dd,
J = 7.6, 7.6 Hz, 1H), 5.26 (ddd, J = 9.9, 5.6, 5.6 Hz, 1H),
4.44-4.38 (m, 1H), 4.32 (br s, 1H), 4.04-3.86 (m, 2H), 3.80-3.66
(m, 2H), 2.51 (ddd, J = 12.8, 5.6, 2.0 Hz, 1H), 1.70 (ddd, J = 12.8,
9.9, 5.8 Hz, 1H); 13C NMR (acetone-d6) δ 145.3, 138.9, 129.1,
128.4, 127.2, 96.1, 88.2, 83.1, 73.0, 62.9, 42.8; IR (film): 3384, 2916,
1650, 1615, 1559, 1512, 1459, 1179, 1047, 754 cm-1; HRMS-FAB
(m/z) [M - H]þ calcd for C11H12IO3 318.9837, found 318.9830.
Preparation of 3. C-Nucleoside 2 (42 mg, 0.13 mmol) was
coevaporated with pyridine three times and then dissolved in
pyridine (1.2 mL). To the solution was added 4,4-dimethoxy-
trityl chloride (67 mg, 0.2 mmol). The mixture was stirred at
25 °C for 16 h and concentrated. The residue was loaded onto a
silica gel (oven-dried) column and eluted (2:1 hexanes/EtOAc)
to give 3 as a colorless foam (75 mg, 91%): 1H NMR (acetone-
d6) δ 7.85 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H),
7.60-7.52 (m, 2H), 7.47-6.62 (m, 13H), 5.31 (ddd, J = 9.6, 5.9,
5.9 Hz, 1H), 4.46-4.33 (m, 2H), 4.18-4.10 (m, 1H), 3.79 (s, 6H),
3.41-3.35 (m, 2H), 2.55 (ddd, J = 12.8, 5.8, 2.2 Hz, 1H), 1.78
(ddd, J = 15.5, 9.6, 5.9 Hz, 1H); 13C NMR (acetone-d6) δ 158.7,
145.4, 145.3, 139.1, 136.1, 130.2, 129.2, 128.5, 128.2, 127.7,
126.7, 113.0, 96.1, 86.7, 85.9, 83.2, 73.1, 64.2, 54.6, 42.9; IR
(film): 3446, 2933, 2836, 1608, 1509, 1463, 1300, 1251, 1177,
1034, 754 cm-1; HRMS-FAB (m/z) [M - H]þ calcd for
C32H30IO5 621.1137, found 621.1174.
Preparation of 4. To a solution of 3 (74 mg, 0.118 mmol) in
CH2Cl2 (1.2 mL) were added diisopropylethylamine (31 mg,
0.24 mmol) and 2-cyanoethyl N,N0-diisopropylchlorophos-
phoramidite (45 mg, 0.19 mmol). The reaction mixture was
stirred at 25 °C for 4 h and concentrated. The residue was loaded
onto a silica gel (oven-dried) column and eluted (3:1 hexanes/
EtOAc) to afford 4 as a colorless foam (62 mg, 64%): 1H NMR
(acetone-d6) δ 7.85 (d, J = 8.0 Hz), 7.76-7.68 (m), 7.61-7.54
(m), 7.49-7.21 (m), 7.10-7.03 (m), 6.97-6.87 (m), 5.34-5.26
(m), 4.67-4.55 (m), 4.32-4.23 (m), 3.99-3.61 (m), 3.49-3.34
(m), 2.83-2.62 (m), 1.92-1.77 (m), 1.33-0.85(m); 31P NMR
(acetone-d6) δ 148.0, 147.9; IR (film) 2966, 2836, 2252, 1607,
1508, 1462, 1178, 1034, 895, 754 cm-1; HRMS-FAB (m/z) [M þ
Na]þ calcd for C41H48N2O6NaPI 845.2187, found 845.2203.
Preparation of 7. n-BuLi (1.6 M in hexanes, 2.1 mL,
3.36 mmol) was added via syringe to a solution of the 1,4-
diiodobezene (1.07 g, 3.25 mmol) in anhydrous THF (3.0 mL)
cooled at -78 °C under argon. After 30 min, a solution of
30,50-O-((1,1,3,3-tetraisopropyl)disiloxanediyl)-20-deoxy-D-ribo-
no-1,4-lactone40 (1.2 g, 3.12 mmol) in anhydrous THF (5.0 mL)
was added to the solution via cannula at -78 °C. After 1 h,
the reaction mixture was quenched at -78 °C with saturated
Experimental Section
General Methods. Oligonucleotides were synthesized via
standard automated DNA synthesis on an Applied Biosystems
model 394 instrument. The coupling time for the phosphorami-
dites of modified nucleotides IT, I2, I3, and I4 was 5 min. For
synthesis of oligonucleotides containing IT, fast-deprotecting
phosphoramidites were used and 1 M TBHP in toluene was used
as the oxidation reagent. Oligonucleotides were deprotected
using 1:1 methylamine (40% in water)- concentrated NH4OH
at 65 °C for 75 min (oligonucleotides containing I2, I3, and I4),
or concentrated NH4OH at 25 °C for 9 h (oligonucleotides
containing IT). Oligonucleotides were purified by 20% denatur-
ing polyacrylamide gel electrophoresis (PAGE). All oligo-
nucleotides containing modified nucleotides were characterized
by ESI-MS. Radiolabeled oligonucleotides were hybridized
with 1.5 equiv of complementary oligonucleotides in 10 mM
potassium phosphate (pH 7.2) and 100 mM NaCl at 90 °C for 5
min and cooled to room temperature. The concentration of
duplexes was 50 nM in all experiments involving radiolabeled
samples. All anaerobic reactions were carried out in sealed Pyrex
tubes, which were degassed and sealed using freeze-pump-
thaw (three cycles, 3 min each) degassing techniques. Experi-
ments involving radiolabeled oligonucleotides were analyzed
following PAGE using a Storm 840 phosphorimager.
(57) Loakes, D.; Gallego, J.; Pinheiro, V. B.; Kool, E. T.; Holliger, P.
J. Am. Chem. Soc. 2009, 131, 14827–14837.
(58) Hirao, I.; Kimoto, M.; Mitsui, T.; Fujiwara, T.; Kawai, R.; Sato, A.;
Harada, Y.; Yokoyama, S. Nature Methods 2006, 3, 729–735.
(59) Silverman, A. P.; Jiang, Q.; Goodman, M. F.; Kool, E. T. Biochem-
istry 2007, 46, 13874–13881.
(60) Leconte, A. M.; Matsuda, S.; Romesberg, F. E. J. Am. Chem. Soc.
2006, 128, 6780–6781.
(61) Young Jun, S.; Floyd, E. R. ChemBioChem 2009, 10, 2394–2400.
(62) Hwang, G. T.; Romesberg, F. E. J. Am. Chem. Soc. 2008, 130,
14872–14882.
(63) Seo, Y. J.; Hwang, G. T.; Ordoukhanian, P.; Romesberg, F. E.
J. Am. Chem. Soc. 2009, 131, 3246–3252.
J. Org. Chem. Vol. 75, No. 3, 2010 543

Ding and Greenberg
JOCFeatured Article
aqueous NH4Cl (3 mL). The mixture was extracted with diethyl
ether. The combined ether phases were washed with saturated
aqueous NH4Cl, water, and brine. The organic phase was dried
over anhydrous Na2SO4 and concentrated to give an oil that was
used without further purification.
0.33 mmol) and 2-cyanoethyl N,N0-diisopropylchlorophospho-
ramidite (59 mg, 0.25 mmol). The reaction mixture was stirred at
25 °C for 4 h and concentrated. The residue was loaded onto a
silica gel (oven-dried) column and eluted (3:1 hexanes/EtOAc)
to afford 10 as a colorless foam (73 mg, 54%): 1H NMR (CDCl3)
δ 7.76-7.62 (m), 7.56-7.45 (m), 7.44-7.15 (m), 6.92-6.76 (m),
5.22-5.08 (m), 4.61-4.49 (m), 4.32-4.19 (m), 3.91-3.54 (m),
3.43-3.20 (m), 2.68-2.55 (m), 2.54-2.28 (m), 2.10-1.95 (m),
1.32-0.88 (m); 31P NMR (CDCl3) δ 148.1, 148.0; IR (film) 2967,
2932, 2250, 1608, 1509, 1462, 1365, 1251, 1178, 1034, 1003, 828
cm-1; HRMS-FAB (m/z) [M þ Na]þ calcd for C41H48N2O6Na-
PI 845.2187, found 845.2181.
Photoreactions. Photoreactions of the duplexes were carried
out in Pyrex tubes in a Rayonet photoreactor fitted with 16
lamps having a maximum output at 300 nm. All photoreactions
were carried out for 30 min in 10 mM potassium phosphate (pH
7.2) and 100 mM NaCl. After reaction, each sample (20 μL) was
aliquoted into three 0.6-mL Eppendorf tubes A (6 μL), B (8 μL),
and C (6 μL). Tube A was mixed with formamide loading buffer.
Tube B was incubated with 0.5 M NaOH (2 μL) at 37 °C for
20 min, treated with 1 M AcOH (1 μL), and mixed with form-
amide loading buffer. Tube C was treated with 2 M piperidine
(6 μL) at 90 °C for 20 min, dried, coevaporated with H2O (3 ꢀ
10 μL), and resuspended in formamide loading buffer. Finally,
they were subjected to 20% PAGE analysis.
Thiol Effect on Strand Damage. The appropriate radiolabeled
duplex (11-14) was prepared in 10 mM potassium phosphate
(pH 7.2) and 100 mM NaCl in the presence of varying concen-
trations of BME (0, 1, and 100 mM). Anaerobic samples were
degassed and sealed. Photoreactions were carried out at 300 nm
for 30 min. After reaction, each sample (12 μL) was distributed
in two 0.6 mL Eppendorf tubes A (6 μL) and B (6 μL). Tube A
was mixed with formamide loading buffer. Tube B was dried,
treated with 1 M piperidine at 90 °C for 20 min, dried again,
coevaporated with H2O (3 ꢀ 10 μL), and resuspended in
formamide loading buffer, and subjected to 20% PAGE
analysis.
γ-Radiolysis. γ-Radiolysis of the duplexes were carried out in
Pyrex tubes in a J. L. Shepherd Mark I 137Cs irradiator that has
an output of 25 Gray/min. After reaction (700 Gy), samples
were lyophilized, resuspended in formamide loading buffer, and
subjected to 20% PAGE analysis.
A solution of the crude oil in CH2Cl2 (5.1 mL) under argon
and at -78 °C was treated with Et3SiH (0.72 g, 6.5 mmol) and
BF3 Et2O (0.91 g, 6.5 mmol). The reaction mixture was stirred
3
at -78 °C for 3 h and quenched at -78 °C by the addition of
saturated aqueous NaHCO3 (3 mL). The mixture was extracted
with diethyl ether. The combined organic phases were washed
with saturated aqueous NaHCO3, water, and brine. This solu-
tion was dried over anhydrous Na2SO4, concentrated and
purified by flash chromatography (3:2 to 1:1 hexanes/toluene)
to yield the desired β isomer of 7 (163 mg, 0.29 mmol, 9%) as an
oil (∼6: 1 β/R mixture in the crude product; 1.6% isolated yield
of R-7; the total yield is 13%). β-7: 1H NMR (CDCl3) δ 7.67 (d,
J = 8.2 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 5.06 (dd, J = 14.4, 7.2
Hz, 1H), 4.52 (ddd, J = 7.7, 4.7, 4.7 Hz, 1H), 4.18-4.10 (m, 1H),
3.94-3.86 (m, 2H), 3.39 (ddd, J = 12.4, 7.0, 4.7 Hz, 1H), 2.03
(ddd, J = 15.4, 7.7, 7.7 Hz, 1H), 1.28-0.76 (m, 28H); 13C NMR
(CDCl3) δ 142.0, 137.4, 127.8, 92.8, 86.4, 78.4, 72.9, 63.5, 43.0,
17.6, 17.5, 17.39, 17.3, 17.11, 17.08, 16.99, 13.5, 13.4, 13.3, 12.6;
IR (film) 2994, 2867, 1464, 1386, 1116, 1089, 1037, 885 cm-1
;
HRMS-FAB (m/z) [M - H]þ calcd. for C23H39IO4Si2 561.1353,
found 561.1355.
R-7: 1H NMR (CDCl3) δ 7.68 (d, J = 8.3 Hz, 2H), 7.14 (d,
J = 8.3 Hz, 2H), 4.98 (ddd, J = 9.6, 6.4, 6.4 Hz, 1H), 4.63-4.56
(m, 1H), 4.09-4.02 (m, 1H), 3.97-3.88 (m, 2H), 2.66 (ddd, J =
12.8, 6.4, 6.4 Hz, 1H), 2.09 (ddd, J = 9.6, 9.6, 9.6 Hz, 1H),
1.24-0.88 (m, 28H); 13C NMR (CDCl3) δ 142.6, 137.4, 127.9,
92.7, 83.7 77.8, 73.5, 63.1, 42.9, 17.5, 17.44, 17.43, 17.4, 17.3,
17.2, 17.0, 13.5, 13.3, 12.9, 12.6; IR (film): 2944, 2867, 1462,
1388, 1247, 1141, 1034, 1005, 782 cm-1; HRMS-FAB (m/z)
[M - H]þ calcd for C23H39IO4Si2 561.1353, found 561.1336.
Preparation of 8. To a solution of 7 (163 mg, 0.29 mmol) in
THF (5 mL) was added Bu4NþF- hydrate (280 mg, 0.89 mmol).
The resulting mixture was stirred at 25 °C for 18 h. It was
concentrated and purified by flash column chromatography (15:
1 CH2Cl2-MeOH) to give 8 as a colorless oil (79 mg, 85%): 1H
NMR (MeOH-d4) δ 7.68 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.4
Hz, 2H), 5.09 (ddd, J = 10.8, 5.4, 5.4 Hz, 1H), 4.35-4.29 (m,
1H), 3.99-3.93 (m, 1H), 3.68 (d, J = 3.8 Hz, 2H), 2.24-2.15 (m,
1H), 1.97-1.84 (m, 1H); 13C NMR (MeOH-d4) δ 141.9, 137.1,
127.8, 92.0, 87.9, 79.5, 73.0, 62.6, 43.6; IR (film) 3333, 2923,
2891, 1682, 1559, 1458, 1066, 997, 815 cm-1; HRMS-FAB (m/z)
[M þ NH4]þ calcd for C11H17NO3I, 338.0248, found 338.0248.
Preparation of 9. C-Nucleoside 8 (79 mg, 0.246 mmol) was
coevaporated with pyridine three times and dissolved in pyridine
(2.0 mL). To the solution was added 4,4-dimethoxytrityl chlor-
ide (114 mg, 0.34 mmol). The mixture was stirred at 25 °C for 20
h and concentrated. The residue was loaded onto a silica gel
(oven-dried) column and eluted (2:1 hexanes/EtOAc) to give 9 as
a colorless foam (102 mg, 67%): 1H NMR (acetone-d6) δ 7.72 (d,
J = 8.1 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.42-7.19 (m, 9H),
6.91-6.87 (m, 4H), 5.14 (ddd, J = 9.6, 4.8, 4.8 Hz, 1H), 4.39 (s,
1H), 4.34-4.26 (m, 1H), 4.13-4.05 (m, 1H), 3.80 (s, 6H),
3.28-3.24 (m, 2H), 2.31-2.23 (m, 1H), 1.98-1.88 (m, 1H);
13C NMR (acetone-d6) δ 158.7, 145.4, 143.1, 137.2, 136.1, 130.1,
128.2, 127.7, 126.6, 113.0, 91.8, 86.9, 85.9, 792, 73.4, 64.6, 54.5,
44.2; IR (film) 3425,2967, 1607, 1508, 1459, 1300, 1250, 1177,
1080, 1034, 1004, 827 cm-1; HRMS-FAB (m/z) [MþNa]þ calcd
for C32H31IO5Na 645.1108, found 645.1099.
Fe(II)-EDTA Digestion of Cross-Linked DNA. Fe(II)-EDTA
cleavage reactions of ICLs were carried out in 50 μM
(NH4)2Fe(SO4)2, 100 μM EDTA, 1 mM sodium ascorbate,
5.0 mM H2O2, 100 mM NaCl, and 10 mM potassium phosphate
(pH 7.2) for 1 min at 25 °C (total volume of 20 μL each). The
reactions were quenched with 100 mM thiourea (10 μL). Sam-
ples were lyophilized, resuspended in formamide loading buffer,
and subjected to 20% PAGE analysis.
Acknowledgment. We are grateful for support of this
research from the National Institute of General Medical
Sciences (GM-054996).
Supporting Information Available: Strand damage data for
30-32P-labeled duplexes. Hydroxyl radical digestion analysis of
cross-linked products. Sample autoradiogram of UV-irradia-
tion of 50- and 30-32P-11 showing cleavage pattern and comigra-
tion with Maxam-Gilbert sequencing reactions. Spectral data
for previously unreported compounds, UV absorption spectra
of aryl iodide nucleosides, and ESI-MS for oligonucleotides
containing nucleotide analogues. This material is available free
of charge via the Internet at http://pubs.acs.org.
Preparation of 10. To a solution of 9 (102 mg, 0.16 mmol) in
CH2Cl2 (1.6 mL) were added diisopropylethylamine (42 mg,
544 J. Org. Chem. Vol. 75, No. 3, 2010