Photochemistry of benzophenone immobilized in a major groove of DNA: Formation of thermally reversible interstrand cross-link

Article abstract of DOI:10.1021/ja017611r

We here report a highly site and sequence selective formation of an interstrand cross-link of _BPU-containing oligomer duplexes. The cross-link was found spontaneously reverted to original oligomers upon heating, providing a new method for the temporary connection of two DNA strands. Copyright

Full text of DOI:10.1021/ja017611r

Published on Web 02/16/2002
Photochemistry of Benzophenone Immobilized in a Major Groove of DNA:
Formation of Thermally Reversible Interstrand Cross-link
Kazuhiko Nakatani,* Takashi Yoshida, and Isao Saito*
Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering,
Kyoto UniVersity, Kyoto 606-8501, Japan
Received November 26, 2001
Scheme 1
The excited triplet state of benzophenone undergoes [2+2]
cycloaddition to alkenes producing oxetanes (Paterno-Bu¨chi reac-
tion)1 and hydrogen abstraction from accessible H donors eventually
forming a covalent bond between the resulting geminate alkyl and
ketyl radicals.2 These photoreactions of benzophenone are used in
photoaffinity labeling studies of protein-protein and protein-ligand
interactions.3 Benzophenone-mediated interstrand cross-link ef-
ficiently proceeds when the reactant is present near the benzophe-
none chromophore with a required geometry within a lifetime of
benzophenone triplet. Otherwise, triplet benzophenone readily
relaxes to the ground state without any reactions. Such cage and
proximity effects in fact played important roles in one-electron
oxidation of the nearest guanine from the 4-cyanobenzophenone-
substituted uridine (CNBPU) in duplex DNA.4,5 We have synthesized
a different type of benzophenone-substituted uridine (BPU) in which
the chromophore is tethered to uridine at C5 by a flexible linker
Table 1. Oligomers Used for the Photoreactions
rather than the structurally rigid ethynyl linker as used for CNBPU.
The benzophenone chromophore was located in a major groove of
DNA upon duplex formation. The linker structure connecting C5
of uridine and benzophenone was designed so that the carbonyl
group of benzophenone did not protrude out of the major groove
but pointed toward the sugar-phosphate backbone of the opposite
strand. We here report a highly site and sequence selective formation
of an interstrand cross-link of BPU-containing oligomer duplexes.6
The cross-link was found spontaneously reverted to the original
at 29.0 min was identified as a cross-link of two oligomers (BPUAA-
TTA) (18% yield, conversion yield 51%) as evidenced from
MALDI-TOF MS (obsd 7548.04, calcd 7549.17). To eliminate the
formation of the undesired thymine dimer, oligomers UTA and
oligomers upon heating, providing a new method for the temporary
connection of two DNA strands.7
Synthesis of 5′-dimethoxytrityl-BPU 1 was accomplished as
TUA containing uridine in place of thymine in the 5′-TTA-3′
shown in Schemes 1 and S1. The modified base was incorporated
sequence were used for the analysis. While neither cross-link
into 12-mer 5′-d(GCA TABPU AAT TCG)-3′ (BPUAA) (Table 1)
by automated DNA synthesis using phosphoramidite 4. Comple-
mentary strands are 5′-d(CGA ATT ATA TGC)-3′ (TTA), 5′-
d(CGA AUT ATA TGC)-3′ (UTA), 5′-d(CGA ATU ATA TGC)-
3′ (TUA), and 5′-d(CGA Ad3TU ATA TGC)-3′ (d3TUA) containing
5-triduteriomethyluridine d3T.8 CD spectra of duplex BPUAA/TTA
showed a B-form structure. The melting temperature of the duplex
was 37.8 °C (2.1 mM duplex, NaCl 100 mM, Na cacodylate 10
mM, pH 7.0), 3.4 °C lower than that of the unmodified duplex
containing thymine.
Duplexes (83 µM for each strand) in 30 mM Na cacodylate (pH
7.0) were irradiated at room temperature with a 312 nm light
obtained from a monochromator, and the reaction was monitored
by HPLC. Irradiation of duplex BPUAA/TTA produced two
products (Figure 1a). A minor product eluted at 23.5 min was
identified as an oligomer 5′-d(CGA AT)T ATA TGC)-3′ (T)TA)
containing a thymine dimer (T)T) by both ESI-TOF MS (obsd
3642.66, calcd 3642.66) and a nucleoside analysis. The formation
of T)TA is likely due to an energy transfer from the triplet
benzophenone to neighboring thymines.9 The major product eluted
formation nor destruction of the duplex was observed for BPUAA/
UTA, cross-link BPUAA-TUA was efficiently produced from
BPUAA/TUA (Figure 1b). The quantum yield for the formation of
BPUAA-TUA was 0.0012 at 312 nm using phenylglyoxylic acid
as an actinometer.10 A considerable difference in the cross-link
formation between UTA and TUA suggests that T in the 5′-TUA-
3′ of duplex BPUAA/TUA is the site of a covalent bond formation.
Only a small amount of cross-link was produced from the oligomer
duplex containing the BPUTA/TAA sequence, probably due to the
steric repulsion between the methyl group of the 3′ side T of BPU
and the linker connecting the chromophore.11
The quantum yield for the formation of the cross-link from
duplex BPUAA/d3TUA containing d3T at the cross-linking site was
virtually the same as that obtained for the photoreaction of BPUAA/
TUA, showing no first-order kinetic isotope effect on the cross-
link formation by incorporating deuterium at the T methyl group.
This strongly suggested that a direct H abstraction of methyl
hydrogens of T by triplet benzophenone was not involved in the
cross-link formation. Both cross-linked oligomers BPUAA-TTA and
BPUAA-TUA were thermally reverted to original oligomers. Thus,
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2118 VOL. 124, NO. 10, 2002 J. AM. CHEM. SOC.
10.1021/ja017611r CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
In marked contrast to a highly efficient cross-link formation from
duplex BPUAA/TTA, photoirradiation of single-stranded BPUAA
resulted in a complete destruction of the oligomer without formation
of any significant detectable products. While triplet benzophenone
oxidizes guanine in DNA by one electron transfer,13 guanine
oxidation was at the best a very minor reaction in the photoinduced
cross-link formation of BPUAA/TTA. It is likely that a restricted
dynamic motion of the chromophore in the major groove of DNA
effectively suppressed nonspecific reactions leading to the destruc-
tion of BPUAA, but promoted the specific photocross-linking with
a high site selectivity. These results described here demonstrated
the significance of the cage and proximity effects attained by
immobilizing a benzophenone chromophore in a major groove of
the duplex DNA. Furthermore, thermally reversible interstrand
cross-link may be useful for a temporary connection of two
oligomers in DNA manipulations.
Acknowledgment. We thank Dr. Falvey of the University of
1
Maryland for sending H NMR spectra of 6.
Figure 1. Reversed-phase HPLC profiles for the photoreactions of duplexes
(a) BPUAA/TTA and (b) BPUAA/TUA and (c) for a heat-induced splitting
of cross-links of BPUAA-TUA. Photoirradiation was carried out at 312 nm
for (a) 3000 and (b) 4000 counts with a monochromator (JASCO CRM-
FD, 300 W Xe lamp) in the presence of adenine (A) as an internal standard.
One count of photoirradiation approximately corresponds to a surface energy
of 0.02 J/cm2. Isolated BPUAA-TUA in water was heated at 90 °C for 60
min. A shoulder peak marked with an asterisk is BPUAA containing a
thymine dimer.
Supporting Information Available: Experimental protocol for
photoreactions of duplexes, the detailed synthesis of 4 and BPUAA,
and NMR assignment for 5 (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) For a recent review, see: Bach, T. Synthesis 1998, 683-703.
BPUAA-TUA completely disappeared in HPLC with heating at 90
°C for 1 h in water accompanied by a concomitant formation of
oligomers BPUAA and TUA (Figure 1c). The half-life of BPUAA-
TUA in water (pH 7.0) was 38.5 min at 80 °C and considerably
decreased to 8.9 min at pH 5.2. All attempts to isolate thymidine-
benzophenone adduct by enzymatic digestions of BPUAA-TUA
were unsuccessful due to the thermal instability of the cross-linked
structure, but a model photoreaction of benzophenone with 1,3-
dimethylthymine in aqueous acetonitrile provided significant in-
sights into the cross-linked structure. In addition to the formation
of known oxetane 6 (27% yield),12 we isolated without precedent
oxetane 5, which is a structural isomer of 6, in 38% yield. The
structure of 5 involving a N,O-acetal functionality was unambigu-
ously determined by a complete assignment of the 1H and 13C NMR
signals by HMQC and HMBC spectra (Figure S1). While oxetane
6 was stable under heating at 90 °C in chloroform, isomeric oxetane
5 was decomposed within 1 h to a one-to-one mixture of
1,3-dimethylthymine and benzophenone in an almost quantitative
yield. Thermal dissociation of 5 did not proceed in refluxing
rigorously dried benzene, suggesting that a proton source was
essential for the dissociation process. These observations were in
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BPUAA-TUA to original oligomers and the acceleration of the
reaction rate in acidic solution. On the basis of these data, it is
most likely that the cross-link reaction of BPUAA with TTA and
TUA proceeds via Paterno-Bu¨chi type reaction between a carbonyl
group of benzophenone and a C5-C6 double bond of thymine
producing the oxetane having the N,O-acetal structure.
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