Solid-Phase Synthesis of Oligonucleotides Containing a Site-Specific Psoralen Derivative

Full text of DOI:10.1021/ja9703178

5960
J. Am. Chem. Soc. 1997, 119, 5960-5961
Scheme 1
Solid-Phase Synthesis of Oligonucleotides
Containing a Site-Specific Psoralen Derivative
William R. Kobertz and John M. Essigmann*
Department of Chemistry
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
ReceiVed January 30, 1997
Psoralens are linear furocoumarins used for the treatment of
skin diseases1 and cutaneous T-cell lymphoma.2 DNA is
believed to be the cellular target for the therapeutic activity of
the psoralens. In addition to medical and DNA diagnostic
applications, psoralen-containing oligonucleotides have been
useful probes for determining nucleic acid structure and func-
tion,3 for studying the mechanisms of DNA repair,4 and for
arresting RNA transcription complexes.5 Psoralens react via a
[2 + 2] cycloaddition, in concert with long-wave UV light, to
form photoproducts primarily with thymidines in 5′-TA-3′ sites
in double-stranded DNA. This reaction is highly regio- and
stereospecific, forming three major photoproducts with cis-syn
stereochemistry: an interstand cross-link, a pyrone-side monoad-
duct, and a furan-side monoadduct.6 Traditional methods for
the synthesis of large amounts of psoralen-containing oligo-
nucleotides require the utilization of high intensity lasers and
are best applied when the desired psoralen-containing oligo-
nucleotide contains only one 5′-TA-3′ site.7 It would be
desirable to have total synthetic procedures allowing for the
preparation of psoralen adducts in any sequence context because
recent studies show that context plays an important role in the
replicative bypass, mutagenic, and genotoxic effects of DNA-
damaging agents.8 The inability to design psoralenated oligo-
nucleotides in any desired sequence context has restricted study
of the biological effects of these important therapeutic agents.
Here, we report the synthesis of a 2-carboxypsoralen furan-
side monoadduct phosphoramidite and its incorporation into
DNA.
presence of mild aqueous base.10 It is also known that furan-
side monoadducts can be reversed by treatment with strong base
at elevated temperatures;10 this consideration therefore prompted
us to explore deprotection conditions that avoided the use of
harsh aqueous base. Commercially available PAC-phosphora-
midites have protecting groups that are readily removed with
10% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in ethanol or
methanol.11 Nucleoside 1a when treated with the aforemen-
tioned conditions remained intact for 24 h at room temperature.
Another concern was the observation that nucleoside 1b slowly
hydrolyzes to the carboxylic acid derivative 1a when stored in
neutral aqueous solutions. This hydrolysis did not affect the
photocross-linking capabilities of the psoralen-thymidine
monoadduct; however, for purification purposes it was conve-
nient to have a homogenous oligonucleotide. Therefore, reaction
conditions were developed to saponify the alkyl ester without
destroying the coumarin ring and the photocross-linking ability
of the monoadduct. Treatment of 1b with a 10 mM sodium
carbonate solution at pH 9 for 12 h led to greater than 95%
conversion to 1a, alleviating purification problems.
The synthesis of the suitably protected psoralen-thymidine
phosphoramidite is shown in Scheme 1. The synthesis of
deoxynucleoside 2 has been previously described.12 Protection
of the 5′ hydroxyl of 2 with 4,4-dimethoxytrityl chloride (DMT-
Cl) in the presence of silver nitrate led to rapid conversion to
3.13 Removal of the 3′ acetate with 5% DBU in freshly distilled
methanol afforded 4. Phosphitylation using diisopropylammo-
nium tetrazolide as an activating agent yielded phosphoramidite
5.
Synthesis of the furan-side monoadduct was chosen for two
reasons. First, the furan-side adduct contains an intact coumarin
chromophore, which when hybridized to a complementary strand
can be converted into the cross-link upon irradiation with long-
wave UV light.9 Second, we believed that the furan-side adduct
could survive the basic deprotection conditions used in solid-
phase DNA synthesis, whereas the unsaturated lactone of the
pyrone-side monoadduct is known to transesterify readily in the
* Author to whom correspondence should be addressed.
(1) (a) Fitzpatric, J. B.; Stern, R. S.; Parrish, J. A. Proceedings of the
Third International Symposium. In Psoriasis; Grune and Stratton: New
York, 1982; pp 149-156. (b) Parrish, J. A.; Stern, F. S.; Pathak, M. A.;
Fitzpatric, J. B. Photochemotherapy of skin diseases. In The Science of
Photomedicine; Plenum Press: New York, 1982; pp 595-624.
(2) (a) Knobler, R. M.; Honigsmann, H.; Edelson, R. L. Psoralen
phototherapies. In Psoralen DNA Photobiology; CRC Press: Boca Raton,
FL, 1988; Vol. II, pp 117-134. (b) Edelson, R. L. Sci. Am. 1988, 259, 68.
(3) Cimino, G. D.; Gamper, H. B.; Isaacs, S. T.; Hearst, J. E. Ann. ReV.
Biochem. 1985, 54, 1151.
(4) (a) Raha, M.; Wang, G.; Seidman, M. M.; Glazer, P. M. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 2941. (b) Cheng, S.; Sancar, A.; Hearst, J. E.
Nucleic Acids Res. 1991, 19, 657. (c) Islas, A. L.; Baker, F. J.; Hanawalt,
P. C. Biochemistry 1994, 33, 10794.
(5) (a) Sastry S. S.; Hearst, J. E. J. Mol. Biol. 1991, 221, 1091. (b) Sastry
S. S.; Hearst, J. E. J. Mol. Biol. 1991, 221, 1111. (c) Shi, Y.-B.; Gamper,
H.; Hearst J. E. J. Biol. Chem. 1988, 263, 527.
(6) Hearst, J. E. Chem. Res. Tox. 1989, 2, 69.
(7) Spielmann, H. P.; Sastry, S. S.; Hearst, J. E. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 4514.
(8) (a) Loechler, E. L. Carcinogenesis 1996, 17, 895. (b) Beglguise-
Valladier, P.; Fuchs, R. P. P. J. Mol. Biol. 1995, 249, 903. (c) Dolan, M.
E.; Oplinger, M.; Pegg, A. E. Carcinogenesis 1988, 9, 2139. (d) Burns, P.
A.; Gordon, A. J. E.; Glickman, B. W. J. Mol. Biol. 1987, 194, 385.
(9) Yeung, A. T.; Jones, B. K.; Chu, C. T. Biochemistry 1988, 27, 3204.
To demonstrate the flexibility of our approach, we chose a
prototypical human TATA box sequence to incorporate 1a. Runs
(10) (a) Yeung, A. T.; Dinehart, W. J.; Jones, B. K. Biochemistry 1988,
27, 6332. (b) Shi, Y.-B.; Spielmann, H. P.; Hearst, J. E. Biochemistry, 1988
27, 5174.
(11) Phenoxyacetyl(PAC)-protected phosphoramidites are available from
Pharmacia.
(12) (a) Kobertz, W. R.; Essigmann, J. M. J. Am. Chem. Soc. 1996, 118,
7101. (b) Kobertz, W. R.; Essigmann, J. M. J. Org Chem. 1997, 62, 2630.
(13) Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Can. J. Chem. 1982,
60, 1106.
S0002-7863(97)00317-X CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 25, 1997 5961
of 5′-TA-3′ sequences are known to be mutational hotspots for
psoralen,14 and it is possible that the therapeutic efficacy of
psoralen might be due in part to the inability of transcriptional
complexes to bind to modified TATA boxes. The modified
oligodeoxynucleotide 6 was synthesized on a 1 µmol scale by
using an automated DNA synthesizer. The coupling time for
phosphoramidite 5 was extended to 15 min affording coupling
yields of 90% based on DMT cation release. For unmodified
phosphoramidites, yields are typically higher; our lower yield
is presumably due to the increased bulk of the psoralen-
thymidine phosphoramidite. The solid support was treated with
1 mL of 10% DBU in anhydrous ethanol in the presence of
cetyltrimethyl ammonium bromide, which aided solubility.15
After 24 h at room temperature, the deprotection solution was
neutralized with aqueous acetic acid and the salts were removed
by using a Na+ exchange column. The crude oligonucleotide
was treated with 50 mM sodium carbonate (pH 9.0) solution
for 12 h to saponify the alkyl ester into the carboxylic acid.
Neutralization followed by purification by reversed phase HPLC
afforded oligonucleotide 6.
The integrity of oligonucleotide 6 was established by
enzymatic digestion and electrospray mass spectrometry. En-
zymatic digestion and HPLC analysis of 6 yielded nucleoside
ratios which were within experimental error of the theoretical
composition of oligonucleotide 6.16 The peak corresponding
to the modified nucleoside had identical HPLC retention
characteristics and UV spectrum as the synthesized standard
1a. Electrospray ionization mass spectrometry of 6 revealed
the presence of ions at m/z 1058.05, 846.5, and 705.2 corre-
sponding to 3-, 4-, and 5- ions, respectively. The calculated
molecular weight was 4236.9, which agreed well with the
calculated molecular weight of 4236.7.
One of the valuable applications of psoralen-containing
oligonucleotides is their ability to cross-link to a hybridized
strand.9 The oligonucleotides described herein could be used
as hybridization probes to form a cross-link to a target strand.
Figure 1 shows such photocross-linking experiments of oligo-
nucleotide 6 with its complementary strand 7. By 32P phosphate
labeling the 5′ hydroxyl of only one of the two DNA stands in
each experiment, the reactions of the two DNA stands can be
independently analyzed by denaturing gel electrophoresis.
Oligonucleotide 6 was labeled in lanes 2-7. Oligonucleotide
7 was labeled in lanes 8-13. The presence of the psoralen
derivative caused oligonucleotide 6 to migrate slower than its
unmodified counterpart 8 (lane 1 vs 2). Irradiation of the duplex
with 366 nm light (9.0 J/m2) afforded the slower moving
interstrand cross-link in 5 min (lanes 4 and 10). In fact,
irradiation with a hand-held 4 W long-wave UV lamp yielded
the cross-link in 84% yield in 90 min.16 A well-known chemical
property of psoralen-DNA cross-links is their reversibility with
254 nm light9 or their asymmetric conversion to the pyrone-
side adduct by heating in the presence of base.10 Treatment of
the cross-linked duplex with 254 nm light (16.7 J/m2) for 20
min resulted in photoreversion to the monoadduct (lanes 5 and
11) and eventually complete or near complete reversal to the
unmodified strand (lanes 6 and 12). Again, a low wattage hand-
Figure 1. Photoreactions of the furan-side monoadduct containing
oligonucleotide and the photoreversion and base-catalyzed reversal
(BCR) of the cross-linked oligonucleotides. The photoreactive thymidine
in oligonucleotide 7 is encircled. Oligonucleotide 6 and the comple-
mentary strand 7 were 5′ phosphate labeled with 32P in lanes 2-7 and
lanes 8-13, respectively: (lane 1) unmodified control oligonucleotide
8 labeled with 32P at the 5′ terminus; (lanes 2-4 and 8-10) irradiation
with 366 nm light; (lanes 5-6 and 11-12) photoreversal of the cross-
link with 254 nm light; (lanes 7 and 13) base-catalyzed reversal of
cross-link.
held 254 nm lamp caused near complete reversal in 2 h.16 Base-
catalyzed reversal results in conversion of the interstrand cross-
link into a pyrone-side adduct, essentially transferring the
original psoralen furan-side monoadduct in 6 to the comple-
mentary strand 7.10 Treatment of a cross-linked duplex with
0.1 M NaOH at 90 °C for 30 min efficiently reversed the cross-
link, affording unmodified oligonucleotide (lane 7) and primarily
a pyrone-side monoadduct (lane 13). These experiments
demonstrated that the synthesized psoralen oligonucleotides
posses the useful hybridization/cross-linking/reversal properties
of psoralen-containing oligonucleotides generated by traditional
methods.
The successful site-specific incorporation of a cis-syn furan-
side psoralen-thymidine monoadduct into oligonucleotides will
enable the study of this therapeutic agent in DNA sequence
contexts that contain multiple sites of reactivity. Such sites are
known to be biologically important, and with this methodology,
it will now be possible to design experiments to probe the details
of how sequence context influences biological endpoints. In
addition, this synthetic approach makes it feasible to utilize the
valuable cross-linking properties of oligonucleotides containing
furan-side monoadducts in hybridization assays and other
experiments where site-specific cross-linking is desired.
Acknowledgment. W.R.K. is grateful to the American Chemical
Society Division of Medicinal Chemistry and Abbott Laboratories for
a predoctoral fellowship. Electrospray analyses were provided by the
MIT Division of Toxicology Mass Spectrometry Facility supported by
NIH grants CA26731 and ES05622. This work was supported by NIH
Grants CA52127 and ES07020.
Supporting Information Available: Experimental details for
compounds 3-6 and photocross-linking, photoreversal, base-catalyzed
reversal, and enzymatic digestion conditions of oligonucleotide 6 (8
pages). See any current masthead page for ordering and Internet access
instructions.
(14) (a) Miller, S. S.; Eisenstadt, E. J. Bacteriol. 1987, 169, 2724. (b)
Gunther, E. J.; Yeasky, T. M.; Gasparro, F. P.; Glazer, P. M. Cancer Res.
1995, 55, 1283. (c) Yang, S. C.; Lin, J. G.; Chiou, C. C.; Chen, L. Y.;
Yang, J. L. Carcinogenesis 1994, 15, 201.
(15) Xu, Y.-Z.; Swann, P. F. Nucleic Acids Res. 1990, 18, 4061.
(16) See Supporting Information Figures S1 and S2.
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