An Efficient Synthesis of a Furan-Side Furocoumarin Thymidine Monoadduct

Full text of DOI:10.1021/jo9619084

2630
J . Org. Chem. 1997, 62, 2630-2632
An Efficien t Syn th esis of a F u r a n -Sid e
F u r ocou m a r in Th ym id in e Mon oa d d u ct
William R. Kobertz and J ohn M. Essigmann*
Department of Chemistry, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
psoralen-DNA adducts.6 Out of the monoadducts, the
furan-side adduct is the most appealing for genetic and
DNA repair studies because it contains an intact chro-
mophore that can still photoreact in double stranded
DNA to form a crosslink. Therefore, it was of use to
synthesize a furocoumarin thymidine monoadduct for its
eventual site-specific incorporation into oligonucleotides.
The total synthesis of any psoralen-thymidine or
furocoumarin-thymidine monoadduct has proved par-
ticularly difficult owing to the synthetically challenging
cis-syn stereochemistry in the product. In the absence
of a DNA helix, furocoumarins photodimerize adding an
additional complication.7 Consequently, previous at-
tempts to synthesize furocoumarin-thymidine monoad-
ducts have resulted in photoproducts with the incorrect
stereochemistry8 or a mixture of isomers from which only
small quantities of the desired isomer could be isolated.9
We have recently synthesized a 2-carbomethoxypsor-
alen-thymidine furan-side adduct by utilizing a benzo-
furan derivative in the key photochemical step followed
by a pyrone ring annulation to afford the desired prod-
uct.10 This “two-stage” strategy eliminated photodimer-
ization and yielded a single photoproduct containing a
cis-syn geometry. While this approach gave the desired
monoadduct in reasonable yield, it involved many steps
with a delicate saturated pyrimidine. In this note, we
describe a more direct approach utilizing a full furocou-
marin derivative; this approach is more appealing be-
cause it shortens the synthetic scheme and eliminates
all but one reaction with a saturated pyrimidine. At the
outset, a major concern in using an intact furocoumarin
was that it could lead to self-dimerization instead of a
thymidine-furocoumarin photoproduct. Our earlier stud-
ies suggested, however, that a 2′-ester linkage could favor
the intramolecular reaction between the furocoumarin
and thymidine over furocoumarin photodimerization. We
now report our further exploration of this linkage by
using an intact furocoumarin derivative.
Received October 8, 1996
The construction of oligonucleotides containing a modi-
fied nucleoside at a predefined site has provided new
insights into the mechanisms of DNA repair and muta-
genesis.1 Organic chemists have played a key role by
synthesizing nucleosides damaged by UV and ionizing
radiation, chemical carcinogens, and chemotherapeutic
agents.2 Further incorporation of these modified nucleo-
sides into oligonucleotides has enabled the direct study
of biologically relevant nucleoside adducts in any desired
DNA sequence. Recent studies on anticancer drug-DNA
adducts have suggested a new generation of agents that
have diminished mutagenic potential, while preserving
the desired cytotoxic properties necessary for therapeutic
activity.3 Psoralen (1) and its derivatives are linear
furocoumarins that are highly effective therapeutic agents
to combat skin disorders4 and cutaneous T-cell lym-
phoma.5 Psoralen reacts primarily with thymidines in
helical DNA by a [2 + 2] photocycloaddition. The key
mechanistic step required for reactivity is the intercala-
tion of psoralen between the bases in double-stranded
DNA. The intercalation event positions psoralen in an
orientation to form a cis-syn four-membered ring with
thymidine. Once intercalated, irradiation with long wave
UV-light (320-400 nm) results in a photoreaction be-
tween psoralen and thymidine to give a monoadduct. If
the reaction occurs on the furan-side of psoralen, the
initial adduct can absorb a second photon and react with
an adjacent thymidine in the opposite strand forming an
interstrand crosslink. Pyrone-side adducts do not possess
a chromophore that can absorb long wave UV-light and
therefore do not form crosslinks. Hearst and co-workers
have isolated and characterized a large number of
* Author to whom correspondence should be addressed. Phone (617)
253-6227. Fax (617) 253-5445. email: jessig@mit.edu.
The strategy involves linking a 2′-carboxypsoralen to
the 5′-hydroxyl of thymidine to control the stereochem-
istry of the photoreaction. 2′-Carboxypsoralen deriva-
tives have been previously synthesized to improve pho-
toreactivity and water solubility of furocoumarins.11 On
(1) (a) Hess, M. T.; Schwitter, U.; Petretta, M.; Giese, B.; Naegeli,
H. Chem. Biol. 1996, 3, 121. (b) Bredberg, A.; Sandor, Z.; Brant, M.
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the Third International Symposium. In Psoriasis; Grune and Strat-
ton: New York, 1982; pp 149-156. (b) Parrish, J . A.; Stern, R. S.;
Pathak, M. A.; Fitzpatric, T. B. Photochemotherapy of skin dieases.
In The Science of Photomedicine; Plenum Press: New York, 1982; pp
595-624.
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H. B.; Isaacs, S. T.; Hearst J . E. Annu. Rev. Biochem. 1985, 54, 1151.
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Rev. Biophys. 1984, 17, 1. (e) Peckler, S.; Graves, B.; Kanne, D.;
Rapoport, H.; Hearst, J . E.; Kim, S.-H. J . Mol. Biol. 1982, 162, 157. (f)
Hearst, J . E. Chem. Res. Toxicol. 1989, 2, 69.
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S0022-3263(96)01908-1 CCC: $14.00 © 1997 American Chemical Society

Notes
J . Org. Chem., Vol. 62, No. 8, 1997 2631
Sch em e 1
Sch em e 2
F igu r e 1. (Above) Structure of the furocoumarin-thymidine
photoproduct 8, where dR ) 2′-deoxy-3′-O-acetylribose. (Below)
Circular dichroism spectrum of photoproduct 8. (Inset) Nuclear
Overhauser enhancements for photoproduct 8. (A) Difference
spectrum between sample irradiated at the thymidine methyl
resonance (1.76 ppm) and spectrum B; (B) 4.0-5.0 ppm region
of the proton NMR spectrum.
long wave UV light (366 nm) produced only photodegra-
dation of the starting material, compound 7. However,
the use of 300 nm light and acetone as a photosensitizer
yielded one photoproduct, which underwent a transes-
terification with methanol and silica gel to afford 8 in
24% yield. The low yield was presumably due to some
photodimerization even though the reactions were per-
formed at low concentration (1 mM). Photoproduct 8 has
been previously converted into the nucleoside 9.10
The regio- and stereochemistry of photoproduct 8 was
assigned by NMR and CD spectroscopy. The regiochem-
istry of 8 was determined by the coupling constant
between the H6 proton of thymidine and the H4 proton
of the furocoumarin. Only long range coupling (2 Hz)
was observed, which is indicative of the syn regioisomer.14
Irradiation of the thymidine methyl group in a difference
NOE experiment resulted in positive signals for both
protons on the four-membered ring assigning the stereo-
chemistry as cis (Figure 1, inset). Determination of the
facial selectivity of the photoreaction was ascertained by
CD spectroscopy (Figure 1). Hearst et al. have shown
that the CD spectrum of the two major furan-side
monoadducts behave as enantiomers in the presence of
polarized light.15 The CD spectrum of 8 was identical to
the photoproduct generated by our earlier method10 and
the basis of our earlier work with benzofuran-thymidine
photoproducts,10 we developed a new method for the
construction of 2′-carboethoxypsoralen shown in Scheme
1. Removal of the MOM-protecting group from 210 with
HCl in ethanol yielded phenol 3. Cyclization of com-
pound 3 with dimethylacetamide dimethyl acetal in the
presence of 4 Å sieves gave 2′-(ethoxycarbonyl) psoralen
(4) in 65% yield.12,13 Saponification of 4 afforded acid 5.
The 2-carboxypsoralen was then attached to a suitably
protected thymidine. As shown in Scheme 2, coupling
of psoralen 5 with thymidine 68 yielded photoprecursor
7. Attempts to effect the photochemical reaction by using
(12) (a) Barton, D. H. R.; Hewitt, G.; Sammes, P. G. J . Chem. Soc.
(C) 1969, 16. (b) Begley, M. J .; Crombie, L.; Slack, D. A.; Whiting, D.
A. J . Chem. Soc., Perkin Trans. 1 1977, 2402. (c) Effenberger, F.; Maier,
R. Liebigs Ann. Chem. 1969, 729, 246.
(13) We have found that the addition of 4 Å sieves to remove the
methanol generated during the reaction affords higher yields at room
temperature.
(14) (a) Pretsch, E.; Clerc, T.; Seibl, J .; Simon, W. In Tables of Spec-
tral Data for Structure Determination of Organic Compounds; Springer-
Verlag: New York, 1989; p H185. (b) Silverstein, R. M.; Bassler, G.
C.; Morrill, T. C. In Spectrometric Identification of Organic Compounds;
J ohn Wiley & Sons: New York, 1981; p 209.
(15) Gamper, H.; Piette, J .; Hearst, J . E. Photochem. Photobiol. 1984,
40, 29.

2632 J . Org. Chem., Vol. 62, No. 8, 1997
Notes
to an isolated psoralen-thymidine photoproduct.6f,15 The
synthesized adduct is the equivalent to the biologically
generated photoadduct occurring at a 5′-TpA-3′ site in
DNA.
143.5, 147.6, 153.7, 156.8, 158.9, 160.2; HRMS (EI) calcd for
C
C
14H10O5 (M)+ 258.0528, found 258.0529. Anal. Calcd for
14H10O5: C, 65.12; H, 3.90. Found: C, 65.05; H, 3.72.
2′-Ca r boxyp sor a len (5). A solution of 0.27 g (1.06 mmol)
of compound 4 and 10 mL of aqueous 1 N NaOH in 10 mL of
methanol was heated to reflux for 30 min. The solvents were
removed, and the crude product was dissolved in water and
acidified to pH 1 with 1 N HCl. The resulting precipitate was
filtered, washed with cold 1 N HCl and ether, and then dried
overnight to afford 0.19 g (77%) of a yellowish-white solid: mp
264-266 °C dec; 1H NMR (300 MHz, DMF-d7) δ 6.51 (d, 1 H, J
) 9.6), 7.79 (s, 1 H), 7.83 (s, 1 H), 8.23 (s, 1 H), 8.26 (d, 1 H, J
) 9.6); 13C NMR δ 100.9, 114.4, 116.0, 117.8, 124.1, 125.7, 145.7,
149.7, 154.9, 157.8, 160.9, 160.9; HRMS (EI) calcd for C12H6O5
(M)+ 230.0215, found 230.0214.
5′-O-[2′-P sor a len yl-]-3′-O-a cetylth ym id in e (7). To a solu-
tion of 80 mg (0.35 mmol) of compound 5 in 1.5 mL of dry DMF
and 1.5 mL of dry pyridine was added 150 mg (0.53 mmol) of
3′-O-acetylthymidine (6), and the suspension was sonicated for
5 min. To the milky white solution were added 170 mg (0.88
mmol) of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-
drochloride (EDC) and 5 mg (0.35 mmol) of DMAP. The solution
was stirred at room temperature under an argon atmosphere
for 24 h. The solvent was concentrated, dissolved in CH2Cl2,
washed with 1 N HCl and saturated NaHCO3, and then dried
over MgSO4. The crude product was purified by silica gel
We have described a straightforward synthesis of a
furocoumarin-thymidine furan-side adduct. A 2-car-
boxypsoralen tethered to the 5′-hydroxyl of thymidine
controlled the stereochemical outcome of the photochemi-
cal reaction to give the desired cis-syn isomer. In
addition, this linkage also helped minimize photodimer-
ization. This simple approach to furocoumarin-thymi-
dine adducts should expedite the incorporation of these
adducts into oligonucleotides. The advantage of this total
synthetic approach is that it will allow for the first time
the selective placement of a furocoumarin-thymidine
adduct in sequences where multiple sites of adduction
are possible. Many such sites are mutational hot spots
that occur in proto-oncogenes and tumor suppressor
genes and are thought to play a key role in neoplastic
transformation.16 Incorporation of this adduct into DNA
and its biological ramifications will reported in due
course.
chromatography, eluting with
a gradient of 100:1 to 50:1
dichloromethane/methanol to afford 106 mg (61%) of a white
foam: 1H NMR (300 MHz, CDCl3) δ 1.70 (d, 3 H, J ) 1.0), 2.14
(s, 3 H), 2.39 (ddd, 1 H, J ) 6.6, 8.7, 14), 2.55 (ddd, 1 H, J ) 1.4,
5.5, 14), 4.34-4.40 (m, 1 H), 4.64-4.71 (m, 2 H), 5.36 (app d, 1
H, J ) 6.4), 6.40 (dd, 1 H, J ) 5.5, 8.7), 6.43 (d, 1 H, J ) 9.7),
7.39 (d, 1 H, J ) 1.2), 7.48 (s, 1 H), 7.67 (s, 1 H), 7.80 (d, 1 H, J
) 9.7), 7.83 (s, 1 H), 9.01 (br s, 1 H); 13C NMR δ 12.4, 20.9, 37.4,
65.1, 74.6, 82.1, 85.1, 100.4, 111.5, 114.6, 115.9, 116.9, 122.2,
123.9, 134.7, 143.3, 146.5, 150.3, 154.2, 156.8, 158.1, 159.9, 163.3,
170.5; UVmax (MeOH) 334, 264 nm; HRMS (FAB+, NBA) calcd
for C24H20O10N2 (M + H)+ 497.1196, found 497.1203.
2-Ca r b om et h oxyp sor a len -3′-O-Acet ylt h ym id in e Cis-
Syn P h otop r od u ct 8. A solution of 20 mg (0.04 mmol) of
compound 7 in 40 mL of dry CH3CN was deaerated with argon
bubbling for 30 min. Acetone (2 mL) was added, and the solution
was irradiated with 300 nm light in a 16-bulb Rayonet photo-
reactor for 3 h at room temperature. Removal of the solvents
and adsorption of the crude material onto silica gel using
methanol as the solvent effected lactone ring opening. The
absorbed compound was loaded on top of a silica gel column and
purified by eluting with a gradient of 100:1 to 50:1 dichlo-
romethane/methanol to afford 6 mg (24%) of a clear foam: 1H
NMR (300 MHz, acetone-d6) δ 1.76 (s, 3 H), 2.01-2.05 (m, 1 H),
2.07 (s, 3 H), 2.13 (ddd, 1 H, J ) 2.0, 5.9, 14), 3.71-3.73 (m, 2
H), 3.88 (s, 3 H), 3.90-3.92 (m, 1 H), 4.11 (t, 1 H, J ) 5.4, (-OH)),
4.24 (s, 1 H), 4.97 (d, 1 H, J ) 2.0), 5.20-5.22 (m, 1 H), 6.20 (dd,
1 H, J ) 5.9, 9.8), 6.24 (d, 1 H, J ) 9.3), 6.90 (s, 1 H), 7.39 (s, 1
H), 7.90 (d, 1 H, J ) 9.3); 13C NMR (CDCl3) δ 21.0, 22.9, 35.8,
46.6, 53.7, 55.0, 57.3, 62.5, 74.3, 84.1, 84.9, 87.8, 99.3, 113.8,
114.4, 121.5, 125.8, 143.2, 150.4, 156.3, 160.5, 164.0, 169.2, 169.7,
170.7; UVmax (MeOH) 324, 293; HRMS (FAB+, 3-NBA) calcd for
C25H24O11N2 (M + H)+ 529.1458, found 529.1456.
Exp er im en ta l Section
Gen er a l P r oced u r es. Unless otherwise specified, materials
were obtained from commercial suppliers and were used without
further purification. Tetrahydrofuran (THF) was distilled from
potassium/benzophenone ketyl. Methanol (MeOH) was distilled
from sodium metal and used immediately after distillation.
Anhydrous solvents were otherwise obtained from Aldrich.
Column chromatography was performed on Merck silica gel
(230-400 mesh). 1H NMR spectra were recorded at 500 or 300
MHz on superconducting FT spectrometers. 13C NMR spectra
were proton decoupled and were recorded at 125 or 75 MHz.
Coupling constants are measured in hertz. Melting points
(Pyrex capillary) are uncorrected.
2-Ca r beth oxy-5-for m yl-6-h yd r oxyben zofu r a n (3). A so-
lution of 1.02 g (3.67 mmol) of compound 2, 2 mL of concentrated
HCl, and 100 mL of ethanol was heated at reflux for 1 h. After
cooling to room temperature, the solution was neutralized with
saturated NaHCO3 and diluted with ethyl acetate. The layers
were separated, and the aqueous portion was washed twice with
ethyl acetate. The combined organic washes were washed with
brine and dried over Na2SO4. Removal of the solvents in vacuo
and purification by silica gel chromatography eluting with a
gradient of 10:1 to 2:1 hexanes/ethyl acetate afforded 0.78 g
(91%) of a yellow-white solid. Recrystallization from hexanes/
ethyl acetate afforded yellow plates: mp 158-159 °C; 1H NMR
(300 MHz, CDCl3) δ 1.41 (t, 3 H, J ) 7.2), 4.42 (q, 2 H, J ) 7.2),
7.09 (s, 1 H), 7.49 (s, 1 H), 7.88 (s, 1 H), 9.95 (s, 1 H), 11.23 (s,
1 H); 13C NMR δ 14.3, 61.7, 100.1, 113.7, 119.2, 120.5, 129.9,
147.0, 158.9, 160.2, 161.8, 195.9; HRMS (EI) calcd for C12H10O5
(M)+ 234.0528, found 234.0529. Anal. Calcd for C12H10O5: C,
61.54; H, 4.30. Found: C, 61.17; H, 4.11.
2-Ca r beth oxyp sor a len (4). To a solution of 95 mg (0.41
mmol) of compound 3 in 4 mL of dry THF were added 4 Å sieves
and 120 µL (0.82 mmol) of N,N-dimethylacetamide dimethyl
acetal. The solution was stirred for 5 h at room temperature
under an argon atmosphere. The reaction mixture was diluted
with ethyl acetate, washed with saturated 1 N HCl, saturated
NaHCO3, and brine and then dried over Na2SO4. The crude
product was purified by silica gel chromatography eluting with
a gradient of 3:1 to 1:1 hexanes/ethyl acetate to afford 69 mg
(65%) of a white solid. Recrystallization from hexanes/ethyl
acetate afforded white needles: mp 221-222 °C; 1H NMR (300
MHz, CDCl3) δ 1.44 (t, 3 H, J ) 7.2), 4.46 (q, 2 H, J ) 7.2), 6.42
(d, 1 H, J ) 9.6), 7.55 (s, 1 H), 7.57 (s, 1 H), 7.79-7.82 (m, 2 H);
13C NMR δ 14.3, 61.9, 100.6, 113.2, 115.5, 116.5, 121.8, 124.2,
Ack n ow led gm en t. The American Chemical Society
Division of Medicinal Chemistry and Abbott Laborato-
ries are gratefully acknowledged for support of a pre-
doctoral fellowship to W.R.K. This work was supported
by NIH Grants CA52127 and ES07020.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for compounds 5, 7, and 8 (6 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
(16) Friedberg, E. C.; Walker, G. C.; Siede, W. In DNA Repair and
Mutagenesis; ASM Press: Washington, D.C., 1995.
J O9619084