DNA cross-linking by a phototriggered dehydromonocrotaline progenitor

Article abstract of DOI:10.1021/ja983894k

A wide variety of pyrrolizidine alkaloids, such as monocrotaline, and the clinically significant mitomycins and the related FR-900482, FK 973, and FR-66979 exert their cytotoxicity through the formation of DNA-DNA interstrand cross-links and DNA-protein c

Full text of DOI:10.1021/ja983894k

VOLUME 121, NUMBER 13
APRIL 7, 1999
© Copyright 1999 by the
American Chemical Society
DNA Cross-Linking by a Phototriggered Dehydromonocrotaline
Progenitor
Jetze J. Tepe and Robert M. Williams*
Contribution from the Department of Chemistry, Colorado State UniVersity, Fort Collins, Colorado 80523
ReceiVed NoVember 11, 1998
Abstract: A wide variety of pyrrolizidine alkaloids, such as monocrotaline, and the clinically significant
mitomycins and the related FR-900482, FK 973, and FR-66979 exert their cytotoxicity through the formation
of DNA-DNA interstrand cross-links and DNA-protein cross-links. These naturally occurring antitumor
antibiotics are generally either oxidatively or reductively activated in vivo forming a highly reactive pyrrolic-
type intermediate, which is responsible for the ultimate DNA cross-linking reaction. These oxidative and
reductive pathways, however, often lead to a variety of undesirable toxic events. With the increasing demand
for new and less cytotoxic antitumor agents and the recent success of clinically significant photopheresis
technologies, we describe here the semisynthesis and DNA cross-linking of the first photochemically triggered
progenitor of dehydromonocrotaline.
Introduction
Scheme 1
Pyrrolizidine alkaloids (PAs), such as monocrotaline, 1, are
potent hepatotoxins and carcinogens isolated from a wide variety
of plants.1,2 The mode of action of these toxic agents is through
a cytochrome P450 mediated oxidation forming the highly
reactive dehydropyrrolizidine 2 (dehydromonocrotaline, Scheme
1) which subsequently mediates the interstrand cross-linking
of DNA, forming the bis-alkylated adduct 3.3,4 This oxidation
results in the electrophilic activation of the C-7 and C-9
positions, via conjugation with the pyrrole nitrogen lone pair,
(1) Mattocks, A. R. Chemistry and Toxicology of Pyrrolizidine Alkaloids;
Academic Press: London, UK; 1986.
(2) Smith, L. W.; Culvenor, C. C. J. J. Nat. Prod. 1981, 44, 129.
(3) Niwa, H.; Ogawa, T.; Yamada, K. Tetrahedron Let. 1991, 32, 927.
(4) For excellent general reviews pertaining to the biological relevance
of DNA interstrand cross-linking, see: (a) Kohn, K. W. In Topics in
Molecular and Structural Biology 3. Molecular Aspects of Anti-cancer Drug
Action; Neidle, S., Waring, M., Eds.; Verlag Chemie GmbH: D-6940,
Weinheim, 1994; p 315. (b) Lawley, P. D. BioEssays 1995, 17, 561. (c)
Paustenbach, D. J.; Finley, B. L.; Kacew, S. Proc. Soc. Exp. Biol. Med.
1996, 211, 211. (d) Gniazdowski, M.; Cera, C. Chem. ReV. 1996, 96, 619.
(e) Rajski, S. R.; Williams, R. M. Chem. ReV. 1998, 98, 2723.
which are prone to nucleophilic attack by DNA. The DNA cross-
linking specificity of this reaction has been elucidated and
5′
demonstrated to occur at CpG3′ sites via the exocyclic amine
of dG residues in the minor groove.5 The pyrrolic PAs are potent
(5) (a) Weidner, M. F.; Sigurdsson, S. T.; Hopkins, P. B. Biochemistry
1990, 29, 9225. (b) Woo, J.; Sigurdsson, S. T.; Hopkins, P. B. J. Am. Chem.
Soc. 1993, 115, 3407.
10.1021/ja983894k CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/19/1999

2952 J. Am. Chem. Soc., Vol. 121, No. 13, 1999
Tepe and Williams
dehydromonocrotaline. The masked dehydromonocrotaline de-
scribed herein, provides the first example of a new class of
pyrrolizidine alkaloids that can be activated photochemically.
Results and Discussion
The synthesis of the photoactivated dehydromonocrotaline
derivative 10 is shown in Scheme 2. Commercially available11
monocrotaline was condensed with trichloroethylchloroformate
(TrocCl) in the presence of KI in acetonitrile, furnishing the
ring-cleaved product 5.12 Substitution at the tertiary allylic
bridgehead position was not observed, presumably due to steric
interference. A more direct approach using 4,5-dimethoxy-2-
nitrobenzylchloroformate (6-nitroveratrylchloroformate or NVOC-
Cl) proved to be unsuccessful under a wide variety of conditions
and was consequently abandoned. Thus, the allylic iodide 5 was
oxidized with DMSO and AgBF4 to afford the unsaturated
aldehyde 6 in excellent yield. Protection of the aldehyde 6 as
the corresponding ethylene glycol acetal (7) followed by removal
of the 2,2,2-trichloroethyl carbamate with zinc in hot ethanol
(8) and acylation with 6-nitroveratryl chloroformate provided
the corresponding NVOC derivative (9).
Finally, deprotection of the acetal with 1% aqueous HCl in
acetone afforded the masked dehydromonocrotaline derivative
10. As a control experiment the “pro-dehydromonocrotaline”
(10) was dissolved in a DMSO-d6-D2O mixture (pH ) 7.0)
and was exposed to 365-nm UV irradiation. The reaction was
1
monitored by H NMR, and after 5 h, analysis of the reaction
mixture indicated that compound 10 was cleanly converted to
dehydromonocrotaline (2). The cleaved aldehyde intermediate
derived from 10 was not observed by 1H NMR analysis during
the reaction. The dehydromonocrotaline (2) produced in this
experiment was isolated (purified by PTLC silica gel), and the
spectral data for this substance was identical to an authentic
sample of dehydromonocrotaline prepared by oxidation of
monocrotaline as previously described.13
Figure 1.
DNA-DNA and DNA-protein cross-linking agents and are
therefore of interest as potential antitumor and antibacterial
agents.6,7 The clinical utility of such substances has, unfortu-
nately, been obviated by the acute hepatotoxicity that these
compounds display.
The DNA-DNA cross-linking ability of this pro-dehy-
dromonocrotaline (10) was investigated using linear plasmid
(10) For some relevant examples of photochemically activated DNA-
reactive agents, see: (a) Nicolaou, K. C.; Dai, W. M.; Wendeborn, S. V.;
Smith, A. L.; Torisawa, Y.; Maligres, P.; Hwang, C. K. Angew. Chem.,
Int. Ed. Engl. 1991, 30, 1032. (b) Wender, P. A.; Zercher, C. K.; Beckham,
S.; Haubold, E.-M. J. Org. Chem. 1993, 58, 5867. (c) Wender, P. A.;
Beckham, S.; O’Leary, J. G. Synthesis 1994, 1278. (d) Nakatani, K.; Isoe,
S.; Maekawa, S.; Saito, I. Tetrahedron Lett. 1994, 35, 605. (e) Funk, R. L.;
Young, E. R. R.; Williams, R. M.; Flanagan, M. A.; Cecil, T. R. J. Am.
Chem. Soc. 1996, 118, 3291-3292. DNA photocleavage by dynemicin A
involves an initial photoreduction step, see: (f) Shiraki, T.; Sugiura, Y.
Biochemistry 1990, 29, 9795. DNA photocleavage by esperamicin and
neocarzinostatin chromophore has also been reported, see: (g) Sugiura, Y.;
Kuwahara, J.; Vesawa, Y. Biochem. Biophys. Res. Commun. 1989, 164,
903. The latter undergoes a Norrisch type II cleavage to produce a fulvene
derivative which may be responsible for the DNA cleavage, see: (h) Hirama,
M.; Nehira, T.; Fujiwara, K.; Gomibuche, T. Tetrahedron Lett. 1993, 34,
5753.
Similar to the pyrrolizidine alkaloids, the mitomycins and
the related FR-900482, FK973, and FR-66979 also exert their
cytotoxicity through the formation of DNA-DNA interstrand
cross-links and DNA-protein cross-links.4e,8 However, the
irreversible formation of these DNA cross-links in these systems,
are the result of an in vivo reductive activation, forming the
highly reactive mitosene intermediate (4, Figure 1), which is
structurally similar to the aforementioned pyrrolic PAs.
Most families of naturally occurring antitumor antibiotics are
activated in vivo by either reduction or oxidation.4 Reductive
activation pathways often lead to adventitious production of
superoxide and related oxygen radical species which manifest
in a variety of undesirable toxic species and events. Oxidative
activation, which requires liver cytochrome P450 enzymes, is
typically accompanied by undesirable liver toxicity where the
reactive drug molecule is initially formed.4 As part of a program
aimed at diversifying the repertoire of chemical reactions that
can be applied to selectively activating antitumor anitbiotics,9,10
we describe here the semisynthesis and DNA cross-linking
reactivity of the first photochemically triggered progenitor of
(11) Monocrotaline used in this study was purchased from Aldrich
Chemical Co.
(12) Cooley, J. H.; Evain, E. J. Synthesis 1989, 1. Compound 5 was
obtained as a 1:2 mixture with the alternate regioisomer i:
(6) Kim, H. Y.; Stermitz, F. R.; Coulombe, R. A., Jr. Carcinogenesis
1995, 16, 2691.
(7) Hurley, L. H. J. Med. Chem. 1989, 32, 2027.
(8) (a) Williams, R. M.; Rajski, S. R.; Rollins, S. B. Chem. Biol. 1997,
4, 127. (b) Williams, R. M.; Rajski, S. R. J. Am. Chem. Soc. 1998, 120,
2192.
(13) An authentic specimen of dehydromonocrotalin (2) was prepared
by oxidation of commercially available monocrotalin (1) with o-chloranil,
as previously described in ref 6.
(9) Rollins, S. B.; Williams, R. M. Tetrahedron Lett. 1997, 38, 4033.

PhotoactiVation of Dehydromonocrotaline Progenitor
J. Am. Chem. Soc., Vol. 121, No. 13, 1999 2953
Scheme 2a
a Reagents and conditions: (a) TrocCl, KI, CH3CN, rt, 10 h (30%); (b) AgBF4, DMSO, TEA, rt, 20 min (95%); (c) TMSCl, HOCH2CH2OH,
CH2Cl2, rt, 5 h (95%); (d) Zn0, EtOH, reflux, 8 h, (63%); (e) NVOCCl, Hunig’s base, CH2Cl2, rt, 30 min (88%); (f) 1% aqueous HCl, acetone, rt,
5 h (75%).
DNA by denaturing alkaline agarose gel electrophoresis ac-
cording to Cech.14 The duplex DNA substrate employed in this
study was pBR322 plasmid DNA which was linearized by
restriction endonuclease digestion with EcoR1. The amount of
linearized pBR322 was quantitated by UV analysis at 260 nm
as described by P. N. Borer.15 Due to the poor water solubility
of compound 10, DNA cross-linking experiments were per-
formed in a dilute DMSO-H2O solvent mixture (10% DMSO).
Compound 10 (various concentrations of a 10 mM stock solution
made from 2.6 mg of 10 dissolved in 460 mL of DMSO) and
0.5 mg of DNA (EcoR1 linearized pBR322) in a DMSO/H2O
solution (10 mL, 10% DMSO/H2O final volume) was exposed
to 365-nm irradiation at 23 °C for 2 h (longer UV exposure
times did not result in an increased amount of cross-linking),
followed by 3 h incubation at 37 °C. The crude reaction mixture
was loaded onto a denaturing 1.2% alkaline agarose gel14 and
provided the results shown in Figure 2.
Lambda Hind III was employed as a molecular weight
standard (lane 1). Control reactions were performed with
monocrotaline (10 µM, 1) in the dark (lane 4) and with an
Figure 2. All dark (control) reactions were incubated at 37 °C for 5
authentic sample of dehydromonocrotaline (10 µM, 2)13 irradi-
h. The reactions exposed to UV irradiation (2 h) were incubated an
ated for 2 h at 365 nm (lane 6) as well as in the dark (lane 5).
additional 3 h after UV irradiation. Lane 1 0.5 µg lambda Hind III
As illustrated in Figure 2, incubation of compound 10 with
the DNA duplex in the dark leads to no detectable cross-linked
product (lane 7 and 9). Lanes 7 and 9 contain mixtures of 0.5
mg linearized pBR322 with compound 10 at 10 and 1.0 mM
concentrations, respectively and were not exposed to UV
irradiation. Lanes 8 and 10 contain mixtures of 0.5 mg linearized
pBR322 with compound 10 at 10 mM and 1.0 mM concentra-
tions, respectively and were exposed to UV irradiation for 2 h.
As shown, only the reactions depicted in lanes 5, 6, 8, and 10
produced the interstrand DNA-DNA cross-link product pro-
duced by dehydromonocrotaline (lane 5 and 6).
(molecular weight standard); lane 2 0.5 µg pBR322 (control); lane 3
0.5 µg pBR322 + hν, 2 h (light control); lane 4 0.5 µg pBR322 + 10
µM monocrotaline (1) (dark control); lane 5 0.5 µg pBR322 + 10 µM
dehydromonocrotaline (dark reaction control); lane 6 0.5 µg pBR322
+ 10 µM dehydromonocrotaline + hν, 2 h, (light control); lane 7 0.5
µg pBR322 + 10 µM compound 10 (dark control); lane 8 0.5 µg
pBR322 + 10 µM compound 10 + hν, 2 h; lane 9 0.5 µg pBR322 +
1.0 µM compound 10 (dark control); lane 10 0.5 µg pBR322 + 1.0
µM compound 10 + hν, 2 h.
DNA-protein cross-linking. In addition, these agents may also
provide a conceptual framework upon which the design and
synthesis of a wide variety of pyrrolic PA prodrugs with
potential clinical applications, such as in clinically significant
photopheresis technologies,16 may be examined. It is important
to note that the currently used photodynamic therapies based
on psoralen derivatives target AT-rich sequences in DNA,
whereas the system described herein targets GC-rich regions
These preliminary studies demonstrate the viability of masked
DNA-reactive pyrrolizidine alkaloid progenitors that are capable
of photochemical activation. Such agents hold promise as tools
to further gain insight into the mechanism of DNA-DNA and
(14) Cech T. R. Biochemistry 1981, 20, 1431.
(15) Borer, P. N. Handbook of Biochemistry and Molecular Biology, CRC
Press: 1975.

2954 J. Am. Chem. Soc., Vol. 121, No. 13, 1999
Tepe and Williams
MHz, CDCl3) δ 189, 175, 173, 153, 149, 133, 95, 79, 76, 76, 75, 63,
62, 47, 43, 32, 21, 19, 14. IR (NaCl, neat) 3482, 3012, 2959, 2916,
1726 (broad signal), 1678, 1411, 1171, 1109 cm-1; exact mass calcd
for C19H25NO9Cl3 m/z 516.059490, found m/z 516.058233.
of duplex DNA; this difference in chemical selectivity coupled
with selective chemical activation is being examined as a cellular
targeting vehicle. Studies toward these ends are under investiga-
tion in these laboratories and will be reported on in due course.
Protection of the Unsaturated Aldehyde. Compound 7. An excess
ethylene glycol (0.34 mmol, 19 mL) was added to a solution of the
aldehyde 6 (0.0677 mmol, 35 mg) in 1.0 mL of dry CH2Cl2. After 5
min TMSCl (0.20 mmol, 26 µL) was added, and the solution was stirred
for 5 h at room temperature. The mixture was diluted with ether (10
mL) and was extracted with saturated NaHCO3(aq) (10 mL) and brine
(10 mL). The ether layer was dried over Na2SO4 and filtered, and the
product was purified by column chromatography on silical gel (eluted
with 1:1 ether/ ethyl acetate) (Rf ) 0.42) to yield 36 mg of 7 as a
Experimental Section
1H NMR and 13C spectra were obtained using a Varian 300 at 300
MHz. NMR spectra were recorded at room temperature unless otherwise
noted. All compounds were further analyzed by HMQC and/or APT
spectra obtained using a Varian 400 MHz spectrometer. All chemical
shifts are reported in ppm. Analytical thin-layer chromatography (TLC)
was carried out on Merck precoated silica gel 60 F-254 plated and
were visualized with a phosphomolybdic acid/ethanol solution. All
solvents were distilled prior to use.
Dehydromonocrotaline 2. Chloranil (0.05 mmol, 12 mg) was
dissolved in chloroform (2.0 mL) in a separatory funnel. Monocrotaline
(Aldrich Chemical Co., 0.031 mmol, 10 mg) in 2.0 mL of chloroform
was added, and the solution was gently stirred for 2 min. A mixture of
NaBH4 (0.08 mmol, 3 mg) and an excess of NaOH (700 mg) in 2.0
mL of H2O were added. A green precipitate formed, and the organic
layer was drained through a filter of decolorizing charcoal into an
Erlenmeyer flask. The organic layer was dried over Na2SO4, filtered,
and concentrated to yield 10 mg of pure dehydromonocrotaline (100%).
1H NMR (300 MHz, CDCl3) δ 6.62 (d, J ) 2.4 Hz, 1H), 6.32 (d, J )
3.0 Hz 1H), 6.13 (dd, J ) 3.0 Hz, 7.8 Hz, 1H), 5.72 (d, J ) 12 Hz,
1H), 4.61 (d, J ) 12 Hz, 1H), 4.20 M, 1H), 4.06 (m, 1H), 3.03 (m,
3H), 2.56 (m, 1H), 2.45 (m, 1H), 1.52 (s, 3H), 1.49 (s, 3H), 1.37 (d, J
) 6.9 Hz, 3H).
1
colorless oil (95%). H NMR (300 MHz, CDCl3) δ 5.99 (d, J ) 3.0
Hz, Hz, 1H), 5.66 (m, 1H), 5.31 (d, J ) 8.7 Hz, 1H), 4.98 (m, 1H),
4.87-4.50 (AB, J ) 9 Hz, 2H), 4.10 (m, 1H), 4.00-3.88 (m, 3H),
3.86(m, 2H), 3.62 (m, 1H), 2.85 (q, J ) 5.1 Hz, 10.5 Hz, 1H), 2.72
(bs, 1H, OH), 2.05 (m, 1H), 1.88 (m, 1H), 1.45 (s, 3H), 1.20 (s, 3H),
1.12 (d, J ) 5.4 Hz, 3H); exact mass calcd for C21H29NO10Cl3 m/z
560.085705, found m/z 560.082972.
Deprotection of the 2,2,2-Trichloroethylene Carbamate. Com-
pound 8. A suspension of the acetal 7 (0.0642 mmol, 36 mg) in dry
ethanol (2.0 mL) containing an excess (0.642 mmol, 42 mg) of zinc
was refluxed for 8 h. The suspension was cooled to room temperature
and was filtered through a Celite plug. The solvent was evaporated off
under reduced pressure, and the product was purified by column
chromatography on silica gel (eluted with 5% diisopropylamine in
ethanol) (Rf ) 0.37) to yield 16 mg of 8 (63%) as a white solid; mp
55-57 °C (recryst ether). 1H NMR (300 MHz, CDCl3) δ 5.87 (d, J )
3.9 Hz, 1H), 5.57 (m, 2H), 5.33 (t, J ) 4.2 Hz, 1H), 4.13 (d, J ) 8.4
Hz, 1H), 3.99-3.84 (m, 6H), 3.36 (m, 1H), 2.84 (m, 1H), 2.83 (q, J )
5.7 Hz,11.2 Hz, 1H), 2.13 (m, 1H), 1.75 (m, 1H), 1.44 (s, 3H), 1.25 (s,
3H), 1.09 (d, J ) 5.7 Hz, 3H). 13C NMR and APT (400 MHz, CDCl3)
δ 175.05 (C), 173.50 (C), 136.41 (C), 136.42 (CH), 98.95 (CH), 78.96
(C), 77.33 (CH), 75.63 (C), 65.02 (2 × CH3), 64.88 (CH2), 62.33 (CH),
44.45 (CH2), 43.63 (CH), 33.75 (CH2), 21.81 (CH3), 18.40 (CH3), 13.87
(CH3). IR (NaCl, neat) 3421, 2983, 1732, 1115 cm-1; exact mass calcd
for C18H28NO8 m/z 386.181492, found m/z 386.181998.
Ring Opening of Monocrotaline. Compound 5. KI (0.675, 112
mg) was added to a suspension of monocrotaline (0.135 mmol, 44 mg)
in freshly distilled acetonitrile (2.0 mL). The suspension was stirred
for 10 min at room temperature after which 2,2,2-trichloroethylchlo-
roformate (0.162 mmol, 22 µL) was added. The suspension slowly
turned yellow and was allowed to stir overnight. The mixture was
diluted with ether (10 mL) and was extracted with saturated NaHCO3(aq)
(10 mL) and brine (10 mL). The ether layer was dried over Na2SO4
and filtered, and the product was purified by column chromatography
on silica gel (eluted with ether) (Rf ) 0.4 for compound 5) to yield 26
mg of compound 5, (30%) as a white foam, and 58 mg of compound
i, (68%) as a white solid. The NMR spectrum of product 5 revealed
that this substance was as a mixture of rotamers at room temperature
NVOC Protection of the Pyrrole. Compound 9. Hunig’s base
(0.065 mmol, 11 µL) was added to a solution of the pyrrole 8 (0.013
mmol, 5 mg) in dry CH2Cl2 (2.0 mL). The solution was cooled to 0
°C, and NVOCCl (0.026 mmol, 7 mg) was added. The solution was
slowly warmed to room temperature. The solution was stirred for 30
min, and the mixture was diluted with ether (10 mL) and was extracted
with saturated NaHCO3(aq) (10 mL) and brine (10 mL). The ether layer
was dried over Na2SO4 and filtered, and the product was purified by
column chromatography on silica gel (eluted with 1:1 ether/ethyl
acetate) (Rf ) 0.27) to yield 7 mg of 9 (88%) as a light yellow solid;
1
on the NMR time scale. H NMR (300 MHz, CDCl3) δ 6.14 (t, J )
9.3 Hz, 1H), 5.68 (bm, 1H), 5.43 (dd, J ) 11.7 Hz, 12 Hz, 1H), 5.0-
4.5 (m, 3H), 4.17-3.95(m, 6H), 3.91 (bs, 1H, OH), 3.66 (m, 1H), 2.93
(q, J ) 7.2 Hz, 14.1 Hz, 1H), 2.75 (bs, 1H, OH), 2.07 (m, 1H), 1.98
(m, 1H), 1.48 (s, 3H), 1.32 (s, 3H), 1.17 (d, J ) 6.9 Hz, 3H). 13C
NMR (300 MHz, CDCl3) δ 175, 174, 153, 136, 133, 95, 79, 76, 75,
74, 63, 61, 46, 43, 31, 21, 19, 14, -3. IR (NaCl, neat) 3485, 3017,
2987, 1721, 1273, 1161, 1111 cm-1; exact mass calcd for C19H26NO8-
Cl3I m/z 627.976877, found m/z 627.975475.
1
mp 94-96 °C (recryst ether). H NMR (400 MHz, DMSO-d6, 75 °C)
δ 7.64 (s, 1H), 7.10 (s, 1H), 5.73 (d, J ) 6.0 Hz, 1H), 5.51 (d, J ) 6.0
Hz, 1H), 5.33-5.29 (m, 3H), 5.09 (s, 1H, OH), 4.92-4.88 (m, 2H),
4.06-3.98 (m, 1H), 3.89 (s, 3H), 3.84 (s, 3H), 3.82-3.68 (m, 5H),
3.43 (m, 1H), 3.05 (s, 1H, OH), 2.97 (q, J ) 5.4, 10.5 Hz, 1H), 2.05
(m, 1H), 1.75 (m, 1H), 1.30 (s, 3H), 1.08 (s, 3H), 0.99 (d, J ) 5.4 Hz,
3H). 13C NMR and APT (400 MHz, DMSO-d6, 75 °C) d 173.70 (C),
173.58 (C), 170.23 (C), 153.99 (C), 153.57 (C), 148.37 (C), 140.15
(C), 136.57 (C), 134.64 (CH), 111.85 (CH), 108.88 (CH), 98.33 (CH),
79.09 (C), 76.02 (CH), 74.88 (C), 64.38 (2 × CH2), 63.41 (CH2), 61.42
(CH), 59.74 (CH2), 56.52 (CH2), 56.47 (CH3), 45.35 (CH2), 43.10 (CH),
30.83 (CH2), 21.67 (CH3), 19.44 (CH3), 13.88 (CH3). IR (NaCl, neat)
3484, 2987, 1731, 1706, 1518, 1324, 1274, 1111 cm-1; exact mass
calcd for C28H37N2O14 m/z 625.224479, found m/z 625.223328.
Deprotection of the Acetal. Compound 10. An aqueous solution
of 1% HCl (0.5 mL) was added to a solution of acetal 9 (0.010 mmol,
6 mg) in 2.0 mL of acetone and was stirred at room temperature for 5
h. The clear solution was diluted with ether (10 mL) and was extracted
with saturated NaHCO3(aq) (10 mL) and brine (10 mL). The ether layer
was dried over Na2SO4 and filtered, and the product was purified by
column chromatography on silica gel (eluted with 1:1 ether/ethyl
acetate) (Rf ) 0.42) to yield 4 mg of 10 (75%) as a light yellow solid;
Oxidation of the Allylic Iodide. Compound 6. AgBF4 (0.234 mmol,
46 mg) was added to a solution of the allylic iodide 5 (0.213 mmol,
134 mg) in 1.0 mL of dry DMSO. After 10 min triethylamine (1.065
mmol, 108 mL) was added, and the suspension was stirred at room
temperature for 20 min. The suspension was diluted with ether (10
mL) and extracted with saturated NH4Cl(aq) (10 mL), NaHCO3 (10 mL),
and brine (10 mL). The ether layer was dried over Na2SO4, filtered,
and purified by column chromatography on silica gel (eluted with 1:1
ether/ethyl acetate) (Rf ) 0.42) to yield 105 mg (95%) of a white solid.
1H NMR (300 MHz, CDCl3) δ 9.99 (d, J ) 3.0 Hz, 1H), 6.50 (d, J )
5.1 Hz, 1H), 5.77 (m, 1H), 5.64 (m, 1H), 5.54 (m, 1H), 4.80 (m, 2H),
4.26 (dd, J ) 11.7 Hz, 17.4 Hz, 1H), 4.02 (m, 1H), 3.86 (bs, 1H, OH),
3.72 (m, 1H), 2.89 (q, J ) 7.2 Hz, 14.1 Hz, 1H), 2.76 (bs, 1H, OH),
1.47 (s, 3H), 1.23 (s, 3H), 1.17 (d, J ) 7.2 Hz, 3H). 13C NMR (300
(16) (a) Dougherty, T. J.; Gomer, C. J.; Henderson, B. w.; Jori, G.; Kessel,
D.; Korbelik, M.; Moan, J.; Peng, Q. J. Natl. Cancer Inst. 1998, 90, 889.
(b) Gollnick, H. P. m.; Owsianoswki, M.; Ramaker, J.; Chun, S. C.; Orfanos,
C. E. Recent Results Cancer Res. 1995, 139, 409. (c) Lucvigsson, J.
Diabetes-Metab. ReV. 1993, 9, 329. (d) Rook, A. H.; Cohen, J. H.; Lessin,
S. R.; Vowels, B. R. Dermatol. Clinics 1993, 11, 339.
1
mp 68-70 °C (recryst ether). H NMR (400 MHz, DMSO-d6, 75 °C)

PhotoactiVation of Dehydromonocrotaline Progenitor
J. Am. Chem. Soc., Vol. 121, No. 13, 1999 2955
δ 10.08 (d, J ) 6 Hz, 1H), 7.63 (s, 1H), 7.10 (s, 1H), 6.16 (d, J ) 6.0
Hz, 1H), 5.52 (s, 2H), 5.35 (d, J ) 13.6 Hz, 1H), 5.33 (d, J ) 13.6
Hz, 1H), 5.18 (s, 1H, OH), 5.01 (d, J ) 11.6 Hz, 1H), 4.24 (d, J )
11.6 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.78 (m, 1H), 3.52 (m, 1H),
3.05 (s, 1H, OH), 2.98 (q, J ) 6.8, 14.5 Hz, 1H), 2.15 (m, 1H), 1.87
(m, 1H), 1.30 (s, 3H), 1.06 (s, 3H), 0.99 (d, J ) 57.6 Hz, 3H). 13C
NMR and APT (400 MHz, DMSO-d6, 75 °C) δ 191.35 (CH), 173.86
(C), 173.44 (C), 170.25 (C), 154.11 (C), 153.52 (C), 150.97 (C), 148.46
(C), 140.15 (C), 134.41 (CH), 111.85 (CH), 108.91 (CH), 79.07 (C),
76.20 (CH), 74.95 (C), 63.73 (CH2), 61.83 (CH2), 61.83 (CH) 56.53
(CH3), 56.50 (CH3), 45.62 (CH2), 43.02 (CH), 31.19 (CH2), 21.94 (CH3),
19.10 (CH3), 14.15 (CH3). IR (NaCl, neat) 3505, 3027, 2915, 1736,
1705, 1685, 1522, 1279, 1106 cm-1; exact mass calcd for C26H33N2O13
m/z 581.198265, found m/z 581.198011.
General Procedure for Linearization of Plasmid pBR322 by
EcoR1. Supercoiled pBR322 (30 µL, 30 µg) was incubated with EcoR1
(New England Biolabs) (10 µL), EcoR1 buffer (10×, 20 µL), and 144
µL of H2O (sterile) for 1 h and 20 min at 37 °C. NaOAc (20 mL, 3M)
and ethanol (440 µL) were added, and the solution was cooled at -70
°C for 10 min. The mixture was centrifuged for 15 min, and the ethanol
was decanted off. The remainder of the ethanol was evaporated off in
vacuo, and the remaining linearized DNA was suspended in 60 µL of
sterile H2O. The amount of linearized pBR322 was quantitated by UV
analysis as described by P. N. Borer in the Handbook of Biochemistry
and Molecular Biology, CRC Press, 1975, in conjunction with the
optical density measurements obtained by UV absorption at 260 nm.
For this plasmid DNA substrate the conversion factor was determined
to be an OD260 of 1 ) 50 µg/mL.
EDTA (at pH ) 8) to 0.6 g of agarose. The suspension was heated in
a microwave oven until all of the agarose was dissolved (45 s). The
gel was poured and was allowed to cool and solidify for 1 h at room
temperature. After 1 h the gel was soaked in an alkaline running buffer
(25 mL of 2 N NaOH, 2 mL of 0.5 M EDTA in 1 L of H2O) for an
additional hour. The comb was removed, and the buffer was refreshed
prior to electrophoresis. Agarose loading dye (5 µL) was added to the
samples (10 µL), and the samples were loaded into the wells. The gel
was run for 4 h at 40 mAmps/90 V which resulted in a travelling
distance of approximately 8 cm. The gel was then neutralized for 45
min in a 1 M Tris pH ) 7/1.5 M NaCl solution, which was refreshed
every 15 min. The gel was subsequently stained in an ethidium bromide
solution (200 µL of a 10 mg/mL ethidium bromide solution in 1 L of
1 M Tris/ 1.5 M NaCl buffer at pH ) 7.5) for 1 h. The background
staining was then removed by soaking the gel in a solution of 50 mM
NH4OAc and 10 mM â-mercaptoethanol. Gels were visualized on a
UV transilluminator and photographed using Polaroid black and white
film No. 667.
Acknowledgment. The authors gratefully acknowledge the
financial support provided by the National Institutes of Health
(Grant CA51875). The authors also acknowledge Teri Lansdell
and Brad Herberich for their help with the DNA cross-linking
experiments. We thank Fujisawa Pharmaceutical Co for the
generous gift of FR900482. We would thank Professor Frank
R. Stermitz for an improved procedure to prepare dehydromono-
crotaline.
General Protocol for Alkaline Agarose Gel Electrophoresis. The
agarose gels were prepared by adding 50 mL of a 50 mM NaCl/2 mM
JA983894K