DNA adducts of aristolochic acid II: Total synthesis and site-specific mutagenesis studies in mammalian cells

Article abstract of DOI:10.1093/nar/gkp815

Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In ~50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA→T and AL-II-dG→T transversions. The Author(s) 2009. Published by Oxford University Press.

Full text of DOI:10.1093/nar/gkp815

Published online 23 October 2009
Nucleic Acids Research, 2010, Vol. 38, No. 1 339–352
doi:10.1093/nar/gkp815
DNA adducts of aristolochic acid II: total synthesis
and site-specific mutagenesis studies in
mammalian cells
Sivaprasad Attaluri, Radha R. Bonala, In-Young Yang, Mark A. Lukin, Yujing Wen,
Arthur P. Grollman, Masaaki Moriya, Charles R. Iden and Francis Johnson*
Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-3400, USA
Received May 22, 2009; Revised September 3, 2009; Accepted September 15, 2009
diarrhea and inflammation (1). A traditional use of this
herb, as its Greek name implies, has been to assist women
in childbirth (2). As part of a screening program for new
anti-tumor agents, Kupchan and Doskovich (3) reported
that aristolochic acid I (AA-I) (1; Figure 1), a principal
chemical constituent of Aristolochia indica, was highly
toxic to cells in culture; in addition, the compound
proved to be nephrotoxic in Phase I clinical trials (4).
Development of aristolochic acid as a drug was aban-
doned after Mengs reported its carcinogenicity in
rodents (5). Earlier reports that Aristolochia sp. might be
nephrotoxic in humans was dramatically confirmed in
1993 (6). Of more than 1800 Belgian women who had
been given pills that contained, by error (7), Aristolochia
fangchi as part of a slimming regimen, more than 100
women later developed chronic renal failure. Shortly
thereafter, Cosyns and his colleagues (8) reported that
these same patients also were at risk for urothelial
carcinomas. The clinical syndrome was initially termed
Chinese herbs nephropathy (CHN); later, it was suggested
(9) that the generic term ‘aristolochic acid nephropathy’
(AAN) be used in place of CHN.
These observations drew attention to an endemic
disease known as Balkan nephropathy (BEN), occurring
exclusively in residents of farming villages in the Danube
river basin (10). In a prescient report, Ivic (11) suggested
that the origins of BEN might lie in the A. clematitis that
grows in the wheat fields in the endemic region. Upper
urinary track carcinomas develop in approximately 50%
of BEN cases, often associated with renal insufficiency
(12,13). That the histopathology and clinical features of
BEN are nearly identical to those of the disease reported
in Belgium was recognized by Cosyns and co-workers
(14). Since then, several groups have used AA-I or a
mixture of AA-I and AA-II to reproduce the main
features of AAN in rodents (15–17), removing any
doubt that the aristolochic acids are responsible for
CHN. In areas where BEN is endemic, A. clematitis
ABSTRACT
Aristolochic acids I and II (AA-I, AA-II) are found
in all Aristolochia species. Ingestion of these
acids either in the form of herbal remedies or as
contaminated wheat flour causes a dose-dependent
chronic kidney failure characterized by renal
tubulointerstitial fibrosis. In ꢀ50% of these cases,
the condition is accompanied by an upper urinary
tract malignancy. The disease is now termed aris-
tolochic acid nephropathy (AAN). AA-I is largely
responsible for the nephrotoxicity while both AA-I
and AA-II are genotoxic. DNA adducts derived from
AA-I and AA-II have been isolated from renal tissues
of patients suffering from AAN. We describe the total
synthesis, de novo, of the dA and dG adducts derived
from AA-II, their incorporation site-specifically into
DNA oligomers and the splicing of these modified
oligomers into a plasmid construct followed by
transfection into mouse embryonic fibroblasts.
Analysis of the plasmid progeny revealed that both
adducts blocked replication but were still partly
processed by DNA polymerase(s). Although the
majority of coding events involved insertion of
correct nucleotides, substantial misincorporation
of bases also was noted. The dA adduct is
significantly more mutagenic than the dG adduct;
both adducts give rise, almost exclusively, to
misincorporation of dA, which leads to AL-II-dA!T
and AL-II-dG!T transversions.
INTRODUCTION
Various species of Aristolochia have been used as medici-
nal herbs since the time of Hippocrates to treat diverse
disorders including snake-bite, fever, infection, gout,
*To whom correspondence should be addressed. Tel: +1 631 632 8866; Fax: +1 631 632 7394; Email: francis@pharm.sunysb.edu
The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.
ß The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

340 Nucleic Acids Research, 2010, Vol. 38, No. 1
COOH
NO2
O
O
R
AA-I 1 (R = OCH3)
AA-II 2 (R = H)
O
O
O
NH
O
O
O
O
N
NH
N
N
HN
N
N
R
N
N
N
H
HN
O
HO
R
OH
HO
O
4 (R = OCH3)
6 (R = H)
OH
3 (R = OCH3)
5 (R = H)
O
O
O
O
O
O
N
OH
NH
Br
R
R
10 (R = OCH3)
11 (R = H)
7 (R = OCH3)
8 (R = H)
O
O
R2
H3CO
R2
H3CO
CO2H
NO2
NR'
NR1
Br
R3
R3
9
13
12
Figure 1. Structure of the aristolochic acids AA-I and AA-II, the dA and dG adducts of AL-I and AL-II and related compounds discussed in
the text.
grows in the wheat fields, and its seeds, which contain
significant quantities of the AAs, co-mingle with wheat
grain contaminating the flour used for home-baked
bread (18). Although most residents of an endemic
village are potentially exposed to the AAs, <10% suffer
from BEN due to differences in exposure or to the fact
that a subset of the populace is resistant to the effects of
AAs due to individual genetic variation. Sato and his
associates (16) reported significant differences in tissue
responses to various AAs among various strains of mice,
a finding confirmed by Shibutani et al. (17).
The genotoxicity of AAs is supported by the finding of
AA-derived DNA adducts in renal cortex of humans
(19,20). These adducts were identified as 3 and 4,
derived from the aristolactam (AL) metabolite of AA-I,
and the corresponding adducts 5 and 6, derived from the
AL metabolite of AA-II (21). In humans with AAN, AL-
dA adducts are invariably more abundant than AL-dG
(22). Adducts arise by the same metabolic pathways as
do other aromatic nitro compounds (23), in which the
intermediate N-hydroxyamines (in the cases under discus-
sion, the N-hydroxylactams 7 and 8 or their O-acetylated

Nucleic Acids Research, 2010, Vol. 38, No. 1 341
or O-sulfonylated derivatives) are the likely pro-
carcinogens. Surprisingly, C-8 purine adducts are not
formed, and only products of attack at the exocyclic
amino groups of dA and dG have been detected.
Recently Grollman and his group (19) have shown that
‘signature’ A:T!T:A mutations predominate in the p53
tumor-suppressor gene isolated from urothelial cancers
associated with BEN. However, the molecular mechanism
by which AA-I, but not AA-II, induces proximal tubule
damage remains a mystery.
phosphomolybdic acid in ethyl alcohol containing 5%
sulfuric acid. Flash column chromatographic separations
were carried out on 60 A (230–400 mesh) silica gel (TSI
Chemical Co., Cambridge, MA). All experiments dealing
with moisture or air-sensitive compounds were conducted
under dry nitrogen. The starting materials and reagents,
unless otherwise specified, were the best grade commer-
cially available (Sigma-Aldrich, Milwaukee, WI or Fluka
Chemie GmbH, Sigma-Aldrich, Germany) and were used
without further purification.
Cases of AAN have been reported in China and in other
countries where herbal remedies are widely used (24), and
the disease has been described as a problem of global
dimensions (25,26). Studies by Schmeiser and his
associates (21), Nortier et al. (27) and more recently by
Grollman and coworkers (18,19,25) strongly support the
idea that the AAs play a causative role in the upper
urinary track carcinomas in humans exposed to these
toxins. There is a pressing need for public health
authorities to take action to reduce human exposure to
this powerful nephrotoxic carcinogen (28). Recently, in
a comprehensive review of the subject the National
Toxicology Program has designated the aristolochic
acids as established human carcinogens (24).
In this article we describe the total synthesis, in
quantity, of the dA and dG adducts derived from AA-II
(5 and 6, respectively), allowing not only their complete
chemical characterization but also their use as standards
for the identification of AL-DNA adducts in human
tissues by mass spectrometric methods and for their
site-specific incorporation into oligomeric DNA of any
designated sequence. We discuss also some of the
difficulties associated with the chemistry of the AAs and
the reasons that we adopted a ‘total synthesis’ route to the
adducts. Finally, we present the results of site-specific
mutagenesis studies in mouse embryonic cells designed
to establish the mutagenic potential and specificity of
these lesions in vivo.
7H-Furo[30,40:4,5]benzo[1,2-d][1,3]dioxol-5-one (15)
Cuprous cyanide (115.2 g; 1.286 mol) was added to
formamide (800 ml) containing water (19.2 g), and the mix-
ture was heated with stirring to 100ꢁC. 2-Bromopiperonyl
alcohol 14 (147 g; 0.636 mol; mp 89–90ꢁC) (29), easily
obtained by the bromination of piperonyl alcohol in
methanol at 25ꢁC, was then added in portions over a
period of 10 min. The temperature of the mixture was
raised to 160ꢁC, and a vigorous reaction set in, the tem-
perature rising spontaneously to 178ꢁC with the evolution
of ammonia and steam. Over the next 30 min the temper-
ature subsided to 170ꢁC and thereafter was maintained at
165–170ꢁC for 2 h. The mixture was allowed to cool to
100ꢁC, poured into a solution of sodium cyanide (192 g;
3.92 mol) in water (800 ml), stirred for 30 min followed by
the addition of CH2Cl2 (2 l). The mixture was filtered
through diatomaceous earth, the organic phase was
removed and the aqueous phase again was extracted
with CH2Cl2 (400 ml). The organic extracts were
combined and dried over MgSO4. Silica gel (25 g) and
charcoal (5 g) were added to the solution while the
drying agent was still present, the mixture was stirred
for 5 min then filtered, and the filter cake was washed
with boiling CH2Cl2 (200 ml). Removal of the solvent
from the filtrate left a yellow–orange colored residue of
crude 15 (75.6 g; yield 66.8%), mp 185–187ꢁC—Lit. 190–
191ꢁC (30). Recrystallization from EtOAc/CH2Cl2 gave
two crops, 55 g and 10 g, each as a pale yellow solid,
both with mp 190–191ꢁC. Combined yield of pure
lactone 15 was 65 g (57.4%). 1H NMR (CDCl3) ꢀ 5.22
(s, 2H), 6.16 (s, 2H), 6.87 (s, 1H), 7.26 (s, 1H).
MATERIALS AND METHODS
All reagents and solvents employed in this experimental
work were reagent grade and were used as such unless
otherwise specified. Melting points were taken in a
Thomas-Hoover open capillary melting point apparatus
and are uncorrected. 1H NMR spectra were recorded
either on a Varian Gemini 300 or a Varian NOVA 400
spectrometer. Samples prepared for NMR analysis were
dissolved in CDCl3 or DMSO-d6. Chemical shifts are
reported in parts per million (ppm) relative to TMS.
Mass spectra were recorded on either a Thermo Electron
DSQ GC/MS equipped with a solid probe inlet and EI
ionization or a Micromass Platform mass spectrometer
using electrospray ionization. Thin-layer chromatography
4-Nitro-7H-furo[30,40:4,5]benzo[1,2-d][1,3]dioxol-5-one (16)
Concentrated sulfuric acid (320 ml) was cooled in water-
ice bath (20ꢁC), and lactone 15 (63.5 g, 35.9 mmol)
was added in small portions with stirring over 10 min
while keeping the temperature <20ꢁC. The solution was
cooled to 5ꢁC, and concentrated (70%) nitric acid
(24.7 ml; 37.8 mmol) was added drop-wise over a period
of 30 min (exothermic) while maintaining the reaction
temperature <5ꢁC (ice-MeOH bath). Stirring was
continued for 2 h at 7–10ꢁC, then the mixture was
poured on to ice (2 kg). The resulting yellow precipitate
was removed by filtration and washed with water until free
of acid. The product was air dried and recrystallized from
EtOH to give 16 as a yellow solid (70.5 g, 88%); mp 186–
(TLC) was performed on silica gel sheets (Tiedel-deHaen,
¨
Sleeze, Germany). After appropriate purification all new
products showed a single spot on TLC analysis in two
solvent systems: (i) 30% EtOAc in hexanes and (ii) 5%
MeOH in CH2Cl2. Components were visualized by UV
light (l = 254 nm) or by spraying with a solution of 2%
1
187ꢁC; H NMR (DMSO-d6) ꢀ 7.39 (s, 1H), 6.41 (s, 2H),
5.26 (s, 2H); electrospray mass spectrometry (ESI-MS)
(M + H)+ 224.1.

342 Nucleic Acids Research, 2010, Vol. 38, No. 1
4-Amino-7H-furo[30,40:4,5]benzo[1,2-d][1,3]
dioxol-5-one (17)
an additional 10 min, then a solution of Na2CO3 (3.5 g)
in water (120 ml; previously degassed with nitrogen)
was added, and nitrogen was bubbled through reaction
mixture for an additional 30 min. To this mixture
2-formylphenylboronic acid 20 (7.02 g, 46.8 mmol) was
added, and the mixture was refluxed for 6 h after which
TLC analysis (EtOAc: hexanes/3:7) showed the reaction
to be complete. The mixture was cooled, diluted with
EtOAc and then filtered. Solvent removal gave a solid
residue which was dissolved in CH2Cl2, and the solution
was washed with water then dried over anhydrous
Na2SO4. The isolated product was purified by column
chromatography over silica gel (elution with
hexane:EtOAc/70:30) which afforded pure compound 21
Nitro-lactone 16 (15 g, 67.3 mmol) was dissolved in DMF
(120 ml), and 5% Pd/C catalyst (1.5 g) was added under
nitrogen. The mixture was then shaken in a Parr
hydrogenator at 60 psi overnight. The reaction mixture
was then warmed to dissolve some precipitated product,
and the catalyst was removed by filtration. The filtrate was
concentrated under vacuum, and the residual liquid was
poured into water. The resulting solid was collected and
recrystallized from EtOH to give 17 as white crystals (12 g,
1
92%); mp 238–239ꢁC; H NMR (DMSO-d6) ꢀ 6.36 (s,
1H), 6.04 (s, 2H), 5.88 (s, 2H), 5.11 (s, 2H); EI-MS
M+ 193.2.
1
(7 g, 63.7%); mp 145–146ꢁC; H NMR (CDCl3) ꢀ 9.94
4-Amino-8-bromo-7H-furo[30,40:4,5]benzo[1,2-d][1,3]
dioxol-5-one (18)
(s, 1H), 8.08–8.03 (m, 1H), 7.73–7.66 (m, 1H), 7.42–7.39
(m,1H), 7.30 (s, 1H), 6.13–6.09 (d, 2H), 5.20–4.88 (dd,
2H); ESI-MS (M + H)+ 283.3.
Amino-lactone 17 (3.8 g, 19.8 mmol) was dissolved in dry
pyridine (100 ml), and bis(N-methyl-2-pyrrolidinone)-
hydrogen tribromide (8 g, 23.7 mmol) was added under a
nitrogen atmosphere with magnetic stirring. After 15 h,
TLC analysis (EtOAc:hexanes/3:7) showed complete
absence of the starting material. The pyridine was
removed under reduced pressure, and the residue, after
dissolution in CH2Cl2, was washed with 10% NaHCO3
solution. The organic layer was washed with water then
dried over anhydrous Na2SO4. After removal of the
solvent, the resulting solid was suspended in a minimum
amount of CH2Cl2, triturated well, filtered and dried to
give almost pure 18. Recrystallization from isopropyl
ether gave the pure material (4.6 g, 86%); mp 210–
212ꢁC; 1H NMR, (DMSO-d6) ꢀ 6.16 (s, 2H), 6.03
(s, 2H), 5.06 (s, 2H); ESI-MS (M + H)+ 272.2.
6H-Benzo[f]1,3]dioxolo[40,50:4,5]benzo[1,2,3-cd]
furan-5-one:Aristolactone II (22)
The lactonic aldehyde 21 (7.3 g, 25.9 mmol) was dissolved
in anhydrous THF, and anhydrous potassium t-butoxide
(5.7 g) was added under nitrogen. The mixture was
refluxed with stirring for 4 h, then cooled and the solvent
removed under reduced pressure. The residue was taken
up in MeOH (100 ml), acidified with 12 N HCl (20 ml) and
the solution was refluxed for 1 h. After cooling and
removal of the MeOH, the residue was dissolved in
CH2Cl2, and the solution was washed with water
(50 ml), then with saturated NaHCO3 solution (50 ml),
again with water (2 ꢃ 50 ml) and finally with brine
(50 ml). The solution was dried over anhydrous Na2SO4,
filtered and evaporated to dryness. The resulting yellow
solid was purified by column chromatography on silica
gel. Elution with 1% MeOH in CH2Cl2 gave the pure
desired product AA-II lactone 22 (3.7 g, 55%); mp 183–
8-Bromo-7H-furo[30,40:4,5]benzo[1,2-d][1,3]
dioxol-5-one (19)
Amino-lactone 18 (4.9 g, 18.08 mmol) was dissolved in
concentrated HCl (100 ml), and the resulting solution
was diluted with cold water (200 ml) then cooled to
ꢂ5ꢁC. To this mixture sodium nitrite solution (1.2 g/
180 ml) was added dropwise while maintaining the
internal temperature <0ꢁC. After the addition was
completed, the mixture was stirred for 2 h at 0ꢁC, then
cold hypophosphorous acid (30%) (68 ml) was added
dropwise while maintaining the internal temperature at
<0ꢁC. Thereafter, stirring was continued for 2 h, then
the mixture was held at 5ꢁC overnight. The pale pink pre-
cipitate was removed by filtration, washed with cold water
and dried to give the pure desired product 19 (4.3 g,
73.7%); mp. 173–174ꢁC; 1HNMR (DMSO-d6) ꢀ 7.27
(s, 1H), 6.28 (s, 2H), 5.19 (s, 2H); ESI-MS (M + H)+
257.2.
1
184ꢁC (31); H NMR (DMSO-d6) ꢀ 8.50–8.47, (m, 1H),
8.05–8.04 (m, 1H), 7.86 (s, 1H), 7.69–7.68 (m, 2H), 7.50
(s,1H), 6.56 (s, 2H); ESI-MS (M + H)+ 265.3.
6H-Benzo[f][1,3]dioxolo[40,50:4,5]benzo[1,2,3-cd]
indol-5-one:Aristolactam II (23)
A mixture of lactone 22 (600 mg, 2.3 mmol), concentrated
aqueous ammonium hydroxide (8 ml), sodium sulfite
(450 mg) and ammonium chloride (350 mg) dissolved in
water (1 ml) was heated in a sealed tube at 140ꢁC over-
night. The lactone went into solution at 110–115ꢁC, then
gradually, as the reaction proceeded, a solid separated.
The mixture was cooled, filtered and the solid product
was washed with water then dried to give an almost
quantitative yield of pure AL-II (23); mp 297–298ꢁC
(32); 1H NMR (DMSO-d6) ꢀ 10.72 (s, 1H), 8.60–8.58
(d, 6 Hz, 1H), 7.94–7.92 (d, 6 Hz, 1H), 7.61 (s, 1H),
7.60–7.58 (m, 2H), 7.09 (s, 1H), 6.46 (s, 1H) essentially
identical with the published spectrum (32); ESI
(M + H)+ 264.3.
2-(7-Oxo-5,7-dihydro-furo[30,40:4,5]benzo[1,2-d][1,3]
dioxol-4-yl)-benzaldehyde (21)
A solution of bromolactone 19 (10 g, 39 mmol) in dioxane
(150 ml) was degassed with nitrogen for 10 min and 1,
10-bis(diphenylphosphino)ferrocene]palladium dichloride
catalyst (0.85 g) was added. Degassing was continued for

Nucleic Acids Research, 2010, Vol. 38, No. 1 343
7-Bromo-6H-benzo[f][1,3]dioxolo[40,50:4,5]
benzo[1,2,3-cd]indol-5-one (11)
7.45–7.58 (m, 3H), 6.51 (t, 1H), 6.46 (s, 2H), 5.80 (s, 1H),
5.19 (s, 1H), 4.66 (m, 1H), 4.04 (m, 1H), 3.95–3.78 (m, 2H),
2.70 (m, 1H), 2.47 (m, 1H), 0.94–0.90 (m, 27H), ꢂ0.10
to 0.21 (m, 18H); ESI-MS (M + H)+ 885.5.
To a solution of 23 (1.5 g, 5.7 mmol) dissolved in glacial
HOAc (10 ml) and cooled in an ice bath was added anhy-
drous NaOAc (500 mg, 6 mmol) followed by the drop-wise
addition of bromine (1.18 g) in HOAc (5 ml). After the
addition was complete, the reaction mixture was stirred
for 15 min and filtered. The collected solid was washed
with CH2Cl2, then water and dried to give compound 11
(1.9 g, quantitative) mp 301–302ꢁC. It was virtually
insoluble in any of the usual organic solvents and very
7-[9-(4-Hydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-
1,9-dihydro-purin-6-ylideneamino]-6H-benzo[f][1,3]dioxolo
[40,50:4,5]benzo[1,2,3-cd]indol-5-one (5)
To an ice-cold solution of the silyl-protected compound
26, (700 mg, 0.81 mmol) in pyridine (15 ml) was added
70% HF in pyridine (2 ml) over a period of 3 min. The
mixture was stirred at room temperature overnight, then
poured into aqueous 10% NaHCO3 (50 ml) and stirred for
2 h. The solid that precipitated was collected, washed with
water and then added to THF (10 ml) and concentrated
aqueous ammonia (5 ml). This mixture was heated at 70ꢁC
in a closed vial overnight, then taken to dryness, and the
remaining solid was chromatographed over silica gel using
7.5% MeOH in CH2Cl2 to elute the pure deprotected
adduct 5 (0.35 g; 84%) whose UV absorbance spectrum
1
sparingly soluble in hot DMSO. H NMR (hot DMSO-
d6) ꢀ 11.19 (s, 1H), 8.61–8.59 (d, 6 Hz, 1H), 7.82–7.80
(m, 3H), 6.52 (s, 2H); EI-MS M+ 341.2.
7-Bromo-6-(tert-butyl-dimethylsilanyloxymethyl)-6H-
benzo[f][1,3]dioxolo[40,50:4,5]benzo[1,2,3-cd]indol-5-one (24)
To a solution of sodium hydride (120 mg, 3.2 mmol; 60%
suspended in mineral oil) in dry DMF (20 ml), AA-II
bromolactam (11), (900 mg, 2.6 mmol) was added, and
the mixture was heated at 50ꢁC for 30 min under a
nitrogen atmosphere. After cooling to ꢀ5ꢁC, freshly
prepared (33) tert-butylchloromethoxydimethylsilane
(0.5 g, 3 mmol) was added by syringe. The ice bath was
removed after 10 min, and the reaction mixture was stirred
at 24ꢁC for 0.5 h when TLC analysis (EtOAc: hexanes/2:8)
showed completion of the reaction. The DMF was
removed under reduced pressure, and the residue taken
up in CH2Cl2 (50 ml).This solution was washed with
water, dried over anhydrous Na2SO4, and the solvent
was removed under reduced pressure to give the crude
product. This was purified by column chromatography
(EtOAc: hexanes/2:8) on silica gel to afford the pure
desired N-protected lactam 24 (800 mg, 63%). mp
1
was identical to that published by Pfau et al. (34). H
NMR (DMSO-d6): ꢀ 10.80 (s, 1H), 9.83 (br s, 1H), 8.70
(m, 2H), 8.41 (s, 1H), 8.00 (m, 1H), 7.55 (m, 3H), 6.59 (s,
2H), 6.49 (t, 1H), 5.19 (br s, 1H), 4.49 (m, 1H), 3.39 (br s,
1H), 3.75–3.51 (m, 2H), 3.21 (m, 1H), 2.83 (m, 1H), 2.37
(m, 1H); ESI-MS (M + H)+ 513.3.
7-(9-{5-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-
4-hydroxy-tetrahydro-furan-2-yl}-1,9-dihydro-purin-6-
ylideneamino)-6H-benzo[f][1,3]dioxolo[40,50:4,5]benzo
[1,2,3-cd]indol-5-one (27)
To a solution of compound 5 (165 mg, 0.32 mmol) in
pyridine (5 ml) was added solid dimethoxytrityl (DMT)
chloride (165 mg, 0.48 mmol), and the mixture was
stirred at room temperature for 3 h at which time TLC
analysis (CH2Cl2/MeOH 92:8) showed the reaction to be
ꢀ90% complete. MeOH (5 ml) was added, and the
mixture was stirred for 30 min at room temperature,
then concentrated under reduced pressure. To the
residue was added aqueous 10% NaHCO3 (25 ml),
and the mixture was extracted with CH2Cl2. The extract
was dried over anhydrous MgSO4, filtered and
concentrated to give the crude product which was
purified by chromatography over silica gel. Elution with
5–10% MeOH in CHCl2 afforded pure DMT-protected
compound 27 again a glassy solid (120 mg; 66% based
1
199–200ꢁC; H NMR (CDCl3) ꢀ 8.62 (dd, 6H, 1H), 8.5
(dd, 6 Hz, 1H), 7.79 (m, 2H), 7.6 (s, 1H), 6.01 ((s, 2H), 5.95
(s, 2H), 0.0 (s, 9H), 5.95 (s, 6H); ESI-MS (M + H)+ 486.5.
7-{9-[4-(tert-Butyl-dimethyl-silanyloxy)-5-(tert-butyl-
dimethyl-silanyloxymethyl)-tetrahydro-furan-2-yl]-1,9-
dihydro-purin-6-ylideneamino}-6-(tert-butyl-dimethyl-
silanyloxymethyl)-6H-benzo[f][1,3]dioxolo[40,50:4,5]
benzo[1,2,3-cd]indol-5-one (26)
An oven-dried 100 ml two-necked flask was charged
with the protected dA 25 (920 mg, 1.9 mmol), cesium
carbonate (1.82 g, 5.6 mmol), trisdibenzylideneacetone
dipalladium catalyst (220 mg, 0.24 mmol), xantphos
(460 mg, 0.79 mmol), the protected 7-bromo AL-II (24,
465 mg, 0.96 mmol) and toluene (30 ml). This mixture
was stirred under a nitrogen atmosphere for 30 min at
room temperature and then heated at 100ꢁC for 5 h.
After cooling to room temperature, the solids were
removed by filtration and washed with EtOAc. The
filtrate was evaporated to dryness, and the residual
crude product was purified by chromatography over
silica gel using 5% EtOAc in CH2Cl2 as the eluent to
afford pure compound 26 as a colorless glassy solid
1
on unrecovered 5; 50 mg of 5 was recovered). H NMR
(CDCl3): ꢀ 9.80 (br s, 1H), 8.58 (s, 1H), 8.23 (s, 1H), 8.08
(s, 1H), 8.01 (br s, 1H), 7.59 (m, 2H), 7.42 (m, 3H), 7.38–
7.18 (m,8H), 6.79 (m, 4H), 6.38 (t, 1H), 6.23 (s, 2H), 4.70
(br s, 1H), 4.20 (m, 1H), 3.79 (s, 6H), 3.40 (m, 3H), 2.81
(m, 1H), 2.47 (m, 1H); ESI-MS (M + H)+ 815.5.
Diisopropyl-phosphoramidous acid, 2-[bis-(4-methoxy-
phenyl)-phenyl-methoxymethyl]-5-[6-(5-oxo-5,6-dihydro-
benzo[f][1,3]dioxolo[40,50:4,5]benzo[1,2,3-cd]indol-7-
ylimino)-1,6-dihydro-purin-9-yl]-tetrahydro-furan-3-yl
ester, 2-cyano-ethyl ester (28)
1
(750 mg, 88%). H NMR (CDCl3): ꢀ 8.67 (d, 1H), 8.58
(s, 1H), 8.32 (d, 1H), 8.25 (br s, 1H), 7.89 (d, 1H),
DMT compound 27 (110 mg, 0.14 mmol) was
co-evaporated with dry toluene (3 ꢃ 10 ml) and then

344 Nucleic Acids Research, 2010, Vol. 38, No. 1
dissolved in dry CH2Cl2 (10 ml). Tetrazole (11.3 mg,
0.16 mmol) was added to the solution followed by
2-cyanoethyl N,N,N0,N0-tetraisopropylphosphordiamidite
(57 mg, 0.19 mmol). The reaction mixture was stirred at
room temperature for 2 h under nitrogen and then
diluted with CH2Cl2 (25 ml) containing 2% TEA. The
CH2Cl2 layer was washed with aqueous saturated
NaHCO3 solution, dried over Na2SO4, filtered and
concentrated under reduced pressure to obtain the
desired product 28 as a viscous oil (145 mg, 100%),
which was used as such in the preparation of DNA
(m, 1H), 7.79 (m, 1H), 7.59 (m, 2H), 7.02–7.18 (m, 5H),
6.45 (s, 2H), 6.20 (t, 1H), 5.20 (br s, 1H), 4.79 (br
s, 1H), 4.20 (m,1H), 3.76 (m, 2H), 2.60 (m, 1H), 2.19
(m, 1H); ESI-MS (M + H)+ 619.4.
7-[9-(4-Hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-
6-oxo-6,9-dihydo-1H-purin-2-ylamino]-6H-
benzo[f][1,3]dioxolo[40,50:4,5]benzo[1,2,3-cd]indol-5-one (6)
To a solution of 31 (50 mg, 0.08 mmol) in methanol (50 ml)
was added a 10% palladium-on-carbon catalyst. The flask
was evacuated (50 torr) and flushed with hydrogen thrice.
The reaction mixture was hydrogenated for 16 h at 50 psi
with stirring. It was then filtered through a pad of Celite,
the Celite bed was washed with DMF (20 ml), and the
filtrate was concentrated under reduced pressure. The
residue was diluted with water (20 ml); the separated
solid was filtered and dried in a vacuum oven overnight
1
oligomers. H NMR (CDCl3): ꢀ 9.95 (br s, 1H), 8.60 (s,
1H), 8.25 (s, 1H), 8.09 (s, 1H), 7.99 (br s, 1H), 7.62–7.22
(m, 13H), 6.81 (m, 4H), 6.40 (t, 1H), 6.23 (s, 2H), 4.68 (m,
1H), 3.92 (m, 2H), 3.81 (s, 6H), 3.42 (m, 3H), 2.99 (m, 2H),
2.81 (m, 1H), 2.62 (m, 2H), 2.47 (m, 1H), 1.08 (m, 12H);
31P NMR (CDCl3): 150.2, 149.8.
1
(26 mg, 78%). H NMR (DMSO-d6): ꢀ 12.04 (br s, 1H),
7-{6-Benzyloxy-9-[4-(tert-butyldimethylsilyloxy)-
5-(tert-butyldimethylsilyloxymethyl)-tetrahydrofuran-2-yl]-
9H-purin-2-ylamino}-6-(tert-butyldimethylsilyloxymethyl)-
6H-benzo[f][1,3]dioxolo[4050-4,5]benzo[1,2,3-cd]
indol-5-one (30)
10.98 (br s, 1H), 10.82 (br s, 1H), 8.61 (d, 1H), 8.02
(m, 1H), 7.93 (s, 1H), 7.67–7.61 (m, 3H), 6.50 (s, 2H),
6.22 (s, 1H), 5.83 (t, 1H), 5.05 (m, 1H), 4.59 (br s, 1H),
3.98 (br s, 1H), 3.59 (m, 1H), 3.17 (br s, 1H), 2.44 (m,1H),
1.99 (m,1H); MALDI-MS (M + H)+ 529.1.
A dry 100 ml two-necked flask was charged with the fully
protected dG derivative 29, (418 mg: 0.71 mmol), cesium
carbonate (0.785 g, 2.4 mmol), trisdibenzylideneacetone
dipalladium catalyst (164 mg, 0.18 mmol), xantphos
(343 mg, 0.59 mmol), compound 24 (450 mg, 0.93 mmol)
and toluene (25 ml) under a nitrogen atmosphere. This
mixture was stirred for 30 min at room temperature and
then heated at 100ꢁC for 6 h. After cooling to room tem-
perature, the mixture was filtered, and the collected solids
were washed with EtOAc. After vacuum evaporation of
the solvent, the residue was purified by chromatography
over silica gel. Elution with 5% EtOAc in CHCl2 afforded
7-(6-Benzyloxy-9-{5-[bis(4-methoxyphenyl)-
phenylmethoxymethyl]-4-hydroxy-tetrahydrofuran-2-yl}-
9H-purin-2-ylamino)-6H-benzo[f][1,3]dioxolo[40,50-4,5]
benzo[1,2,3-cd]indole-5-one (32)
To a solution of compound 31 (225 mg, 0.36 mmol) in
pyridine (8 ml) was added solid DMT chloride (200 mg;
0.59 mmol), and the mixture was stirred at room temper-
ature for 3 h. TLC (CH2Cl2/MeOH 92:8) showed the
reaction to be ꢀ90% complete. The product was then
worked up as in the case of 27, noted above, and
purified by chromatography on silica gel. Elution with
5–10% MeOH in CH2Cl2 gave the pure DMT-protected
compound 32 as a colorless glass (190 mg, 57% based on
unrecovered 31; recovered 31 amounted to 99 mg). 1H
NMR (CDCl3): ꢀ 9.62 (br s, 1H), 8.60 (s, 1H), 7.90 (d,
1H), 7.83 (s, 1H), 7.56 (m, 2H), 7.38 (m, 5H), 7.28–7.12
(m, 11H), 6.77 (m, 4H), 6.36 (t, 1H), 6.22 (s, 2H), 5.48
(s, 2H), 4.55 (br s, 1H), 4.23 (m, 1H), 3.73 (s, 6H), 3.20
(m, 2H), 2.62 (m, 2H); ESI-MS (M + H)+ 921.3.
1
pure compound 30 as a solid glass (703 mg, 75%). H
NMR (CDCl3): ꢀ 8.75 (d, 1H), 8.13 (d, 2H), 8.04 (br s,
1H), 7.70 (s, 1H), 7.60 (m, 2H), 7.09–7.18 (m, 5H), 6.42
(s, 2H), 6.29 (t, 1H), 5.93 (s, 1H), 5.19 (s, 3H), 4.42
(m, 1H), 3.98 (m, 1H), 3.78 (m, 2H), 2.62 (m, 1H), 2.27
(m, 1H), 1.05–0.95 (m, 27H), 0.31–0.12(m, 18H); ESI-MS
(M + H)+ 991.5.
7-[6-Benzyloxy-9-(4-hydroxy-5-hydroxymethyl-
tetrahydrofuran-2-yl)-9H-purin-2-ylamino]-6H-benzo[ f ]
[1,3]dioxolo[40,50-4,5]benzo[1,2,3-cd]indol-5-one (31)
7-(9-{5-[bis(4-Methoxyphenyl)phenylmethoxymethyl]-4-
hydroxy-tetrahydrofuran-2-yl}-6-oxo-6,9-dihydro-1H-
purin-2-ylamino)-6H-benzo[f][1,3]dioxolo[4,050-4,5]
benzo[1,2,3-cd]indol-5-one (33)
To an ice-cold solution of compound 30 (530 mg,
0.53 mmol) in pyridine (10 ml) was added 70% HF in
pyridine (1.7 ml) over a period of 3 min. The mixture
was stirred at room temperature overnight, poured into
ice-cold aqueous 10% NaHCO3 solution and again stirred
for 2 h. The separated solids were filtered, washed with
water and added to THF (10 ml) and concentrated
ammonia (5 ml). The mixture was then heated overnight
at 70ꢁC in a closed vial. The solvents were removed by
vacuum evaporation, and the residue was purified chro-
matographically over silica gel. Elution with CH2Cl2/
MeOH (9:1) gave pure coupled product 31 as a glassy
solid (300 mg, 91%). 1H NMR (DMSO-d6): ꢀ 10.62
(s, 1H), 9.21 (s, 1H), 8.98 (br s, 1H), 8.19 (s, 1H), 8.05
To a solution of compound 32 (280 mg, 0.30 mmol)
in EtOAc/MeOH (1:1, 20 ml) was added 10% Pd/C
catalyst (50 mg). The flask was evacuated (50 torr),
flushed thrice with hydrogen and then shaken under
hydrogen for 16 h at 50 psi. The resulting solution was
filtered through a pad of celite and concentrated under
reduced pressure. The residue was purified by silica gel
column chromatography. Elution with 7–10% MeOH in
CHCl21afforded pure debenzylated product 33 (220 mg,
91%). H NMR (CDCl3): ꢀ 12.40 (br s, 1H), 10.62 (br s,
1H), 9.82 (br s, 1H), 8.49 (m, 1H), 8.02 (m, 1H), 7.66
(s, 1H), 7.59 (m, 1H), 7.27 (m, 6H), 6.90 (m, 5H), 6.86

Nucleic Acids Research, 2010, Vol. 38, No. 1 345
(m, 4H), 6.60 (s, 2H), 6.28 (m, 1H), 5.57 (m, 1H), 3.74
(m, 9H), 2.62 (m, 1H), 2.45 (m, 1H); ESI-MS (M + H)+
831.0.
(0.5 mm ꢃ 250 mm) at a flow rate of 12 ml/min. The solvent
gradient was 0% B to 100% B over 20 min (solvent A—
90:10 water:acetonitrile and 0.05% formic acid; solvent
B—95:5 acetonitrile:water with 0.05% formic acid).
Diisopropylphosphoramidous acid, 2-[bis(4-methoxyphenyl)
phenylmethoxymethyl]-5-[6-oxo-2-(5-oxo-5,6-dihydrobenzo
[f][1,3]dioxolo[4,0,50-4,5]benzo[1,2,3-cd]indol-7-ylamino)-
1,6-dihydropurin-9-yl]tetrahydrofuran-3-yl ester-2-
cyano-ethyl ester (34)
Construction of site-specifically modified plasmids
The methods for the construction of the shuttle vector
(pMTEX4) and modified plasmid and the strategy for
the site-specific mutagenesis experiments have been pub-
lished (36). A modified 19-mer, 50-TTCCCTCCAGAAXC
ATCCT, where X represents either the AL-II-dA or AL-
II-dG adduct, and its complementary 19-mer, 50-CCATA
GGATATCTCTGGAG, were annealed following
phosphorylation at their 50-ends to form 4-nucleotide
overhangs on both termini (50-CCAT and 50-TTCC) and
mismatches on both sides of and opposite the adduct
(50-ATC/30-CXA) (Figure 2A). The duplex oligodeoxy-
nucleotide was ligated at 4ꢁC overnight to the pMTEX4
vector, which had been digested with BsaI and BsmBI.
Closed circular constructs containing a site-specific,
single DNA adduct were purified by ultracentrifugation
in a CsCl-ethidium bromide solution. The amount
The DMT derivative 33 (180 mg, 0.22 mmol) was
co-evaporated with dry toluene (3 ꢃ 10 ml), and the
residue was dissolved in dry CH2Cl2 (10 ml). Tetrazole
(18.2 mg, 0.26 mmol) was added, followed by 2-cyanoethyl
N,N,N0,N0-tetraisopropylphosphordiamidite
(91.6 mg,
0.3 mmol), and the reaction mixture was stirred at room
temperature for 2 h under nitrogen after which CH2Cl2
containing 2% TEA (25 ml) was added. This solution
was then washed with aqueous saturated NaHCO3
solution (50 ml), dried over Na2SO4, filtered and
concentrated under reduced pressure to yield the desired
product 34 (245 mg, 100% yield) as a viscous oil. This was
used directly in the synthesis of DNA oligomers. 1H NMR
(CDCl3): ꢀ 12.38 (br s, 1H), 10.62 (br s, 1H), 9.89 (br s,
1H), 8.62 (m, 1H), 8.42 (m, 1H), 8.02 (m, 1H), 7.99 (s, 1H),
7.82 (m, 2H), 7.42 (m, 2H), 7.40–7.05 (m, 9H), 6.66
(m, 4H), 6.37 (s, 2H), 6.22 (m, 1H), 5.60 (m, 1H), 4.62
(m, 1H), 4.02 (m, 2H), 3.83 (s, 6H), 2.97 (m, 2H),
2.60–2.31 (m, 4H), 1.07 (m, 12H); 31P NMR (CDCl3):
149.5, 149.4.
of modified construct was quantified by a
spectrophotometer using a published procedure (36).
UV
Introduction of the modified plasmid into mouse embryonic
fibroblasts and recovery of the progeny plasmid
Immortalized mouse embryo fibroblasts (MEFs) were
cultured in Dulbecco’s modified Eagle’s medium supple-
mented with fetal bovine serum (10%), penicillin
(100 units/ml) and streptomycin (100 mg/ml) under 5%
CO2 at 37ꢁC. Cells (1 ꢃ 106) were plated in a 25-cm2
flask and cultured overnight after which they were
transfected overnight with 500 ng of a modified construct
by the FuGENE6 (Roche) method according to the man-
ufacturer’s instruction. The next day, cells were detached
by treating with trypsin–EDTA, seeded in a 150-cm2 flask
and cultured for 4 days. Progeny plasmids were recovered
by the method of Hirt (37) and analyzed for translesional
events as described below.
DNA synthesis
All oligomers were synthesized at the 1.0 mmol scale using
an Applied Biosystems 394 DNA Synthesizer (Foster City,
CA) with normal phosphoramidite reagents (Glen
Research). Individual oligomers were liberated from the
controlled pore glass (CPG) support by treatment with
aqueous 28% ammonia at 55ꢁC overnight which also
removed all of the nucleobase-protecting groups.
Purification of the DNA was accomplished in two
stages. In the first the deprotected oligomers having a
terminal DMT group were separated from failure
sequences by means of a Waters HPLC system using
Analysis of progeny plasmid for translesion events
a
Luna phenyl-hexyl column (10 ꢃ 250 mm, 5 mm,
To the recovered plasmids, 5 ng of pVgRXR (Invitrogen),
which coded for zeocin resistance, was added. This
plasmid served as an internal control for DpnI digestion.
The mixture was treated with Dpn I (1 unit) for 1 h at
37ꢁC to remove residual nonreplicated input DNA and
then used to transform Escherichia coli DH10BMax
electrocompetent cells (Invitrogen) by an E. coli Pulser
(Bio Rad). Varying portions of a transformation mixture
were plated on YT (1ꢃ) agar plates with ampicillin
(100 mg/ml medium) and blasticidin S (50 mg/ml medium)
or with zeocin (25 mg/ml medium). Since the adduct was
located close to the blasticidin S resistance gene (36),
transformants containing progeny plasmids with large
deletions around the adduct site should not grow on a
blasticidin-containing plate and were thus excluded from
analysis. A marked reduction in the number of colonies on
a zeocin-containing plate assured efficient digestion of
nonreplicated plasmids by DpnI. E. coli transformants
Phenomenex, Torrence, CA) at a solvent flow rate of
4 ml/min. A solvent gradient of 16–36% acetonitrile in
0.1 M TEAA buffer (pH 6.8) was employed over 35 min.
In the second stage the DMT group was removed by treat-
ment with HOAc containing 20% water, and the naked
DNA was again purified on the same column using a
gradient of 5 to 15% acetonitrile in 0.1 M TEAA over a
period of 45 min. Quality control was achieved using two
methods. First, the masses of all of the deprotected
oligomers were measured by ESI-MS using a Micromass
Platform LC/MS. Each oligomer was infused directly
into the ESI source via the autosampler without an
HPLC column present. Second, selected oligomers were
digested to the deoxynucleosides using a published proce-
dure (35) and analyzed by a Thermo Quantum Ultra LC/
MS/MS. Deoxynucleosides were separated on a Hewlett
Packard 1100 HPLC system with an Aquasil C18 column

346 Nucleic Acids Research, 2010, Vol. 38, No. 1
5’–- CCATAGGATATCTCTGGAG GGAA--
3’--GGTA TCCTACXAAGACCTCCCTT –-
A
were picked individually and subjected to oligonucleotide
hybridization as described in detail previously (38).
Examples of hybridization results are shown in Figure
2B. Oligonucleotide probes (Figure 2A) were used to
determine the DNA sequence at the adduct site. L15
(50-GAGGAGCCATAGGAT) and R14 (50-TCTGGAG
GGAAGGG) probes were used to confirm the presence
of the oligonucleotide insert and to detect untargeted
mutations and small deletions around the adduct site.
Plasmids that did not hybridize to both L15 and R14
probes were omitted from a further analysis. U16 (50-TA
GGATATCTCTGGAG) detected progeny derived from
the unmodified complementary strand. A15, T15, G15 and
C15 (50-CTCCAGAANCATCCT, where N represents A,
T, G or C) detected AL-II adduct!dA, !T, !dG and
!dC base substitutions, respectively. When plasmids did
not hybridize to any of these four probes, DNA
sequencing was conducted. Thus, this strategy detected
all types of events including base substitutions,
frameshifts, deletions and insertions without any bias.
BsaI
BsmBI
Replication
U16
5’–CCATAGGATATCTCTGGAGGGAA-
3’-GGTATCCTATAGAGACCTCCCTT-
5’–CCATAGGATGNTTCTGGAGGGAA-
3’-GGTATCCTACXAAGACCTCCCTT-
EcoRV digestion
Replication
L15
R14
R14
L15
5’–CCATAGGAT ATCTCTGGAGGGAA-
3’-GGTATCCTA TAGAGACCTCCCTT-
5’–CCATAGGATGNTTCTGGAGGGAA-
3’-GGTATCCTACNAAGACCTCCCTT-
Probes
A15: TCCTACAAAGACCTC 5’
T15: TCCTACTAAGACCTC
G15: TCCTACGAAGACCTC
C15: TCCTACCAAGACCTC
B
T15
A15
Figure 2. Outline of experimental strategy (36). (A) Modified and
unmodified 19 mers (highlighted), which form 3 consecutive base
mismatches shown in white (X represents adduct) upon annealing,
are ligated to pMTEX4 vector (40) digested with BsaI and BsmBI.
The construct is introduced into MEFs and allowed to replicate.
Progeny plasmids are recovered from transfected MEFs and analyzed
for translesional events. Progeny derived from the unmodified strand
are detected by the hybridization probe, U16 (underlined) while those
derived from the modified strand are detected by the probes, A15, T15,
G15 and C15, which identify nucleotides T, dA, dC and dG, respec-
tively, inserted opposite the adduct. The partial sequences of probes
(L15 and R14), which detect the sequence of the oligonucleotides
ligated, are also underlined. Their full sequences are provided in the
text. Refer to (B) for examples of hybridization. The ratio of progeny
derived from the modified and unmodified strands, presented in Table 2
as a percentage, apparently reflects the blocking effects of DNA
adducts on DNA synthesis. To facilitate the analysis of translesional
coding events, recovered plasmid was digested with EcoRV restriction
enzyme (the recognition sequence is italicized), which inactivates
progeny derived from the unmodified strand. This digested sample
was used to transform E. coli, and transformants were analyzed for
the coding specificity at the adduct site, using A15, T15, G15 and
C15 probes. (B) Filters probed with 32P-lablelled T15 (left panel) and
A15 (right panel). ‘‘N’’ represents one of the four normal
deoxynucleotides.
RESULTS AND DISCUSSION
The chemistry of AA-I and AA-II and total synthesis of
the dA and dG adducts 5 and 6 derived from AA-II
AA-I (1) and AA-II (2) (Figure 1) occur together naturally
in many Aristolochia species and are commercially avail-
able as a mixture. However, they cannot be separated
cleanly by crystallization or by simple column chromatog-
raphy, and their separation by HPLC is an extremely
tedious procedure applicable only to small quantities. By
contrast, the corresponding methyl esters (formed by the
action of diazomethane) can be separated chromatograph-
ically, albeit with difficulty, but when basic hydrolysis
is attempted, <6% of the acid is recovered (39). This
unusual chemistry is a reflection of the steric strain—the
aromatic version of 1,3-allylic strain (40)—that exists
between the nitro and carboxyl functions and parallels
identically the group compression that exists in
8-nitronaphthoic acid 9. The latter acid cannot be
esterified by the standard Fischer–Speier method, and its
methyl ester (formed by diazomethane) undergoes hydro-
lysis extremely slowly under forcing conditions (41).
Similarly, attempts to form the acid chloride (SOCl2) of
9 leads to 8-chloronaphthoyl chloride and other decom-
position products (42). Based on such aberrant chemistry,
the possibility of obtaining multi-gram quantities of the
individual AAs from natural sources seemed limited. We
elected, therefore, to attempt to develop a new synthetic
approach that would not only allow us to obtain the
desired DNA adducts in quantity, but also would permit
the synthesis of a wide range of AA-related substances
potentially including the AAs themselves. The synthesis
of AA-I had previously been accomplished by Kupchan
and Wormser (43), but the method is impracticable; too
many steps are involved, yields at some stages are low, the
route lacks versatility and the final ester hydrolysis is
extremely low-yielding as noted above. In addition the
route did not seem practical for the synthesis of the
7- bromolactams 10 and 11, which we saw as the critical
intermediates for the synthesis of the corresponding DNA
adducts. Satisfactory synthetic routes to this class of
lactam have been published by Estevez et al. (44,45) and
by Couture and associates (46). The first method by the
Estevez group involves a rather low-yielding benzyne
cyclization step, whereas both of the other approaches
involve the linking of the B and D rings using a tributyltin
hydride radical reaction as a critical step in the synthesis
of the basic phenanthrene. In addition the latter approach
utilizes an isoindolenone intermediate (12) which is always
obtained as a mixture of geometric isomers that have to be
separated before conversion to the required penultimate
lactam (13) is possible. These methods are suitable for the
preparation of the aristolactams but not for the synthesis
of the aristolochic acids themselves, one of the ultimate
goals of our research. The approach that we have taken
involves reversing the order of the assembly, first by

Nucleic Acids Research, 2010, Vol. 38, No. 1 347
X
O
O
O
O
b
O
O
OH
O
O
a
O
O
Br
15
16 (X = NO )
2
14
c
17 (X = NH )
2
d
NH
O
O
2
e, f
O
O
O
O
O
O
Br
19
Br
18
Figure 3. Synthetic scheme for the preparation of 8-bromo-7H-furo[30,40:4,5]benzo[1,2-d][1,3]dioxol-5-one (19). Reagents: (a) CuCN/HCONH2/H2O;
(b)HNO3/H2SO4; (c) H2/Pd; (d) bis(N-methylpyrrolidinone) HBr3 and (e) NaNO2/H+; (f) H3PO2.
linking the two principal aromatic rings (B and D) by
means of a Suzuki reaction (tolerant of a nitro group),
then cyclizing the resultant biphenyl intermediate to the
desired phenanthrene. This route avoids the problem of
the double bond isomerism and has proven to be both
efficient and versatile for our purposes. In this report we
present the successful application of the method to the
synthesis of the naturally occurring AA-II adducts of dA
and dG. The dA adduct derived from AA-II had been
synthesized earlier at the milligram level by Pfau and
coworkers (34) by the solvolysis of N-chloroaristolo-
led to compound 18 (86%), which then was converted to
the desired bromolactone 19 (74%) by a standard
diazotization/deamination procedure. The overall yield
for the conversion of 14 to 19 was 37%, acceptable for
further large-scale work.
The synthesis of the required bromolactam 11 was
accomplished according to Figure 4. Coupling of the
bromolactone 19 with the commercially available
boronic acid 20, under Suzuki coupling conditions,
afforded the biphenyl derivative 21 in 64% yield, and
the latter, under the influence of potassium t-butoxide in
boiling t-butanol, led to the lactonic phenanthrene 22 in
55% yield. This was converted by means of the Bucherer
reaction (49) to AL-II (23; ꢀ100%), identical in all
respects with the natural product (32). Bromination of
23 then led smoothly to a quantitative yield of the
desired but extremely insoluble bromolactam 11. The
absence of absorption at or near 7.09 ppm in the 1H
NMR spectrum of 11 (in hot DMSO-d6), characteristic
of the 7-hydrogen atom in AL-II (23), confirmed that
bromination had occurred at this location.
In order to couple 11 with the protected forms 25 and
29, respectively, of dA and dG (Figures 5 and 6) under
Buchwald–Hartwig conditions (50), the nitrogen of the
lactam ring needed to be protected by a group that not
only would block any possible reaction at this site during
the coupling process, but would also provide increased
solubility in common solvents and be easily removable
without causing collateral damage once coupling was
complete. After more than a dozen attempts to accomplish
this with a series of well-established protecting groups,
success was finally achieved with the little-used
t-butyldimethylsilyloxymethyl (TBDMSOM) protecting
group (51). Treatment of the bromolactam 11 with
sodium hydride in dry DMF at 50ꢁC followed by the
addition of t-butyl chloromethyloxydimethylsilane at
0ꢁC for 30 min gave, after chromatographic purification,
lactam II in the presence of
a large excess of
20-deoxyadenosine. The method, although effective,
requires an extensive purification procedure that is not
adaptable to larger-scale work.
The key intermediate in the early phase of the synthetic
work is the bromolactone 19, which was prepared
according to Figure 3. Phthalide 15 had previously been
prepared by a multi-step process (30), but in our hands
was accomplished in a single step by heating 14 with
cuprous cyanide at 160ꢁC in 3% aqueous formamide.
This has the advantages of (i) giving 15 directly without
having to isolate the intermediate nitrile and (ii) avoiding
contamination of the product with solvent by using
formamide instead of DMF as the reaction medium
(formamide has virtually no solubility in non-polar
organic solvents). Surprisingly perhaps, electrophilic
reactions of 15 lead dominantly to substitution at the
7-position (ortho to the carbonyl group), thus blocking a
one-step conversion to 19. Thus, we followed literature
methods that are based largely on the work of Manske
and co-workers (47) in the parallel veratrole series but
with modifications which made the overall route relatively
efficient and manageable on a larger scale. Nitration
of 15 gave 16 (88%) which on catalytic reduction
led to 17 (92%). The latter, on bromination with
bis(N-methylpyrrolidin-2-one) hydrogen tribromide (48),

348 Nucleic Acids Research, 2010, Vol. 38, No. 1
O
O
O
HO
O
O
OH
O
O
B
O
O
O
O
b
O
a
CHO
CHO
Br
19
20
22
21
c
O
O
O
O
O
O
O
O
O
NH
d
NH
NCH2OTBDMS
Br
e
f
Br
23
24
11
Figure 4. Synthetic scheme for the preparation of 7-bromo-6-(tert-butyl-dimethylsilanyloxymethyl)-6H-benzo[f][1,3]dioxolo[40,50:4,5]benzo[1,2,
3-cd]indol-5-one (24). Reagents: (a) PD(dpf)Cl2/K2CO3; (b) KOBut; (c) NaHSO3/NH3; (d) Br2; (e)TBDMSOCH2Cl/NaH/DMF and (f) HF/Pyr,
then NH3/H2O.
O
O
O
O
NH2
NCH OTBDMS
2
O
O
NCH OTBDMS
N
N
2
N
a
N
N
O
Br
TBDMSO
N
HN
N
N
OTBDMS
25
24
TBDMSO
26
O
OTBDMS
b
O
O
O
O
O
O
O
O
O
NH
NH
NH
c
d
N
N
N
N
N
N
N
N
HN
HN
HN
N
N
N
N
5
HO
DMTO
DMTO
O
O
O
OH
O
OH
27
OCH CH CN
2
2
P
28
NPr2i
Figure 5. Synthetic scheme for the preparation of AL-II-dA (5) and its 50-DMT protected phosphoramidite (28). Reagents: (a) PdCl2/Xantphos; (b)
HF/Pyr then NH3/H2O; (c) DMTCl/Pyr and (d) CIP(OCH2CH2CN)N(i-Pr)2/Triazole.

Nucleic Acids Research, 2010, Vol. 38, No. 1 349
O
O
O
O
CH OTBDMS
OBn
2
OBn
N
O
O
N
N
N
NCH OTBDMS
N
N
2
a
H N
N
O
2
N
N
N
TBDMSO
Br
H
TBDMSO
O
OTBDMS
29
24
30
OTBDMS
b
O
O
O
O
O
O
O
O
O
OBn
NH
OBn
NH
O
N
NH
HN
N
N
N
N
N
N
N
c
d
N
H
N
O
N
N
N
O
N
H
H
HO
DMTO
DMTO
O
OH
OH
31
OH
e
32
33
O
O
O
O
O
O
N
NH
HN
O
NH
O
N
N
N
N
HN
N
N
N
H
H
HO
DMTO
O
O
OH
O
OCH CH CN
6
2
2
P
34
i
NPr
2
Figure 6. Synthetic scheme for the preparation of AL-II-dG (6) and its 50-DMT protected phosphoramidite (34). Reagents: (a) PdCl2/Xantphos; (b)
HF/Pyr then NH3/H2O; (c) DMTCl/Pry; (d) Pd/H2 and (e) CIP(OCH2CH2CN)N(i-Pr)2/Triazole.
a 63% yield of the easily soluble, protected lactam 24. This
compound when coupled (Figures 5 and 6) with either 25
or 29 under Buchwald–Hartwig conditions (50) using
Xantphos as the palladium-chelating agent gave excellent
yields of the protected forms 26 and 30 of the dA and dG
adducts 5 and 6, respectively, of AL-II. Deprotection of
the silyl-protecting groups was accomplished in two steps.
Treatment with HF in pyridine removes all of the TBDMS
groups but leaves a hydroxymethyl residue on the lactam
nitrogen. This residue was then easily removed by heating
with aqueous ammonia to give 5 in the case of 26, and 31
in the case of 30. Catalytic hydrogenolysis of the benzyl
group in 31 then led to 6 almost quantitatively. The UV
absorption spectrum of 5 proved to be identical with that
of the published spectrum (34). Also, the 1HNMR spectral
data of 5 was identical to those already published (34),
and in agreement it contained a peak at 9.83 that was
assigned by previous workers to the hydrogen at the 1
position of the purine ring. Surprisingly, however, no
evidence for geometrical isomerism was noted at the N6
position.
Although it proved possible to convert 5 smoothly to its
DMT derivative (27) and subsequently to the desired
phosphoramidite 28 by standard procedures, insolubility
problems plagued adduct 6 and its 50-O-DMT derivative
could not be prepared directly. This difficulty was
overcome by introducing the DMT group at an earlier
stage. Reaction of 31 with DMT chloride in pyridine
afforded the much more soluble 50-O-DMT derivative
32. Removal of the benzyl group from 32 by catalytic

350 Nucleic Acids Research, 2010, Vol. 38, No. 1
Table 1. Sequence and mass data for the synthesized DNA oligomers
Entry Sequence
1.
Calc Mass (Da)
Meas Mass (Da)
50-CTC CTC A*AT ACC T-30
4091
5929
4702
3251
7345
7439
7448
3579
2050
4107
5945
4718
7439
7439
4090.2
5927.3
4701.1
3250.3
7344.2
7439.7
7447.9
3577.6
2048.9
4106.1
5944.0
4717.6
7438.6
7436.9
2.
3.
4.
50-TTC CCT CCA GAA A*CA TCC T-30
50-CCA TTC ACA CA*A TCC-30
50-TTT TTA* TTT T-30
5.
6.
7.
8.
50-CCT TCA* CTT CTT TCC TCT CCC TTT-30
50-TCT TCT TCT GTG CA*C TCT TCT TCT-30
50-TCT TCT TCT GCA* GAC TCT TCT TCT-30
50-CGT ACG* CAT GC-30
9.
50-TTG* TTT-30
10.
11.
12.
13.
14.
50-CTC CTC G*AT ACC T-30
50-TTC CCT CCA GAA G8CA TCC T-30
50-CCA TTC ACA CG*A TCC-30
50-TCT TCT TCT GCG*TAC TCT TCT TCT-30
50-TCT TCT TCT GTG* CAC TCT TCT TCT-30
A*, AL-II-dA; G*, AL-II-dG.
hydrogenolysis followed by phosphoramidite formation
then afforded the required compound 34. Thus, the
DMT-phosphoramidites 28 and 34 of adducts 5 and 6,
respectively, became available for site-specific incorpora-
tion into oligomeric DNA by automated solid-state
methods. Further applications of this methodology to
AL-I and related substances are under study.
ratio should be 50:50, as revealed with a construct con-
taining three base mismatches without a lesion (52). DNA
repair (removal of a DNA lesion and the two flanking
mismatches followed by gap-filling synthesis) converts
the three nucleotide sequence of the modified strand to
the sequence complementary to the unmodified strand,
thus losing the strand tag. Thus, DNA repair could influ-
ence the apparent blocking effect of a DNA adduct in
experiments using repair-proficient MEFs. This possibility
should be considered in determing the ratio of progeny.
Nevertheless, translesion DNA synthesis (TLS) occurred,
giving rise to a progeny plasmid from the modified strand.
Both adducts block DNA synthesis strongly. When
fractions of progeny for the AL-II-dG and AL-II-dA
adducts were compared, the dA adduct yielded about
half of the progeny produced by the dG adduct. This
suggests that the dA adduct is more effective at blocking
DNA synthesis than is the dG adduct.
Synthesis of DNA oligomers containing adducts 5 and 6
Both DMT-phosphoramidites (28 and 34) were used
successfully in the synthesis of a series of oligomers.
Table 1 contains the sequences and masses obtained by
ESI/MS for the oligomers containing respectively the
xenonucleosides AL-II-dA (entries 1–7) and AL-II-dG
(entries 8–14) adducts. All these oligomers were
synthesized at the 1.0 mmol scale on an Applied
Biosystems 394 DNA Synthesizer (Foster City, CA). In
all cases the coupling time was 15 min for the modified
deoxynucleoside
phosphoramidites,
and
coupling
Miscoding properties of the two adducts
efficiencies at the point of introduction varied from 93 to
98%. To verify that the modified deoxynucleosides were
incorporated without further modification by reagents
during DNA synthesis, two HPLC-purified oligomers
were digested enzymatically to the deoxynucleosides
using previously published procedures (35). The first was
entry #5 in Table 1; the second was the same sequence
in which the AL-II-dA was replaced by AL-II-dG.
Products for both reactions were analyzed by LC/ESI/
MS/MS, and in both cases the retention time and the
MS/MS spectrum for the modified deoxynucleoside
matched that for the synthetic standard (data not
shown) indicating that AL-II-dA and AL-II-dG were
stable to the conditions of DNA synthesis and were
present in the oligomers.
In MEFs, the major coding events were the insertion of
the correct nucleotides opposite the adducts: T and dC for
the dA and dG adducts, respectively. However, substan-
tial frequencies of misincorporation were observed for
both adducts; 22% for the dA adduct and 9% for the
dG adduct. The nucleotide mis-inserted opposite both
adducts was almost exclusively dA, leading to AL-II-
dA!T and AL-II-dG!T transversions. The insertion
of T opposite the dG adduct was observed once, leading
to an AL-II-dG!dA transition.
The Schmeiser group reported, using an in-vitro
primer extension system, that both dA and dG adducts
strongly blocked DNA synthesis, mainly one nucleotide
30 to the adduct, and that dA and T were inserted
equally well opposite the dA adduct whereas the dG
adduct primarily directed insertion of the correct dC
(53). In general, our site-specific mutagenesis results for
the dA adduct are in accord with the findings obtained
in the in-vitro system and other studies in cells, animals
and humans (19,24,54–56). However, we find that the
dG adduct is less miscoding than is the dA adduct in
cells (Table 2).
Blocking of DNA synthesis in cells
For the biological experiments, lesions were positioned in
the middle of three consecutive base mismatches. This
made it possible to determine the number of progeny
plasmids derived from modified and unmodified strands;
the ratio of progeny reflects the degree to which DNA
synthesis is blocked. In the absence of blocking, the

Nucleic Acids Research, 2010, Vol. 38, No. 1 351
Table 2. Translesional events induced by a site-specific AL-II-dA and AL-II-dG adducts in mouse cells
DNA adduct
No. of progeny from
Nucleotide inserted opposite adducta
MFb (%)
Others
UMSc
MSc
T
A
C
G
AAII-dA
AAII-dG
429 (95)d
416 (91)
22 (5)
41 (9)
191 (78)
1 (0.4)
53 (22)
25 (8.8)
0
257 (90.8)
0
0
22
9
3e
2f
aNumbers were determined following removal of progeny derived from the unmodified strand by EcoRV treatment.
bMF, miscoding frequency.
cUMS, unmodified strand; MS, modified strand; numbers were determined before removal of progeny derived from the unmodified strand by
digesting with EcoRV.
dThe numbers in parentheses represent percentages.
eTGXTT ! AGTTT, TATGT, TATAT.
fTGXTT ! TAACT, TACTT.
2. Grieve,M. (1971) A Modern Herbal: the Medicinal, Culinary,
Cosmetic and Economic Properties, Cultivation and Folk-lore of
SUMMARY
The aristolochic acids I and II have been implicated in
the development of urothelial cancer via DNA adduc-
tion of their metabolites. We have developed a method
for the large-scale synthesis of the dA and dG adducts
derived from AA-II and, after facile conversion to the
50-dimethoxytrityl-protected phosphoramidites, have
incorporated these adducts into DNA oligomers using
automated synthesis techniques. Adducts were chemically
stable to the conditions of oligomer synthesis and were
isolated intact from selected oligomers by enzymatic diges-
tion. After rigorous HPLC purification, DNA oligomers
containing dA or dG adducts were used for site-specific
mutagenesis studies in mouse embryonic cells designed to
establish the mutagenic potential and specificity of these
lesions in vivo. Both adducts block DNA synthesis, but the
dA adduct is the more effective inhibitor. The major
coding events are the insertion of the correct nucleotides
opposite the dA or dG adducts; however, misincorpora-
tion is also observed, and the nucleotide mis-inserted
opposite both adducts is almost exclusively dA, leading
to AL-II-dA!T and AL-II-dG!T transversions.
Herbs, Grasses, Fungi, Shrubs and Trees with all their Modern
Scientific Uses. Dover Pubs, New York, p. 104.
3. Kupchan,S.M. and Doskovitch,R.W. (1962) Tumor inhibitors I.
Aristolochic acid, the active principle of Aristolochia indica.
J. Med. Pharm. Chem., 91, 657–659.
4. Jackson,L., Kofman,S., Weiss,A. and Brodovsky,H. (1964)
Aristolochic acid (NSC-50413): Phase I clinical study. Cancer
Chemother. Rep., 42, 35–37.
5. Mengs,U. (1988) Tumor induction in mice following exposure to
aristolochic acid. Arch Toxicol., 61, 504–505.
6. Vanherweghem,J.L., Depierreux,M., Tielemans,C., Abramowicz,D.,
Dratwa,M., Jadoul,M., Richard,C., Vandervelde,D., Verbeelen,D.,
Vanhaelen-Fastre,R. et al. (1993) Rapidly progressive interstitial
renal fibrosis in young women: association with slimming regimen
including Chinese herbs. Lancet, 341, 387–391.
7. Vanhaelen,M., Vanhaelen-Fastre,R., But,P. and
Vanherweghem,J.L. (1994) Identification of aristolochic acid in
Chinese herbs. Lancet, 343, 174.
8. Cosyns,J.P., Jadoul,M., Squifflet,J.P., Wese,F.X. and van Ypersele
de Strihou,C. (1999) Urothelial lesions in Chinese herb
nephropathy. Am. J. Kidney Dis., 33, 1011–1017.
9. Dillerot,G., Jadoul,M., Arlt,V.M., van Ypersele De Strihou,C.,
Schmeiser,H.H., But,P.P.H., Bierler,C.A. and Cosyns,J. (2001)
Aristolochic acid nephropathy in a Chinese patient: time to abandon
the term ‘‘Chinese herbs nephropathy’’. Am J. Kidney Dis., 38, E26.
10. Djukanovic,L. and Radovanovic,Z. (2003) In De Broe,M.E.,
´ ´
Porter,G.A., Bennett,W.M. and Verpooten,G.A. (eds), Balkan
Endemic Nephropathy in Clinical Nephrotoxins, 2nd edn. Kluwer
Academic Publishers, Dordrecht, The Netherlands, pp. 588–601.
11. Ivic,M. (1969) The problem of etiology of endemic nephropathy.
Lijetc Vjes, 91, 1278–1281.
ACKNOWLEDGEMENTS
The authors would like to thank Mr Robert Rieger
for obtaining mass spectra of intermediates and final
products, Ms Naomi Suzuki and Dr Shinya Shibutani
for assistance in obtaining UV spectra and Dr Robert
J. Turesky for the enzymatic digestion and MS/MS
analysis of two synthetic oligomers.
12. Nikolic
Tumors Izdavacko preduzec
13. Petronic,V. (2000) Tumors of the upper urothelium and endemic
nephropathy. In Radovanovic,Z., Sindic,M., Polenakovic,M.,
Djukanovic,L. and Petronic,V. (eds), Endemic Nephropathy.
´
,J. (2006) Epidemic Nephropathy and Upper Urothelial
´
e Belgrade, Serbia.
´
´
Zavod Za Udzbenike I Nastavna Sredstva, Belgrade, Serbia,
pp. 350–439.
14. Cosyns,J.P., Jadoul,M., Squifflet,J.P., De Plaen,J.F., Ferluga,D.
and van Ypersele de Strihou,C. (1994) Chinese herbs nephropathy:
a clue to Balkan endemic nephropathy? Kidney Int., 45, 1680–1688.
15. Cosyns,J.-P., Dehoux,J.-P., Guiot,Y., Goebbels,R.-M., Robert,A.,
Bernard,A.M. and Van Ypersele De Strihou,C. (2001) Chronic
aristolochic acid toxicity in rabbits: a model of Chinese herb
nephropathy? Kidney Int., 59, 2164–2173.
FUNDING
National Institute for Environmental Health Sciences
grant [ES04068]. Funding for open access charge:
National Institute for Environmental Health Sciences
grant [ES04068].
16. Sato,N., Takahashi,D., Chen,S.M., Tsuchiya,R., Mukoyama,T. and
Yamagata,S. (2004) Acute nephrotoxicity of aristolochic acids in
mice. J. Pharm. Pharmacol., 56, 221–229.
Conflict of interest statement. None declared.
17. Shibutani,S., Dong,H., Suzuki,N., Ueda,S., Miller,F. and
Grollman,A.P. (2007) Selective toxicity of aristolochic I and II.
Drug Metab. Dispos., 35, 1217–1222.
REFERENCES
18. Hranjec,T., Kovac,A., Kos,J., Mao,W., Chen,J.J., Grollman,A.P.
and Jelakovic,B. (2005) Endemic nephropathy: the case for chronic
poisoning by aristolochia. Croat. Med. J., 46, 116–125.
1. Dawson,W.R. (1927) Birthwort: a study of the progress of
medical botany through twenty-two centuries. Pharm. J Pharmacist,
396–397, 427–430.

352 Nucleic Acids Research, 2010, Vol. 38, No. 1
19. Grollman,A.P., Shibutani,S., Moryia,M., Miller,F., Wu,L. and
Moll,U. (2007) Aristolochic acid and the etiology of endemic
(Balkan) nephropathy. Proc. Natl Acad. Sci., 104, 12129–12134.
20. Schmeiser,H.H., Bieler,C.A., Weissler,M., van Ypersele de
Strihou,C. and Cosyns,J.P. (1996) Detection of DNA adducts
formed by aristolochic acid in renal tissue from patients with
Chinese herbs nephropathy. Cancer Res., 56, 2025–2028.
21. Arlt,V.M., Stiborova,M. and Schmeiser,H.H. (2002) Aristolochic
acid as a probable human hazard in herbal remedies: a review.
Mutagenesis, 17, 265–277.
38. Stein,S., Lao,Y., Yang,I.-Y., Hecht,S.S. and Moriya,M. (2006)
Genotoxicity of acetaldehyde- and crotonaldehyde-induced
1,N2-propanodeoxyguanosine DNA adducts in human cells.
Mutation Res., 608, 1–7.
39. Pailer,M. and Schleppnik,A. (1957) Plantzliche naturstoffe mit
einer nitrogruppe II. Die konstitution der aristolochiasaure II.
Monat., 88, 367–387.
40. Johnson,F. (1968) Allylic strain in six-membered rings. Chem. Rev.,
68, 374–413.
41. Acevedo,S. and Bowden,K. (1986) Transmission of polar effects.
Part 16. Ionization of 8-Substituted 1-Naphthoic acids and Alkaline
Hydrolysis of their Methyl Esters. J. Chem. Soc. Perkin Trans II,
2049–2050.
42. Rule,H.G. and Barnett,A.J.G. (1932) Displacement of the nitro
group in 8-nitro-1-naphthoic acid by thionyl halides to form
8-chloro- and 8-bromonaphthoic acids. J. Chem. Soc., 175–79.
43. Kupchan,S.M. and Wormser,H.C. (1965) Tumor inhibitors X.
Photochemical synthesis of phenanthrenes. Synthesis of aristolochic
acid and related compounds. J. Org. Chem., 30, 3792–3800.
44. Estevez,J.C., Estevez,R.J. and Castedo,L. (1995) The intramolecular
aryne cycloaddition approach to aporphinoids. A new total
synthesis of aristolactams and phenanthrene alkaloids. Tetrahedron,
51, 10801–10810.
45. Estevez,J.C., Villaverede,M.C., Estevez,R.J. and Castedo,L. (1995)
Tributyltin (IV) hydride mediated free-radical syntheses of
dehydrodibenzochromanones, dihydrodibenzocoumaranones and
aristolactams. Tetrahedron, 14, 4075–4082.
46. Couture,A., Deniau,E., Grandclaudon,P., Rybalko-Rosen,H.,
Leonce,S., Pfeiffer,B. and Renard,P. (2002) Synthesis and
biological evaluation of aristolactams. Bioorg. Med. Chem. Lett.,
12, 3557–3559.
22. Pfau,W., Schmeiser,H.H. and Wiessler,M. (1990) 32P-postlabeling
analysis of the DNA adducts formed by aristolochic acid I and II.
Carcinogenesis, 11, 1627–1633.
23. Stiborova,M., Frei,E., Arlt,V.M. and Schmeiser,H.H. (2008)
´
Metabolic activation of carcinogenic aristolochic acid, a risk factor
for Balkan endemic nephropathy. Mutat. Res., 658, 55–67.
24. Jameson,C.W., Lunn,R., Jeter,S., Garner,S., Atwood,S., Carter,G.,
Levy,D. and Cosyns,J.-P. (2008) Background document for
aristolochic acid-related exposures, National Toxicology Program
Report on Carcinogens. 12th edn. US Department of Health and
Human Services, Public Health Service, Research Triangle Park,
NC, pp. 1–228.
25. Grollman,A.P., Scarborough,J. and Jelakovic,B. (2009) Aristolochic
acid nephropathy: an environmental and iatrogenic disease.
Adv. Mol. Tox., 3, 211–227.
26. Debelle,F.D., Vanherweghem,J.-L. and Nortier,J.L. (2008)
Aristolochic acid nephropathy: a worldwide problem. Kidney Int.,
74, 158–169.
27. Nortier,J.L., Martinez,M.C., Schmeiser,H.H., Arlt,V.M.,
Bieler,C.A., Petein,M., Depierreux,M.F., DePauw,L.,
Abramowicz,D., Vereerstraeten,P. et al. (2000) Urothelial
carcinoma associated with the use of a Chinese herb
(Aristolochia fangchi). N. Engl. J. Med., 342, 1686–1692.
28. Grollman,A.P. and Jelakovic,B. (2009) Role of environmental
toxins in endemic (Balkan) nephropathy. J. Am. Soc. Nephrol., 18,
2817–2823.
29. Bressy,C., Menant,C. and Piva,O. (2005) Synthesis of polycyclic
lactams and sultams by a cascade ring-closure metathesis/
isomerization and subsequent radical cyclization. Synlett., 4, 577–582.
30. Sinhababu,A.K. and Borchardt,R.T. (1983) General method for
the synthesis of phthaldehydic acids from o-bromobenzaldehydes.
J Org. Chem., 48, 2356–2358.
47. Manske,R.H., McRae,J.A. and Moir,R.Y. (1951)
3-Bromometameconine. Can. J. Chem., 29, 526–535.
48. Daniels,W.E., Chiddix,M.E. and Glickman,S.A. (1963) Lactam
complexes with bromine-hydrogen bromide. J. Org. Chem., 28,
573–574.
49. Bucherer,H.T. (1904) Effect of sulfurous acid salts on aromatic
amido and hydroxyl compounds. J. Prakt. Chem., 69, 49–91.
50. De Riccardis,F., Bonala,R.R. and Johnson,F. (1999) A general
method for the synthesis of the N2- and N6-carcinogenic amine
adducts of 20-deodyguanosine and 20-deoxyadenosine. J. Am. Chem.
Soc., 121, 10453–10460.
31. Schmeiser,H.H., Frei,E., Wiessler,M. and Stiborova,M. (1997)
Comparison of DNA adduct formation by aristolochic acids in
various in vitro activation systems by 32P-post-labelling: evidence
for reductive activation by peroxidases. Carcinogenesis, 18,
1055–1062.
51. Benneche,T., Gundersen,L-L. and Undheim,K. (1988)
(tert-Butyldimethylsilyloxy)methyl chloride: synthesis and use as
N-protecting group in pyrimidinones. Acta Chem. Scand., 42,
384–389.
52. Yang,I.-Y., Johnson,F., Grollman,A.P. and Moryia,M. (2002)
Genotoxic mechanism for the major acrolein-derived
deoxyguanosine adduct in human cells. Chem. Res. Toxicol., 15,
160–164.
53. Broschard,T.H., Wiessler,M., von der Lieth,C.-W. and
Schmeiser,H.H. (1994) Translesional synthesis on DNA templates
containing site-specifically placed deoxyadenosine and
deoxyguanosine adducts formed by the plant carcinogen
aristolochic acid. Carcinogenesis, 15, 2331–2340.
54. Kohara,A., Suzuki,T., Honma,M., Ohwada,T. and Hayashi,M.
(2002) Mutagenicity of aristolochic acid in the lambda/lacZ
transgenic mouse (MutaMouse). Mutation Res., 515, 63–72.
55. Chen,L., Mei,N., Yao,L. and Chen,T. (2006) Mutations induced
by carcinogenic doses of aristolochic acid in kidney of Big Blue
transgenic rats. Toxicol Lett., 165, 250–256.
56. Nedelko,T., Arlt,V.M., Phillips,D.H. and Hollstein,M. (2009)
TP53 mutation signature supports involvement of aristolochic
acid in the aetiology of endemic nephropathy-associated tumours.
Int. J. Cancer, 124, 987–990.
32. Priestap,H.A. (1985) Seven aristololactams from Aristolochia
argentina. Phytochemistry, 24, 849–852.
33. Cosyns,J.P. (2003) Aristolochic acid Chinese herbs nephropathy.
A review of the evidence to date. Drug Safety, 26, 33–48.
34. Pfau,W., Schmeiser,H.H. and Wiessler,M. (1991) N6-
Adenylarylation of aristolochic II and a synthetic model for the
putative proximate carcinogen. Chem. Res. Toxicol., 4, 581–586.
35. Goodenough,A.K., Schut,H.A.J. and Turesky,R.J. (2007) Novel
LC-ESI/MS/MSn method for the characterization and
quantification of 20-deosyguanosine adducts of the dietary
carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by
2-D linear quadrupole ion trap mass spectrometry. Chem. Res.
Toxicol., 20, 263–276.
36. Yang,I.-Y., Hashimoto,K., de Wind,N., Blair,I.A. and Moriya,M.
(2009) Two distinct translesion synthesis pathways across a lipid
peroxidation-derived DNA adduct in mammalian cells. J. Biol.
Chem., 284, 191–198.
37. Hirt,B. (1967) Selective extraction of polyoma DNA from infected
mouse cell cultures. J. Mol. Biol., 26, 365–369.