Synthesis of the PhIP adduct of 2′-deoxyguanosine and its incorporation into oligomeric DNA

Article abstract of DOI:10.1021/tx0600191

The 2′-deoxyguanosine adduct of the dietary mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been synthesized and incorporated into DNA using solid state synthesis technology. The key step to obtaining the C8-dG adduct is a palladium (Xa

Full text of DOI:10.1021/tx0600191

734
Chem. Res. Toxicol. 2006, 19, 734-738
Communications
Synthesis of the PhIP Adduct of 2′-Deoxyguanosine and Its
Incorporation into Oligomeric DNA
Radha Bonala, M. Cecilia Torres, Charles R. Iden, and Francis Johnson*
Department of Pharmacological Sciences, Stony Brook UniVersity, Stony Brook, New York 11794-3400
ReceiVed January 27, 2006
The 2′-deoxyguanosine adduct of the dietary mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) has been synthesized and incorporated into DNA using solid state synthesis technology. The key
step to obtaining the C8-dG adduct is a palladium (Xantphos-chelated)-catalyzed N-arylation (Buchwald-
Hartwig reaction) of PhIP by a suitably protected 8-bromo-2′-deoxyguanosine derivative. The reaction
proceeded in good yield without complicating side products, and the adduct was converted to the required
5′-O-DMT-3′-O-phosphoramidite by standard methods. This modified deoxynucleoside was used to
synthesize three oligodeoxynucleotides in which the C8-PhIP-dG adduct was incorporated at a single
site. The oligomers were purified by reverse phase HPLC and characterized by mass spectrometry.
Introduction
A considerable number of polycyclic aromatic amines
(PAAs)1 are now well-established as precursors to mutagenic
species that are strongly implicated as some of the causative
factors in mammalian cancer (1). The amines themselves have
little physiological activity, but by well-understood hepatic
metabolic processes, they are first converted by microsomal
enzymes to the N-hydroxy derivatives, which in turn undergo
transformation to the O-acetyl or O-sulfonate esters by conju-
gating transferases. These esters, on solvolysis, give rise to
highly reactive electrophilic nitrenium and/or isomeric carboca-
tions (2), which are the true promutagens in that they react with
DNA, mainly with the purine bases, to give stable adducts. In
general, the nitrenium ions attack these bases at the C8 position,
whereas the carbocations are largely restricted to reaction at
the exocyclic amino groups.
The recently discovered (1) dietary mutagens also fall into
the carcinogenic amine class, being polycyclic heteroaromatic
amines in nature. Although a wide array of premutagens is found
in the human diet, of great interest currently are the extremely
potent precursor carcinogens 2-amino-1-methyl-6-phenylimi-
dazo[4,5-b]pyridine (PhIP) (1), 2-amino-3-methylimidazo[4,5-
f]quinoline (IQ) (2), and 2-amino-3,4-dimethylimidazo[4,5-
f]quinoline (MeIQ) (3), all members of the condensed 2-amino-
imidazole class of premutagens (Figure 1). These particular
substances are thought to arise (1) from the condensation of
amino acids with creatinine, when proteinaceous food such as
meat, fish, and poultry is cooked above 120 °C. Interestingly,
they have also been found in cigarette smoke and in rainwater
Figure 1. Chemical structures of the food mutagens 1-3.
Figure 2. Chemical structures of the C8-dG adduct of PhIP 4 and
two reactive PhIP metabolites 5 and 6.
(3, 4). Because of the presence of PhIP in cigarette smoke
condensate (5), it is not surprising that this amine is a major
mutagenic component recovered from the urine of tobacco
smokers (6). It has been estimated that the average American
citizen is exposed daily to PhIP, at the level of 16-200 ng/kg
(6). The metabolic transformations of these substances are
identical to those of the PAAs; however, the mutagenicities
associated with the metabolites of these byproducts of cooking
appear, on a molar basis, to be substantially greater (1) than
those derived from the simpler carcinogenic amines.
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine or PhIP
(1) continues to receive significant attention because not only
is it the most mass abundant of the dietary mutagens that occur
in cooked meat and fish, but in mammals, it is one of the most
potent colon and breast carcinogens isolated to date (7). Interest
in this compound has been further heightened by the identifica-
tion of PhIP adducts in colon tissue that was removed from
* To whom correspondence should be addressed. Tel: 631-632-8866.
Fax: 631-632-7394. E-mail: francis@pharm.sunysb.edu.
1Abbreviations: BH, Buchwald-Hartwig reaction; DMT, dimethoxytri-
tyl; ESI, electrospray ionization; IQ, 2-amino-3-methylimidazo[4,5-f]-
quinoline; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; PAA, poly-
cyclic aromatic amine; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine.
10.1021/tx0600191 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/06/2006

Communications
Chem. Res. Toxicol., Vol. 19, No. 6, 2006 735
Scheme 1
Scheme 2
adult humans undergoing surgery as part of their therapy for
colon cancer (8). The principal adduct is the deoxyguanosine
derivative 4 derived from the metabolite (5 or 6) of PhIP (Figure
2). Adduct 4 was also confirmed as the major DNA lesion in
the tissues of rats that were treated with PhIP, using 32P-
postlabeling analysis (7).
catalyzed solvolysis of the N-acetoxy derivative 5 in the presence
of 2′-deoxyguanosine (9). However, yields of 4 by this method
are exceedingly low, and to permit more extensive biological
and physicochemical studies of this adduct, a much better
synthetic approach was required. We would now like to report
a significantly more efficient procedure for the synthesis of 4
and its incorporation into oligomeric DNA using standard
automated methods (10).
Our interest in PhIP and its dG adduct arose because methods
of synthesis of the latter substance were limited to the acid-

736 Chem. Res. Toxicol., Vol. 19, No. 6, 2006
Communications
Scheme 3
Table 1
Results and Discussion
oligomer sequence X )
C8-PhIP-dG residue
calculated observed average
Our approach is similar to that used for the synthesis and
incorporation of the benzo[a]pyrene adducts into DNA in which
the key procedure is a Buchwald-Hartwig (BH) reaction (11).
Initial attempts to use the standard BH coupling of the protected
form of 8-bromo-dG (7) with PhIP using the conditions outlined
in Scheme 1 did not proceed well. Although the desired coupled
product 8 was obtained in 30% yield, it was contaminated with
22% of the unusual substance 9, in which we have assigned a
PhIP residue at C6 in place of the benzyloxy function and the
bromine atom is retained at C8. Separation of these substances
was not difficult by column chromatography, but the occurrence
of 9 was an unwanted complication. This difficulty was resolved
by changing the 2-amino protecting group on the deoxygua-
nosine from isobutyryl to the tetramethyl-bis-silylethyl group
used by Wang and Rizzo (12) in their synthesis of the dG adduct
of IQ. Thus, when 10 was used in the coupling procedure
(Scheme 2) with PhIP employing 4,5-bis(diphenylphosphino)-
9,9-dimethyl xanthene (Xantphos) as the Pd ligand under the
conditions described by Yin et al. (13), the required bis-
heteroarylamine 11 was obtained in 58% yield (after chroma-
tography) with virtually no significant contamination by the
analogue corresponding to 9 or by other coupled products2 (14).
Importantly, this procedure proved to be highly successful when
applied to the coupling reaction of 10 with a series of other
related 2-aminoimidazoles. 1-Methyl-2-aminobenzimidazole led
mass (Da)
mass (Da)
a. 5′-TT X TTT-3′
b. 5′-CTC CTC XAT ACC T-3′
c. 5′-TTC CCT CCT CXA TAC CT-3′
2010.2
4067.6
5254.4
2011.2 ( 0.07
4068.8 ( 0.33
5256.5 ( 0.42
to the adduct 12 in 95% yield, whereas IQ and MeIQ gave
adducts 13 and 14, respectively, both in 94% yield. This yield
is considerably better than that reported (12) previously for the
coupling reaction of IQ with a similarly protected dG intermedi-
ate but in which BINAP was used as the Pd ligand rather than
Xantphos.
The bis-silyl protecting group on the 2-amino group of 11
was immediately removed by mild acid hydrolysis to give 15,
which was then treated with isobutyryl chloride to give the more
stable 8. The resulting product 8 (Scheme 3) was then desilylated
by means of HF in anhydrous pyridine, and the benzyl group
on the six-oxygen atom of the deoxyguanosine was removed
by hydrogenation over a Pd catalyst to give 16 (15) in an overall
yield of 80%. Compound 16 was heated in NH4OH at 60 °C to
remove the isobutyryl group yielding the xenodeoxynucleoside
17, which was identical to the PhIP-dG adduct reported in the
literature (16). Conversion of 16 in two steps (60%) to the
dimethoxytrityl (DMT) phosphoramidite 19, suitable for incor-
poration into DNA, was accomplished via 18 using conventional
methods.
2After this work was completed, Dr. Takeji Takamura of the National
Cancer Center Research Institute, Tokyo, Japan, informed us privately that
he also had been able to synthesize 4 by means of a BH reaction using
different protecting groups on the 2′-deoxyguanosine moiety.
Compound 19 was then utilized in the automated synthesis
(17) of the three oligomers listed in Table 1 in which X
represents the C8-PhIP-dG residue.

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Chem. Res. Toxicol., Vol. 19, No. 6, 2006 737
Figure 3. Final HPLC chromatogram for the fully deprotected oligomer b (DMT-off).
Figure 4. Electrospray mass spectrum for oligomer b yielding an average mass of 4068.8 ( 0.3 Da (calcd mass, 4067.6 Da).
The coupling yield was 78-80% for the small oligomer when
a reaction time of 15 min was used at the point of introduction
of the xenonucleoside; increasing this time to 30 min effected
no improvement in yield. However, under similar conditions,
the coupling yields for the synthesis of the larger oligomers
were reduced to approximately 40%. Deprotection of the
oligomers was accomplished first by means of 28% NH3/water
at 50 °C in a sealed tube yielding the DMT-protected form of
the oligomers. Purification was accomplished by two consecu-
tive HPLC passes under conditions reported elsewhere (11, 18).
In the first pass, the DMT-protected oligomer was separated
from the failure sequences lacking DMT residues and collected
as a pure fraction. This fraction was dried, and the DMT group
was removed by treatment with 80% acetic acid in water at
room temperature. Thereafter, the oligomers (DMT-off) were
again subjected to final HPLC purification (Figure 3, oligomer
b). Removal of the solvent under vacuum afforded the dry,
purified oligomers, which are stable for at least 6 months when
stored at -20 °C. The molecular weights of the oligomers were
confirmed by electrospray ionization (ESI) mass spectrometry
(Table 1) using a Waters Platform LC-MS system. A typical
mass spectrum (oligomer b) is shown in Figure 4. The use of
these oligomers for physicochemical and mutagenic studies will
be reported in separate publications.
Acknowledgment. We thank the National Institutes of
Health, Institute of Environmental Health Sciences, for a Grant
(ES04068) that supported this work. We thank also Avalyn
Lewis who obtained the mass spectral data and Maryann Wente
for the HPLC analyses.

738 Chem. Res. Toxicol., Vol. 19, No. 6, 2006
Communications
poration into DNA. Chem. Res. Toxicol. 15, 1489-1494.
(12) Wang, Z. W., and Rizzo, C. J. (1991) Synthesis of the C8-
deoxyguanosine adduct of the food mutagen IQ. Org. Lett. 3, 565-
568.
(13) Yin, J. J., Zhao, M. M., Huffman, M. A., and McNamara, J. M. (2002)
Pd-catalyzed N-arylation of heteroarylamines. Org. Lett. 4, 3481-
3484.
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10H), 6.78 (m, 1H), 5.62 (s, 2H), 4.64 (m, 1H), 4.06 (m, 1H), 3.92
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TX0600191