Synthesis of oligonucleotides containing Fapy·dG (N6- (2-deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5- formamidopyrimidine) [22]

Full text of DOI:10.1021/ja0160952

8636
J. Am. Chem. Soc. 2001, 123, 8636-8637
a defined site was reported.7 These methods are not applicable
to the synthesis of DNA containing the parent Fapy‚dG. Conse-
quently, studies on Fapy‚dG are limited to DNA in which it and
other lesions are produced via photolysis, γ-radiolysis, or radio-
mimetic methods.2-6
Synthesis of Oligonucleotides Containing Fapy‚dG
(N6- (2-Deoxy-r,â-D-erythro-pentofuranosyl)-2,6-
diamino-4-hydroxy-5-formamidopyrimidine)
Kazuhiro Haraguchi‡ and Marc M. Greenberg*
Solid-phase oligonucleotide synthesis is extremely useful for
providing oligonucleotides containing DNA lesions that are useful
in physicochemical and biological studies.8-11 However, the
chemical properties of Fapy‚dG presented unique challenges for
oligonucleotide synthesis. The greatest anticipated hurdle for the
successful synthesis of oligonucleotides containing Fapy‚dG was
the facile epimerization of N-(2-deoxy-R,â-D-erythro-pento-
furanosyl)formamidopyrimidine nucleosides and their rearrange-
ment to pyranose isomers, a process that cannot occur when the
lesion is produced within a biopolymer.12 Facile epimerization
of formamidopyrimidines obviated the need for stereoselective
synthesis of oligonucleotides containing R- or â-Fapy‚dG.
Consequently, we adopted a synthetic strategy that was uncon-
cerned with stereochemistry at the anomeric center, but avoided
exposing the primary hydroxyl group of the sugar in the presence
of the formamidopyrimidine. For oligonucleotide synthesis this
required incorporating Fapy‚dG as part of a dinucleotide phos-
phoramidite (1).13
Department of Chemistry, Colorado State UniVersity
Fort Collins, Colorado 80523
ReceiVed April 26, 2001
ReVised Manuscript ReceiVed July 13, 2001
Exposure of DNA to various forms of oxidative stress results
in its structural alteration. The effects of the lesions produced on
the structure of DNA and their interaction with repair and
polymerase enzymes have important consequences in aging and
in the etiology of diseases, including cancer and neurodegenerative
diseases such as Cockayne syndrome and xeroderma pigmento-
sum.1 The formamidopyrimidine lesions (e.g. Fapy‚dG) are
produced from the purine nucleotides in DNA due to the effects
of ionizing irradiation and agents that produce reactive oxygen
species.2,3 Under O2 limiting conditions the yield of Fapy‚dG
formed via γ-radiolysis is greater than that of the well-studied
lesion, OxodG.2a An indication that Fapy‚dG formation is
biologically significant is its excision by the base excision repair
enzyme that bears its name, formamidopyrimidine glycosylase
(fpg, mutM).4 Furthermore, there is evidence that related mol-
ecules MeFapy‚dG and Fapy‚dA adversely affect DNA poly-
merase activity.5,6 However, examination of the effects of Fapy‚dG
on DNA structure and function is limited by the inability to
prepare nucleic acids containing this lesion at a defined site. We
wish to report the first synthesis of oligonucleotides containing
Fapy‚dG.
The nitro group also served to accelerate nucleophilic aromatic
substitution by the requisite 2-deoxyribosylamine, which was
prepared from the triacetate (3) and used crude (Scheme 1).15
The dimethoxytrityl group was used to protect the C3-hydroxyl
because we wanted to unmask the primary hydroxyl under
nonacidic conditions in order to reduce the likelihood that 6
rearranges and minimize protecting group manipulations later in
the synthesis. This required that we synthesize the oligonucleotides
in the 5′f3′ direction using reverse phosphoramidites.16 Since
Fapy nucleosides epimerize in water, it was not necessary to
separate anomers following glycosylation.12 But the anomers of
6 were separated following protection of the N2-amino group and
desilylation, to facilitate characterization. Neither anomer of 6
rearranged to the pyranose isomer, indicating that the electron
(7) Asagoshi, K.; Yamada, T.; Terato, H.; Ohyama, Y.; Monden, Y.; Arai,
T.; Nishimura, S.; Aburatani, H.; Lindahl, T.; Ide, H. J. Biol. Chem. 2000,
275, 4956.
DNA containing N-methylformamidopyrimidines (e.g. MeFapy‚
dG) has been prepared via random methylation by dimethyl
sulfate, followed by alkaline hydrolysis.5a Recently, a chemo-
enzymatic method for preparing DNA containing MeFapy‚dG at
‡ Present address: School of Pharmaceutical Sciences, Showa University,
Hatanodai 1-5-8, Shinagawa-ku, Tokyo 142-8555, Japan.
(1) (a) Le Page, F.; Kwoh, E. E.; Avrutskaya, A.; Gentil, A.; Leadon, S.
A.; Sarasin, A.; Cooper, P. K. Cell 2000, 101, 159. (b) Hanawalt, P. C. Science
1994, 266, 1957. (c) Lindahl, T. Nature 1993, 362, 709.
(2) (a) Gajewski, E.; Rao, G.; Nackerdien, Z.; Dizdaroglu, M. Biochemistry
1990, 29, 7876. (b) Doetsch, P. W.; Zastawny, T. H.; Martin, A. M.;
Dizdaroglu, M. Biochemistry 1995, 34, 737.
(3) (a) Aruoma, O. I.; Halliwell, B.; Gajewski, E.; Dizdaroglu, M. J. Biol.
Chem. 1989, 264, 20509. (b) Dizdaroglu, M.; Aruoma, O. I.; Halliwell, B.
Biochemistry 1990, 29, 8447.
(4) Boiteux, S.; Gajewski, E.; Laval, J.; Dizdaroglu, M. Biochemistry 1992,
31, 106.
(5) (a) Tudek, B.; Boiteux, S.; Laval, J. Nucleic Acids Res. 1992, 20, 3079.
(b) Tudek, B.; Graziewicz, M.-A.; Kazanova, O.; Zastawny, T. H.; Obtulowicz,
T.; Laval, J. Acta Biochim. Pol. 1999, 46, 785.
(6) Graziewicz, M.-A.; Zastawny, T. H.; Olinski, R.; Tudek, B. Mutation
Res. 1999, 434, 41.
(8) For a recent review see: Butenandt, J.; Burgdorf, L. T.; Carrell, T.
Synthesis 1999, 1085.
(9) Taylor, J.-S.; O’Day, C. L. J. Am. Chem. Soc. 1989, 111, 401.
(10) (a) Shibutani, S.; Takeshita, M.; Grollman, A. P. Nature 1991, 349,
431. (b) Iwai, S.; Shimizu, M.; Kamiya, H.; Ohtsuka, E. J. Am. Chem. Soc.
1996, 118, 7642. (c) Sambandam, A.; Greenberg, M. M. Nucleic Acids Res.
1999, 27, 3597.
(11) Iyer, R. P.; Beaucage, S. L. In ComprehensiVe Natural Products
Chemistry; Kool, E. T., Ed.; Pergamon: Amsterdam, The Netherlands, 1999;
Vol. 7, pp 105-152.
(12) (a) Raoul, S.; Bardet, M.; Cadet, J. Chem. Res. Toxicol. 1995, 8, 924.
(b) Berger, M.; Cadet, J. Z. Naturforsch. 1985, 40b, 1519. (c) Greenberg, M.
M.; Hantosi, Z.; Wiederholt, C. J.; Rithner, C. D. Biochemistry. Submitted
for publication.
(13) DMT ) dimethoxytrityl; Pac ) phenoxyacetyl
(14) Al-Hassan, S. S.; Cameron, R. J.; Curran, A. W. C.; Lyall, W. J. S.;
Nicholson, S. H.; Robinson, D. R.; Stuart, A.; Suckling, C. J.; Stirling, I.;
Wood, H. C. S. J. Chem. Soc., Perkin Trans. 1 1985, 1645.
(15) Gold, A.; Sangaiah, R. Nucleosides & Nucleotides 1990, 9, 907.
(16) Rippe, K.; Jovin, T. M. Methods in Enzymology; Lilley, D. M. J.,
Dahlberg, J. E., Eds.; Academic Press: San Diego, 1992; Vol. 211, pp 199-
231.
10.1021/ja0160952 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/11/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 35, 2001 8637
Scheme 1a
groups on the exocyclic amines of dA, dC, and dG.11 Native
nucleotide phosphoramidites (0.05 M) were coupled by using
automated synthesis cycles comparable to those previously
reported for reverse phosphoramidites,16 but 1 (0.05 M) required
double-coupling for extended times (30 min/coupling) to achieve
a 70% yield. Unreacted hydroxyl groups on the growing oligo-
nucleotide were capped by using trimethylacetic anhydride/lutidine
instead of acetic anhydride to guard against transamidation of
deoxyguanosine and deformylation of Fapy‚dG.18 Deprotection
was carried out in two steps. Demethylation of the phosphate
a Key: (a) TMSN3, TMSOTf, CH2Cl2. (b) NaOMe, MeOH. (c)
TBDMSCl, pyridine. (d) DMTCl, pyridine. (e) H2, Pd/CaCO3, EtOH. (f)
2, diisopropylethylamine, EtOH. (g) PhOCH2CO2H, PyBOP, CH2Cl2. (h)
Bu4N+ F-, AcOH, THF.
Scheme 2
triester introduced along with 1 was achieved with disodium
2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (0.2 M, 20
min).19 The remainder of the protecting groups were removed
and the oligonucleotide cleaved from the support by using
anhydrous K2CO3 (0.05 M)/MeOH (4 h). Denaturing polyacryl-
amide gel electrophoresis (PAGE) purified oligonucleotides (10,
11) exhibited the expected molecular weight by ESI-MS and
showed no evidence for deformylation, dehydration, or deglyco-
sylation of Fapy‚dG.20 Due to the modest coupling yield of 1, a
36mer (12) was prepared enzymatically from 10 on a 5 nmol
scale.21 Oligonucleotides 10 and 13 were 5′-phosphorylated by
using polynucleotide T4 kinase and then hybridized along with
14 to a DNA template (15). Following incubation with T4 DNA
ligase, 12 was purified by PAGE and analyzed by MALDI-TOF
MS.20 The 36mer was characterized further by reacting 5′-32P-12
hybridized to its complement with fpg protein (200 nM).
Quantitative cleavage within 5 min provided additional evidence
that none of the Fapy‚dG underwent dehydration to form dG
during any of the synthesis or purification procedures.20
Scheme 3a
a Key: (a) â-6, tetrazole, then t-BuOOH. (b) H2, Pd/C, MeOH. (c)
HC(O)OAc, pyridine, THF. (d) Bu4N+ F-, AcOH, THF. (e) 2-cyanoethyl
phosphoramidic chloride, diisopropylethylamine, CH2Cl2.
withdrawing nitro group inhibited the ability of the N6 nitrogen
to stabilize the acyclic intermediate necessary for rearrangement
and epimerization (Scheme 2).
In summary, we have developed chemical and enzymatic
methods for synthesizing oligonucleotides containing Fapy‚dG
at a defined site. These biopolymers will facilitate elucidation of
the physical, chemical, and biological effects of this lesion.
Dinucleotide couplings were carried out by using single
diastereomers of 6 for characterization purposes (Scheme 3).
However, for large-scale synthesis anomeric mixtures of 6 were
employed. The O-methyl phosphoramidite (7) was employed
because the alternative â-cyanoethyl group was unstable to
subsequent transformations. Formylation of the N5-amino group
obtained upon reduction of the nitro group (8) produced 9 as a
mixture of diastereomers and formamide rotamers. Diastereomeric
products differing at the anomeric center of Fapy‚dG (â:R ) 2:1)
were separated from one another.17 Desilylation of the â-anomer
of 9 yielded a mixture of dinucleotides epimeric at the Fapy‚dG
anomeric center (â:R ) 1.7:1).17 The major isomer assigned as
the â-Fapy‚dG dinucleotide was carried on to 1 by using standard
phosphitylation methods.11
Acknowledgment. We are grateful for support of this research by
the National Institutes of Health (CA-74954). We thank Michael Delaney
for helpful discussions and technical assistance and Jamie Milligan
(UCSD) for providing us with fpg protein.
Supporting Information Available: Procedures for the synthesis of
1, synthesis/purification of oligonucleotides 10-12, and the cleavage of
5′-32P-12 by fpg protein; mass spectra of 10-12; and phosphor image
showing the cleavage of 5′-32P-12 by fpg protein (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA0160952
(18) Control experiments on protected monomers revealed that acetic
anhydride/lutidine/N-methyl imidazole capping conditions resulted in acylation
of Fapy‚dG. For the effects of capping conditions using “fast-deprotecting”
phosphoramidites see: Zhu, Q.; Delaney, M. O.; Greenberg, M. M. Bioorg.
Med. Chem. Lett. 2001, 11, 1105.
(19) Scaringe, S. A.; Wincott, F. E.; Carruthers, M. H. J. Am. Chem. Soc.
1998, 120, 11820.
(20) See Supporting Information.
(21) Ordoukhanian, P.; Taylor, J.-S. Nucleic Acids Res. 1997, 25, 3783.
Oligonucleotides (10, 11) were prepared by using 1 and reverse
â-cyanoethyl phosphoramidites containing “fast-deprotecting”
(17) Stereochemical assignments of anomers in 9 and the desilylated product
were based upon the chemical shift of the formyl protons. Shielding by the
phenyl rings of the dimethoxytrityl protecting group results in an upfield shift
of this proton in the respective R-isomers (9, 0.52 ppm; desilylated product,
0.42 ppm).