Preparation of C8-amine and acetylamine adducts of 2′-deoxyguanosine suitably protected for DNA synthesis

Article abstract of DOI:10.1021/ol026474f

(Equation presented) C8-Amine and acetylamine adducts of 2′-deoxyguanosine were synthesized. Our approach provides solutions for the coupling of aromatic amines to a protected 8-bromo-2′-deoxyguanosine derivative, for the selective acetylation of the coup

Full text of DOI:10.1021/ol026474f

ORGANIC
LETTERS
2002
Vol. 4, No. 24
4205-4208
Preparation of C8-Amine and
Acetylamine Adducts of
2′-Deoxyguanosine Suitably Protected
for DNA Synthesis
Ludovic C. J. Gillet and Orlando D. Scha1rer*
Institute of Medical Radiobiology, UniVersity of Zu¨rich, August Forel Str. 7,
8008 Zu¨rich, Switzerland
scharer@imr.unizh.ch
Received July 8, 2002
ABSTRACT
C8-Amine and acetylamine adducts of 2′-deoxyguanosine were synthesized. Our approach provides solutions for the coupling of aromatic
8
amines to a protected 8-bromo-2′-deoxyguanosine derivative, for the selective acetylation of the coupled adduct at N and for a protecting
8
group scheme preserving the integrity of the base-labile N acetyl group during DNA synthesis.
A number of carcinogenic aryl-amino and -nitro compounds
are known to produce C8-arylamine adducts of 2′-deoxy-
guanosine (dG).1 If not corrected, these covalent DNA
modifications can trigger mutagenesis and eventually induce
cancer. Among these adducts, 8-(N-acetyl-aminofluorene)-
2′-deoxyguanosine (dG-AAF) (1) has been intensively used
as a model compound (Figure 1) for studying mutagenesis,
carcinogenesis, and DNA repair.1a Interestingly, although
8-(N-aminofluorene)-2′-deoxyguanosine (dG-AF) (2) and
dG-AAF (1) only differ by one acetyl group, they exhibit
very different physicochemical properties and biological
effects: dG-AAF causes much more severe local distortion
of DNA than dG-AF1a,2 and is thus a much more potent block
to replication and transcription,1a,3 as well as a better substrate
for DNA repair enzymes.4 Despite its importance, a straight-
forward method for the site-specific incorporation of dG-
AAF and related adducts into DNA using solid-phase
synthesis is still not available. Two major issues need to be
addressed to achieve this goal. (i) The base labile N8 acetyl
group of dG-AAF is known to be unstable upon the standard
NH4OH deprotection step after solid-phase DNA synthesis.5
Therefore, protecting groups that can be removed under
conditions compatible with the maintenance of the N8 acetyl
group are required. Zhou and Romano have reported a
(1) (a) Heflich, R. H.; Neft, R. E. Mut. Res. 1994, 318, 73-174. (b)
Schut, H. A. J.; Snyderwine, E. G. Carcinogenesis 1999, 20, 353-368. (c)
Heflich, R. H.; Howard, P. C.; Beland, F. A. Mut. Res. 1985, 149, 25-32.
(2) O’Handley, S. F.; Sanford, D. G.; Xu, R.; Lester, C. C.; Hingerty,
B. E.; Broyde, S.; Krugh, T. R. Biochemistry 1993, 32, 2481-2497.
Figure 1. C8-Arylamine adducts of 2′-deoxyguanosine naturally
formed by carcinogenic aminofluorene derivatives: 1, N-acetyl-2-
aminofluorene (AAF); 2, 2-aminofluorene (AF).
10.1021/ol026474f CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/05/2002

solution to this problem using an Fmoc protecting group
strategy and milder deprotection conditions that ensured the
stability of the dG-AAF.6,7 A limitation of this strategy,
however, is that Fmoc-protected nucleotide phosphoramidites
are not commercially available. (ii) The syntheses of C8-
arylamine and acetylarylamine adducts of dG have tradition-
ally been based on the modification of nucleosides and
oligonucleotides with the corresponding N-hydroxy or N-
acetoxy arylamine derivates. This reaction is severely limited
in yield and scope;1b,6 in particular, a single site-specific
modification cannot be introduced into oligonucleotides that
contain multiple guanines, therefore restricting the sequence
context in which dG-AAF can be introduced. An alternative
strategy for obtaining these adducts is a Buchwald-Hartwig
coupling reaction8 of protected 8-bromo-2′-deoxyguanosine
(Br-dG) derivates with aromatic amines. Indeed the Buch-
wald-Hartwig coupling reaction has been reported for the
formation of N6 adducts of dA,9 N2 adducts of dG,10 and
while this work was in progress, C8 adducts of dA11 and
dG.12,13 Previous syntheses of C8-arylamine adducts of dG
have shown that protections of the hydroxyl groups of the
deoxyribose and of the N2 and O6 positions of the base of
Br-dG were required for the palladium-mediated coupling
reactions. Depending on the amine to be coupled, Wang and
Rizzo used either bis-BOC or STABASE protection for the
N2 position,12 while Meier and Gra¨sl reported that an N2-
isobutyryl protecting group allowed the coupling of a number
of simple amines.13 Although the isobutyryl group is the
standard N2 protecting group for dG in DNA synthesis, the
conditions required for its removal are incompatible with
the base sensitive N8 acetyl group of dG-AAF.
acetylated at the N8 position, and the facile replacement of
the N2-DMTr with the N2-isopropylphenoxyacetyl (iPrPac)
group yielded monomers suitable for solid-phase DNA
synthesis in which the integrity of the base labile N8 acetyl
group should be preserved by using the commercial “ultra-
mild” phosphoramidites.14 Our approach thus provides a
general strategy for the synthesis of C8-arylamine and acetyl
arylamine adducts of dG suitably protected for incorporation
into DNA.
Our synthesis starts by bromination of dG (3) with NBS15
and precipitation in acetone as a convenient purification step.
Silylation of the 5′- and 3′-hydroxyl groups and protection
of the O6 position as benzyl ether were achieved using
standard methods (Scheme 1). In search for a generally
Scheme 1. Preparation of the Fully Protected Br-dGa
a Reaction conditions: (a) NBS, water, acetonitrile (80%); (b)
tBDMS-Cl, imidazole, DMF (98%); (c) Bn-OH, PPh3, DIAD,
dioxane (78%); (d) DMTr-Cl, pyridine (94%).
applicable N2 protection, we examined the DMTr group,
which is stable under basic conditions and should thus
survive the Buchwald-Hartwig coupling reaction. The fully
protected Br-dG derivative (4) was obtained in good yields
on a multigram scale, using chromatography on aluminum
oxide for all compounds carrying the N2-DMTr group to
prevent loss of this acid-labile group on silica gel. Among
the various conditions tested for the coupling reaction, we
found that Pd2(dba)3/BINAP as a catalyst, NaOtBu as a base,
and toluene as a solvent gave good coupling yields for a
number of structurally diverse aromatic amines (Scheme
2).8d,16 As expected, protection of both the N2 and O6
positions was required for successful coupling reactions. Our
method allowed the coupling of 2-aminofluorene (5a) and
the synthesis of several new C8 adducts of dG such as those
with 2-amino-1-methylimidazo[4,5-f]-quinoline (isoIQ) (5b),
1-aminopyrene (5c), the electron-poor p-aminobenzophenone
(5d) and its isomer m-aminobenzophenone (5e). To get direct
access to dG-AAF, the acetylated form of 5a, we attempted
a direct coupling between 4 and AAF, but we only observed
the formation of the deacetylated product in low yields.17
Despite these advances, an efficient method for the
synthesis of N8-acetyl arylamine adducts of dG and a
protecting group strategy compatible with their incorporation
into DNA are still missing. Here, we disclose our approach
to address these problems. The use of a transient dimethoxy-
trityl (DMTr) protecting group for the N2 position of Br-dG
derivatives allowed the efficient coupling of a wide variety
of amines under Buchwald-Hartwig conditions. The prod-
ucts of this coupling reaction were subsequently selectively
(3) (a) van Oosterwijk, M. F.; Filon, R.; de Groot, A. J. L.; van Zeeland,
A. A.; Mullenders, L. H. F. J. Biol. Chem. 1998, 273, 13599-13604. (b)
Shibutani, S.; Suzuki, N.; Grollman, A. P. Biochemistry 1998, 37, 12034-
12041.
(4) (a) Gunz, D.; Hess, M. T.; Naegeli, H. J. Biol. Chem. 1996, 271,
25089-25098. (b) Batty, D. P.; Wood, R. D. Gene 2000, 241, 193-204.
(5) Sto¨hrer, G.; Osband, J. A.; Alvarado-Urbina, G. Nucleic Acids Res.
1983, 11, 5093-5101.
(6) Zhou, Y.; Chladek, S.; Romano, L. J. J. Org. Chem. 1994, 59, 556-
563.
(7) Zhou, Y.; Romano, L. J. Biochemistry 1993, 32, 14043-14052.
(8) (a) Louie, J.; Hartwig, J. F. Tetrahedron Lett. 1995, 36, 3609-3612.
(b) Hartwig, J. F. Angew. Chem., Int. Ed. 1998, 37, 2046-2067. (c) Wolfe,
J. P.; Wagaw, S.; Marcoux, J. F.; Buchwald, S. L. Acc. Chem. Res. 1998,
31, 805-818. (d) Wolfe, J. P.; Buchwald, S. L. J. Org. Chem. 2000, 65,
1144-1157.
(9) Lakshman, M. K.; Keeler, J. C.; Hilmer, J. H.; Martin, J. Q. J. Am.
Chem. Soc. 1999, 121, 6090-6091.
(14) Schulhof, J. C.; Molko, D.; Teoule, R. Nucleic Acids Res. 1987,
15, 397-416.
(10) (a) De Riccardis, F.; Bonala, R. R.; Johnson, F. J. Am. Chem. Soc.
1999, 121, 10453-10460. (b) Harwood: E. A.; Hopkins, P. B.; Sigurdsson,
S. T. J. Org. Chem. 2000, 65, 2959-2964.
(15) Gannett, P. M.; Sura, T. P. Synth. Com. 1993, 23, 1611-1615.
(16) We tested a number of other base and solvent systems for this
reaction, including: K3PO4 in DME and Cs2CO3 in dioxane. The reactions
using NaOtBu/toluene were complete after 1 h at 100 °C; reactions with
weaker bases such as K3PO4 or Cs2CO3 yielded no detectable product or
took several days to go to completion.
(11) Schoffers, E.; Olsen, P. D.; Means, J. C. Org. Lett. 2001, 3, 4221-
4223.
(12) Wang, Z.; Rizzo, C. J. Org. Lett. 2001, 3, 565-568.
(13) Meier, C.; Gra¨sl, S. Synlett 2002, 802-804.
4206
Org. Lett., Vol. 4, No. 24, 2002

Scheme 2. Buchwald-Hartwig Coupling Reactionsa
Scheme 3. Selective Acetylation and Deprotection of the
DG-AF Adducta
a Reaction conditions: (g) Ac2O, Et3N, DMAP, pyridine; (h) HCl
i
0.01 M, MeOH (78% for g-h); (i) PrPac-Cl, pyridine (97%); (j)
CH3COOH, TBAF, THF (79%); (k) Pd/C, cyclohexene, EtOH
(88%).
a Reaction conditions: (e) Pd2(dba)3, BINAP, toluene, NaOtBu
and the corresponding amines 2-aminofluorene (5a, 72%), isoIQ
(5b, 68%), 1-aminopyrene (5c, 51%), 4-aminobenzophenone (5d,
83%), or 3-aminobenzophenone (5e, 61%); (f) HCl, MeOH (89%).
N2-iPrPac without compromising the integrity of the N8 acetyl
group (Figure 2).20 Only after 24-48 h under these condi-
More recently developed catalysts described by Buchwald
and co-workers might be used for this purpose.18 dG-AAF
could, however, readily be obtained from 5a by selective
acetylation of the N8 position (Scheme 3). The bulky DMTr
group apparently prevented further acylation reaction at the
N2 position successfully. The product of acetylation turned
out to be rather acid- and base-sensitive, and the position of
the acetyl group was verified by extensive NMR analysis
after removal of the DMTr group using very dilute hydro-
chloric acid in methanol. The silyl protecting groups of 7
were removed using TBAF buffered with acetic acid to
preserve the base-sensitive N8 acetyl group and benzyl group
deprotection was achieved smoothly by transfer hydro-
genolysis to yield the AAF modified nucleoside 1. For
purposes of oligonucleotide synthesis, the N2 position of 7
could also be selectively protected by reaction with iPrPac-
Cl to yield 8. The removal of the silyl and benzyl groups
was achieved using the same conditions employed for the
unprotected compound (7) and yielded the iPrPac protected
form of the AAF modified nucleoside 9 (Scheme 3).19 This
iPrPac group of 9 could be deprotected under very mild basic
conditions: treatment with iPr2NH in MeOH (1:20) at room
temperature for 1-12 h led to complete deprotection of the
Figure 2. Time course of the reaction of iPrPac-dG-AAF in 20/1
i
MeOH/iPr2NH. Elution times on reverse-phase HPLC are PrPac-
dG-AAF (9) t ) 23.2′; dG-AAF (1) t ) 15.9′; and dG-AF (2) t )
17.2′.
tions did we observe the appearance of the deacetylated
product (2) in noticeable amounts. We furthermore confirmed
that both the N2-iPrPac and N8 acetyl groups of 9 were
compatible with conditions used in DNA synthesis as shown
by Zhou and Romano with N2-Fmoc-dG-AAF.6
While we were finishing up this work, the Buchwald-
Hartwig reaction of N2-isobutyryl-protected Br-dG using
K2CO3 as a base was reported.13 We therefore investigated
(17) Failure of efficient coupling of acetyl arylamines under similar
conditions has been reported previously (Wolfe, J. P.; Rennels, R. A.;
Buchwald, S. L. Tetrahedron 1996, 52, 7525-7546. Yang, B. H.; Buchwald,
S. L. Org. Lett. 1999, 1, 35-37).
(18) Klaspars, A.; Huang, X.; Buchwald, S. L. J. Am. Chem. Soc. 2002,
124, 7421-7428.
(19) Compound 9 was further successfully converted to its 5′-O-DMTr-
3′-O-phosphoramidite derivative using standard methods.
Org. Lett., Vol. 4, No. 24, 2002
4207

4): under a variety of conditions, we observed the formation
of a byproduct (12), doubly acetylated at the N2 and N8
positions, that we were unable to separate from the desired
N8 acetylated product 8.22 This approach is apparently not
suited for the generation of C8 aromatic acetylamine adducts
of dG. The N2-DMTr is a preferable protecting group both
for efficient Buchwald-Hartwig coupling reaction and
subsequent acetylation of the N8 position.
Scheme 4. Coupling and Acetylation of the N2-iPrPac
Derivativea
In conclusion, we expanded the scope of the Buchwald-
Hartwig reaction to generate C8-arylamination adducts of
2′-deoxyguanosine through improved protecting group strat-
egy and coupling reactions.23 Our strategy furthermore allows
selective N8-acetylation of the dG adducts and a facile N2
i
reprotection with an PrPac group, yielding building blocks
suitable for ultra-mild DNA synthesis compatible with the
maintenance of the base-labile N8 acetyl group. The incor-
poration of these adducts into DNA and the study of the
repair mechanisms of such DNA lesions are presently
underway in our laboratory.
Acknowledgment. We are grateful to Rolf Ha¨fliger and
Walter Amrein of the MS service of the Department of
Chemistry of the ETH Zu¨rich and to the NMR Service Team
of the Institute of Organic Chemistry of the University of
Zu¨rich for spectral analyses. This work was supported by
the Swiss National Science Foundation and the EMBO
Young Investigator Program.
a Reaction conditions: (e) Pd2(dba)3, BINAP, toluene, NaOtBu,
2-aminofluorene (11, 70%); (g) Ac2O, Et3N, DMAP, pyridine (8,
5%; 12, 95%) or Ac2O, Et3N, pyridine (8, 20%; 12, 80%) or Ac2O,
pyridine (8, 80%; 12, 20%).
whether an analogous coupling reaction could be carried out
with N2-iPrPac-protected Br-dG (10), which might provide
more direct access to the acetyl arylamine-modified building
block for DNA synthesis. We thus investigated the Buch-
wald-Hartwig coupling reaction of the 2-aminofluorene and
N2-iPrPac protected 10 (Scheme 4). Reaction of 10 with
2-aminofluorene, the palladium catalyst, and K2CO3 in
dioxane yielded the coupled product 11 after 5 days at 80
°C without affecting the iPrPac group.21 The N8-acetylation
reaction, however, turned out to be problematic (Scheme
Supporting Information Available: Experimental pro-
cedures for the preparation of all compounds and copies of
1
their H and 13C NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL026474F
(21) Reaction of 10 using the previously established conditions with
i
NaOtBu in toluene led to complete loss of the PrPac group in less than 1
h.
(22) Such transamidation on an N2-iPrPac-protected dG was previously
observed during the capping step of standard oligonucleotide synthesis (Zhu,
Q.; Delaney, M. O.; Greenberg, M. M. Bioorg. Med. Chem. Lett. 2001, 11,
1105-1107).
(20) These new deprotection conditions have been developed in our
laboratory to release oligonucleotides from the ultra-mild Q-supports and
to remove all the protecting groups, including the cyanoethyl groups on
the phosphates and the ultra-mild exocyclic amino protections on the bases,
for oligomers that contained very base labile modifications (Alzeer, J.; Gillet,
L. C. J.; Scha¨rer, O. D. Manuscript in preparation).
(23) Our conditions allow the coupling of amines such as p-aminoben-
zophenone that we found to be too unstable to be activated to their
N-hydroxylamine for direct coupling with dG.
4208
Org. Lett., Vol. 4, No. 24, 2002