Highly efficient synthesis of a phosphoramidite building block of C8-deoxyguanosine adducts of aromatic amines

Article abstract of DOI:10.1055/s-2002-25354

The synthesis of 5′-O-DMTr-3′-O-phosphoramidite-C8-arylamine-dG adducts 11 and 12 is described. The compounds are potential building blocks for the automated synthesis of site-specifically modified oligonucleotides. The C8-adducts were synthesized by a pa

Full text of DOI:10.1055/s-2002-25354

802
LETTER
Highly Efficient Synthesis of a Phosphoramidite Building Block of
C8-Deoxyguanosine Adducts of Aromatic Amines
Synthesis of
a
Ph
h
osphoramidi
r
te
B
uilding Blo
s
c
k
Meier,* Sonja Gräsl
Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
Fax +49(40)428384324; E-mail: chris.meier@chemie.uni-hamburg.de
Received 6 February 2002
Abstract: The synthesis of 5 -O-DMTr-3 -O-phosphoramidite-C8-
arylamine-dG adducts 11 and 12 is described. The compounds are
potential building blocks for the automated synthesis of site-specif-
ically modified oligonucleotides. The C8-adducts were synthesized
by a palladium-catalyzed cross-coupling reaction.
Key words: Pd-cross coupling, amination, nucleoside adducts, pro-
tecting groups, phosphoramidites
Covalent alteration of DNA by electrophiles may be the
reason for the induction of chemical carcinogenesis.1 If
these covalently bonded modifications are not repaired,
they compromise the fidelity of DNA replication, leading
to mutations and possibly cancer. Poly- and monocyclic
aromatic amines belong to the class of chemical carcino-
gens that form covalently bonded adducts with DNA after
metabolic activation.2,3 The ultimate carcinogen is an
arylnitrenium ion, generated by cytochrome P450 oxida-
tion of the arylamine to the corresponding hydroxylamine,
followed by esterification and solvolysis.4 The predomi-
nant site of reaction is the C8-position of 2 -deoxygua-
nosine (dG) 1, although N2-adducts have also been
isolated as minor products (Figure 1). To properly study
the mutagenic effects, structure and DNA-repair of these
lesions, an efficient synthesis of the adducted nucleoside
phosphoramidites would be the prerequisite for the access
Figure 1 C8- and N2-adducts of chemical carcinogens and 2 -dG 1
to site-specifically C8-dG modified oligonucleotides. So
far, only post-synthetic oligonucleotide modification has
been reported in low yields.5 However, only singly modi-
fied oligonucleotides were accessible by this strategy, e.g.
nucleotide strands containing one C8-dG adduct of 2-ami-
nofluorene (AF).6
protected 8-Br-dG with arylamines was unsuccessful due
to depurination.10
Recently, C-N bond formation with palladium catalysts
(Buchwald–Hartwig reaction)11 was introduced by
Lakshman12 and Johnson13 for the synthesis of N6- and
N2-adducts of adenosine. While this work was in progress,
Rizzo published the coupling of a heterocyclic food mu-
tagen (IQ) and a few aromatic amines to the C8-position
of 2 -deoxyguanosine14 and Schoffers prepared C8-aryl-
amine adduct of tris-O-TBDMS-adenosine.15 However,
their approach needs the use of the strong base LiHMDS
and NaO-t-Bu and/or protecting group chemistry that is
not compatible with conditions of automated oligonucle-
otide synthesis. Moreover, no attempts have been made to
prepare the corresponding phosphoramidites and in order
to get suitable DNA-building blocks, both approaches
would need long reaction sequences.
In contrast, our interest is related to DNA-adducts of so-
called borderline carcinogens like toluidine and anisi-
dine.7 We report here on a highly efficient synthesis of the
corresponding C8-adducts using a palladium-catalyzed
cross-coupling reaction and the first synthesis of the cor-
responding 3 -phosphoramidites.
The synthesis of C8-arylamine-dG adducts by electro-
philic amination has been reported in low yields8,9 and
thus this approach was unsuitable for the phosphoramidite
synthesis. Moreover, direct nucleophilic substitution of
Synlett 2002, No. 5, 03 05 2002. Article Identifier:
1437-2096,E;2002,0,05,0802,0804,ftx,en;G03402ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214
Our key reaction step was also a Pd-catalyzed cross-coup-
ling reaction of a 8-bromo-dG derivative with the aroma-

LETTER
Synthesis of a Phosphoramidite Building Block
803
tic amine. From initial studies it became apparent that the
O6-position and the exocyclic 2-amino group of the gua-
nine moiety as well as the hydroxy groups of the 2 -deox-
yribose should be blocked during the cross-coupling
reaction. Although Buchwald and others reported the N-
arylation of amides,16 we decided to introduce the usually
used N2-isobutyryl (i-Bu) group. Thus, N2-i-butyryl-O6-
benzyl-8-bromo-3 ,5 -O-(t-butyldimethylsilyl)-2 -deoxy-
guanosine 2 was used as starting material. Moreover, we
introduced the 4-cyanophenylethyl protecting (CPE)
group to the O6-position17 instead of the benzyl (Bn) resi-
due. This group can be removed after the oligonucleotide
synthesis by DBU treatment and thus would avoid a fur-
ther deprotection step prior to the phosphoramidite syn-
thesis. Hence, also N2-i-butyryl-O6-(4-cyanophenyl)eth-
yl-8-bromo-3 ,5 -O-TBDMS-2 -deoxyguanosine 3 was
prepared as a second starting material. Protected dG-de-
rivatives 2 and 3 were synthesized starting from 2 -dG 1
by NBS-bromination to yield 8-bromo-dG in 78% yield.18
O-Silylation with TBDMS-chloride19 lead to 8-Br-3 ,5 -
O-TBDMS-dG (83% yield). O6-Protection with benzylal-
cohol (Bn) or 4-cyanophenylethanol (CPE)20 and DIAD/
PPh3 was achieved in 72% and 75% yield, respectively.
Finally, the exocyclic amino group was blocked by treat-
ment with i-butyrylchloride to give key intermediates 2
and 3 in 96% and 89% yield (Scheme 1). Fully protected
8-bromo-dG 2 was then subjected to the cross-coupling
reaction. In contrast to the work reported before,14 10
mol% Pd(dba)3 and 30 mol% rac-BINAP instead of 2-di-
cyclohexylphosphino-2 -(N,N-dimethylamino)biphenyl
was used as catalyst in 1,2-dimethoxyethane (1,2-
DME).21 Further, 1.5 equivalents of K3PO4 were added as
base.22 Two equivalents of aniline, toluidine, anisidine, 4-
cyanoaniline, 2-aminofluorene and 4-aminobiphenyl
were used. After heating the reaction mixtures to 80 °C
for 50 hours, silica gel chromatography gave the C8-aryl-
amine adducts 4–9 in yields between 73% and 80%
(Scheme 1; Table).23 Only the acceptor-substituted 4-cy-
anoaniline gave slightly lower yields (7, 66%). The yields
reported here are considerable higher than reported so
far.14,15 These high yields were obtained using the mono-
protected N2-i-Bu-dG derivative. In one experiment we
also studied the aminoarylation with N2-bis(i-Bu)-8-bro-
Scheme 1 Synthesis of the fully protected C8-arylamine adducts 4–
10. i) NBS, H2O, r.t., 15 min; ii) TBDMSCl, imidazole, pyridine, r.t.,
1 h; iii) PhCH2OH (2 equiv) or 4-CN-PhCH2CH2OH (3 equiv), PPh3,
DIAD, 1,4-dioxane, r.t., 1 h; iv) i-butyrylchloride, pyridine, r.t, 1 h; v)
Pd2(dba)3 (10 mol%), rac-BINAP (30 mol%), K3PO4, arylamine (2
equiv), 1,2-DME, 80 °C, 50 h.
(black) debenzylation (83% yield) gave the O6,3 ,5 -O-un-
blocked intermediate. This material was 5 -O-dimethoxy-
tritylated in 72% yield and further converted into the 5 -
O-DMTr-3 -O-phosphoramidite
11
(85%
yield,
Scheme 2).24 Neither during the introduction of the DMTr
group nor the phosphitylation reaction a side reaction at
the N8-atom took place. The same strategy has been fol-
lowed for the O6-CPE adduct 10: 3 ,5 -O-desilylation, 5 -
O-dimethoxytritylation and phosphitylation gave the tar-
get phosphoramidite 12.25
The overall yield of the amidites 11 and 12 were 16% and
21% for the 9- and 8-step synthetic procedure, respective-
ly.
In conclusion, we have accomplished a high yielding syn-
thetic procedure for the access to C8-arylamine-dG ad-
mo-dG. However, yields of the C8-toluidine adduct were ducts using palladium-catalyzed C-N bond formation.
considerably lower as described above and a mixture of The obtained yields are superior to the yields reported so
the bis(i-Bu) and the mono(i-Bu) adduct was isolated. Ob-
Table Yields of C8-Arylamine dG Adducts 4–10
viously, the conversion of the amino group into a guani-
din-type moiety is sufficient to prevent a side reaction of
Adduct 4–10 R1
Ar
Chem. yield [%]
the i-Bu-amide.16
4
5
Bn
Bn
Bn
Bn
Bn
Bn
CPE
Ph
77
75
76
66
73
80
81
The same reaction protocol has been applied to the second
8-bromo-dG derivative 3. Using toluidine, 81% of the dG-
adduct 10 was isolated after 43 hours without cleavage of
the O6-CPE-protecting group. It should be added that no
depurination side products have been observed both start-
ing from 2 or 3.
As a representative reaction, the O6-Bn protected tolui-
dine adduct 5 was first desilylated by tetrabutylammoni-
um fluoride (TBAF) in 97% yield and subsequent Pd
4-Me-Ph
4-MeO-Ph
4-CN-Ph
2-fluorenyl
4-biphenyl
4-Me-Ph
6
7
8
9
10
Synlett 2002, No. 5, 802–804 ISSN 0936-5214 © Thieme Stuttgart · New York

804
C. Meier, S. Gräsl
LETTER
(10) Riehl, H.; Meier, C. 2000, unpublished results.
(11) (a) Wolfe, J. P.; Wagaw, S.; Marcoux, J.-F.; Buchwald, S. L.
Acc. Chem. Res. 1998, 31, 805. (b) Hartwig, J. F. Acc.
Chem. Res. 1998, 31, 852.
(12) (a) Lakshman, M. K.; Hilmer, J. H.; Martin, J. Q.; Keeler, J.
C.; Dinh, Y. Q. V.; Ngassa, F. N.; Russon, L. M. J. Am.
Chem. Soc. 2001, 123, 7779. (b) N6 adducts have also been
synthesized by direct nucleophilic substitution: Véliz, E. A.;
Beal, P. A. J. Org. Chem. 2001, 66, 8592.
(13) (a) De Riccardis, F.; Bonala, R. R.; Johnson, F. J. Am. Chem.
Soc. 1999, 121, 10453. (b) De Riccardis, F.; Johnson, F.
Org. Lett. 2000, 2, 293.
(14) Wang, Z.; Rizzo, C. J. Org. Lett. 2001, 3, 565.
(15) Schoffers, E.; Olsen, P. D.; Means, J. C. Org. Lett. 2001, 3,
4221.
(16) Yin, J.; Buchwald, S. L. Org. Lett. 2000, 2, 1101; and
references cited.
(17) Uhlmann, E.; Pfleiderer, W. Helv. Chim. Acta 1981, 64,
1688.
(18) Gannett, P. M.; Sura, T. P. Synth. Commun. 1993, 23, 1611.
(19) Gao, X.; Jones, R. A. J. Am. Chem. Soc. 1987, 109, 1275.
(20) Harwood, E. A.; Sigurdsson, S. T.; Edfeldt, N. B.; Reid, B.
R.; Hopkins, P. B. J. Am. Chem. Soc. 1999, 121, 5081.
(21) Rac-BINAP is much less expensive than the biphenyl ligand.
(22) Old, D. W.; Wolfe, J. P.; Buchwald, S. L. J. Am. Chem. Soc.
1998, 120, 9722.
Scheme 2 Synthesis of the 5 -O-DMTr-3 -O-phosphoramidites 11,
12. i) n-Bu4NF (3 equiv), THF, r.t., 4 h; ii) Pd black, formamide,
1,4-cyclohexadiene, EtOAc/EtOH/MeOH, r.t., 6 h; iii) DMTrCl
(1.5 equiv), DMAP, pyridine, r.t., 12 h; iv) -cyanoethyl-di(i-
propyl)aminochlorophosphine (1.5 equiv); DIPEA, CH2Cl2, 0 °C,
30 min.
far. These adducts were converted into two potential
monomeric phosphoramidite building blocks in high
yields that should meet the conditions for automated
DNA-oligonucleotide synthesis. This opens the possibili-
ty to incorporate dG-adducts site-specifically into oligo-
nucleotides and/or DNA. Work along this route is current-
ly underway in our laboratories.
(23) Amination of N2-i-Butyryl-O6-benzyl-8-bromo-3 ,5 -bis(t-
butyldimethylsilyl)-2 -deoxyguanosine: 450.0 mg (0.61
mmol) 8-Bromo-2 -deoxyguanosine, 156.0 mg (0.73 mmol)
K3PO4, 56.1 mg (61.0 mol) tris(dibenzylideneacetone)di-
palladium(0) (Pd2dba3), 114.4 mg (0.18 mmol) racemic-
2,2 -bis(diphenylphosphino)-1,1 -binaphthyl (BINAP) and
131.2 mg (1.22 mmol) of p-toluidine was solubilized in
15 mL dry 1,2-DME in an inert atmosphere and stirred at
80 °C until the reaction was complete (TLC analysis). After
cooling to r.t., 1 mL of sat. sodium bicarbonate solution was
added. After addition of 10 mL of brine the layers were
separated and the aq layer was extracted three times with
10 mL of ethyl acetate. The combined organic layers were
washed twice with 10 mL of brine and once with a mixture
of 10 mL brine and 2 mL water. The organic layer was dried
(Na2SO4) and the solvent was removed in vacuo.
Purification by chromatography on silica gel, eluting with
20% ethyl acetate in hexane afforded 350 mg (75%) of the
desired product as a light-yellow foam.
Acknowledgement
This work was supported by Deutsche Forschungsgemeinschaft
(DFG), Fonds der Chemischen Industrie (FCI) and Bundesministe-
rium für Bildung und Forschung (BMBF)
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Synlett 2002, No. 5, 802–804 ISSN 0936-5214 © Thieme Stuttgart · New York