Synthesis and properties of oligonucleotides containing C8-deoxyguanosine arylamine adducts of borderline carcinogens

Article abstract of DOI:10.1021/jo061803t

C8-Arylamine-dG adducts of borderline carcinogens and the bladder and breast carcinogen 4-aminobiphenyl were prepared using cross-coupling chemistry. These adducts were converted into the corresponding C8-arylamine-5′-O- DMTr-2′-deoxyguanosine phosphorami

Full text of DOI:10.1021/jo061803t

Synthesis and Properties of Oligonucleotides Containing
C8-Deoxyguanosine Arylamine Adducts of Borderline Carcinogens†
Nicolas Bo¨ge, Sonja Gra¨sl, and Chris Meier*
Institute of Organic Chemistry, Department of Chemistry, Faculty of Mathematics, Informatics and Natural
Sciences, UniVersity of Hamburg, Martin-Luther-King-Platz 6, D-20149 Hamburg, Germany
chris.meier@chemie.uni-hamburg.de
ReceiVed August 31, 2006
C8-Arylamine-dG adducts of borderline carcinogens and the bladder and breast carcinogen 4-amino-
biphenyl were prepared using cross-coupling chemistry. These adducts were converted into the
corresponding C8-arylamine-5′-O-DMTr-2′-deoxyguanosine phosphoramidites and then used as building
blocks for automated synthesis of site-specifically modified oligonucleotides. The oligonucleotides were
characterized by UV melting temperature analysis, enzymatic digestion, and circular dichroism.
Introduction
Exposure to chemical carcinogens can occur from environ-
mental or industrial sources, diet, smoking, and endogenous
processes. Poly- and monocyclic aromatic amines, e.g., aniline
1, p-toluidine 2, p-anisidine 3, and 4-aminobiphenyl 4 (Figure
1), belong to the class of chemical carcinogens that are known
to form covalent adducts with DNA. If these covalently bonded
modifications are not repaired on the DNA level, they might
compromise the fidelity of DNA replication, leading to muta-
tions, and may be the reason for the induction of chemical
carcinogenesis.1-3
Arylamines like 1-4 require metabolic activation leading to
the ultimate carcinogen, which is an arylnitrenium ion 5 (Scheme
1). The initial step is an oxidation catalyzed by cytochrome P450
of the arylamine to the corresponding N-hydroxylamine 6.4 The
N-arylhydroxylamine is then enzymatically esterified into N-
acetoxyarylamine 7a by N-acetyl transferase (NAT) or to the
corresponding sulfate 7b by sulfotransferase (ST). Such com-
pounds are suspected to act as proximate carcinogens because
FIGURE 1. Potential carcinogenic aniline derivatives.
solvolysis generates a highly reactive arylnitrenium ion 5. The
predominant reaction of the arylnitrenium ion is the C8-position
of 2′-deoxyguanosine (dG) 8 leading to adduct 9 as the major
product. In addition, N2-hydrazino adducts of 2′-dG 10a have
been identified as minor products. Besides N-arylation, C-
arylation 10b was also detected in the case of strong carcinogens
such as 2-(acetyl)aminofluorene and the food carcinogen
2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In vivo studies
this type of adduct showed an even higher persistence as
compared to the C8 adduct, particularly in liver cells.5,6 Other
minor products are the C8- (11) and N6-adducts of 2′-
deoxyadenosine (Scheme 1).7,8
* To whom correspondence should be addressed. Phone: +49-40-42838-
4324. Fax: +49-40-42838-2495.
(5) Cui, X.-S.; Eriksson, L. C.; Mo¨ller, L. Mutat. Res. 1999, 442, 9.
(6) Turesky, R. J.; Box, R.; Markovic, J.; Gremaud, E.; Snyderwine, E.
G. Mutat. Res. 1997, 376, 235.
† Dedicated to Prof. Dr. Joachim Thiem on the occasion of his 65th birthday.
(1) Garner, R. C. Mutat. Res. 1998, 402, 67.
(2) Neumann, H. G. Cancer Res. Clin. Oncol. 1986, 112, 100.
(3) Beland, F. A.; Kadlubur, F. F. EnVir. Health Perspect. 1985, 62, 19.
(4) Guengerich, F. P. Drug Metab. ReV. 2002, 34, 607.
(7) Turesky, R. J.; Rossi, S. C.; Welti, D. H.; Lay, J. O.; Kadlubar, F. F.
Chem. Res. Toxicol. 1992, 5, 479.
(8) Tureky, R. J.; Markovic, J. Chem. Res. Toxicol. 1994, 7, 752.
10.1021/jo061803t CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/29/2006
9728
J. Org. Chem. 2006, 71, 9728-9738

Oligonucleotides Containing C8-Deoxyguanosine Arylamine Adducts
SCHEME 1. Metabolism of Arylamines and the Formed Different Adduct Typesa
a NAT: N-acetyl transferase. ST: sulfotransferase.
So far, the most extensively studied arylamine adducts are
derived from 2-aminofluorene (AF) and N-acetyl-2-aminofluo-
rene (AAF) that were originally used as pesticides.9 Zhou and
Romano reported the synthesis of C8-deoxyguanosine phos-
phoramidite reagents of 2-aminofluorene and its N-acetyl
analogue for the site-specific synthesis of oligonucleotide strands
containing these C8 adducts.10,11 However, they started from
an adduct formed in an electrophilic amination of dG, and the
yield was very poor. Rizzo reported the synthesis of oligo-
nucleotides containing C8 adducts of an heterocyclic amine,
the dietary mutagen IQ.12 However, their approach needs the
use of strong bases like LiHMDS or NaO-tert-Bu, and/or their
protecting group chemistry was not compatible with the
condition of the automated oligonucleotide synthesis. In 2002,
we published a short report on the first high-yielding protocol
for the complete synthesis of phosphoramidites of C8-aryl-
amine-dG adducts using Pd cross-coupling chemistry as the
key step.13 Scha¨rer and Gillet reported the synthesis of C8
adducts of the strong carcinogens 2-aminoflourene and its
acetylated adduct using Pd-catalyzed cross-coupling and its
incorporation into oligonucleotides.14,15 Further work was
reported by Takamura-Enya related to oligonucleotides damaged
by heterocyclic amines.16 Moreover, the same authors described
that adduct formation by Pd-cross-coupling is also possible
starting from protected 8-amino-dG with haloarenes.16
Our interest is related to DNA adducts of monocyclic
aromatic amines like anilines 1-3 that act as so-called borderline
carcinogens.17,18 In contrast to the strong carcinogen 4-amino-
biphenyl 4, these are often used, e.g., as pharmacophores or in
azo-dyes. However, nothing is known so far about the reason
why the monocyclic anilines are less mutagenic/carcinogenic
compared to their polycyclic counterparts like 2-aminofluorene
or 1-naphthylamine. Interestingly, the ultimate carcinogens of
borderline carcinogens formed the same type of adduct in the
same amount as strong carcinogens in biomimetic studies.17-20
In addition, no biochemical studies related to DNA-replication
fidelity or DNA-repair have been conducted in comparison to
the strong carcinogens. Thus, the molecular basis for their
different mutagenic potential remains unclear. Here, we report
on highly efficient syntheses of C8 adducts of borderline
carcinogens using palladium-catalyzed cross-coupling chemis-
tries, the synthesis of the 3′-phosphoramidites and their use in
DNA solid-phase synthesis to give site-specifically modified
oligonucleotides of mixed sequences containing several dG
nucleotides.
(9) Heflich, R. H.; Neft, R. E. Mutat. Res. 1994, 318, 73.
(10) Zhou, Y.; Romano, L. J. Biochemistry 1993, 32, 14043.
(11) Zhou, Y.; Chladek, S.; Romano, L. J. J. Org. Chem. 1994, 59, 556.
(12) Elmquist, C. E.; Stover, J. S.; Wang, Z.; Rizzo, C. J. J. Am. Chem.
Soc. 2004, 126, 11189.
(13) Meier, C.; Gra¨sl, S. Synlett 2002, 5, 802.
(14) Gillet, L. C.; Scha¨rer, O. D. Org. Lett. 2002, 4, 4205.
(15) Gillet, L. C.; Alzeer, J.; Scha¨rer, O. D. Nucl. Acids Res. 2005, 33,
1961.
(16) Takamura-Enya, T.; Ishikawa, S.; Mochizuki, M.; Wakabayashi,
K. Chem. Res. Toxicol. 2006, 19, 770. Takamura-Enya, T.; Ishikawa, S.;
Mochizuki, M.; Wakabayashi Tetrahedron Lett. 2003, 44, 5969.
(17) Meier, C.; Boche, G. Carcinogenesis 1991, 12, 1633.
(18) Meier, C.; Boche, G. Tetrahedron Lett. 1990, 31, 1693.
(19) Meier, C.; Boche, G. Tetrahedron Lett. 1990, 31, 1685.
(20) Famulok, M.; Boche, G. Angew. Chem. 1989, 101, 470; Angew.
Chem., Int. Ed. Engl. 1989, 28, 468. Skipper, P. L.; Trudel, L. J.; Kensler,
T. W.; Groopman, J. D.; Egner, P. A.; Liberman, R. G.; Wogan, G. N.;
Tannenbaum, S. R. Chem. Res. Toxicol. 2006, 19, 1086.
Results and Discussion
Synthesis of the C8-Deoxyguanosine Adducts of Mono-
cyclic Arylamines. The synthesis of C8-arylamine-dG adducts
by electrophilic amination has been reported in very low
yields.19,20 Attempts to optimize the electrophilic amination
J. Org. Chem, Vol. 71, No. 26, 2006 9729

Bo¨ge et al.
SCHEME 2. Synthesis of the Key Intermediates 12 and 13 and the Synthesis of the Adducts
TABLE 1. Yields of C8-Arylamine-dG Adducts 15a-d and 18a,b
reaction used in the biomimetic reaction failed (Meier et al.,
unpublished data), which makes this synthetic approach unsuit-
able for the synthesis of phosphoramidites of the dG adducts.
Moreover, a nucleophilic substitution reaction as reported for
protected 8-bromoguanosine was unsuccessful because extensive
depurination was observed when protected 8-bromo-2′-deoxy-
guanosine was treated with arylamines.21 Recently, C-N bond
formation using palladium catalysis (Buchwald-Hartwig reac-
tion)22 is a suitable method for synthesizing biologically
important nucleoside analogs23 and was introduced by Laksh-
man24 and Johnson25 for the synthesis of N6-aryl adducts of
adenosine and N2-aryl adducts of guanosine, and Schoffers
prepared C8-arylamine adducts of tris-O-TBDMS-(ribo)adenos-
ine.26 However, no suitable process for the corresponding
phosphoramidites was reported. While this work was in progress,
Rizzo published the synthesis of 2′-deoxyguanosine-phosphor-
amidites containing a heterocyclic food mutagen (IQ) in the
C8 position.27
compd
R
arylamine
chem yield (%)
15a
15b
15c
15d
18a
18b
i-Bu
i-Bu
i-Bu
i-Bu
H
aniline
p-toluidine
p-anisidine
4-aminobiphenyl
p-anisidine
77
75
76
80
62
65
H
4-aminobiphenyl
TBDMS-chloride yielded 8-Br-3′,5′-O-TBDMS-2′-dG in 83%.
The blocking of the O6-position was achieved by a Mitsunobu
reaction with benzyl alcohol (Bn), DIAD and PPh3 to give
intermediate 12 in 72% yield. Finally, the exocyclic amino group
was protected by treatment with isobutyryl chloride or phe-
noxyacetyl chloride/1-hydroxybenzotriazole to give the key
intermediates N2-isobutyryl-O6-benzyl-8-bromo-3′,5′-bis(tert-
butyldimethylsilyl)-2′-deoxyguanosine 13 and N2-phenoxy-
acetyl-O6-benzyl-8-bromo-3′,5′-bis(tert-butyldimethylsilyl)-2′-
deoxyguanosine 14 in 96% and 35% yield.
In our approach based also on a palladium cross-coupling as
the key step, it became apparent that the O6-position of guanine
as well as the hydroxyl groups of the 2′-deoxyribose should be
blocked during synthesis. Thus, the benzyl (Bn) group was
introduced to the O6-position, and O-silylation with TBDMS-
chloride was done at the hydroxyl groups. Because the exocyclic
amino group of guanine needs to be protected for the automated
oligonucleotide synthesis and to avoid homo coupling during
arylamination, standard N2-protecting groups like the isobutyryl
(i-Bu) and the more labile phenoxyacetyl group (PAc) were
studied. This fully protected dG-derivatives were synthesized
starting from bromination of 2′-dG 8 using NBS to yield
8-bromo-2′-dG in 78% (Scheme 2).28,14 O-Silylation using
The Buchwald-Hartwig reaction was carried out starting
from intermediates 13 and 14 using conditions that we have
previously published (Scheme 2).12,27 Efficient cross-coupling
was observed for intermediate 13 to give the C8-arylamine
adducts 15a-d in 75-80% yields (see Table 1).
To convert C8-arylamine adducts 15a-d into the correspond-
ing phosphoramidites, the O6-position was deblocked first
(Pd/H2; 99% yield), and then the silyl ethers were cleaved (tetra-
n-butylammonium fluoride; 96-99% yield) to give N2-i-Bu-
protected intermediates 16a-d (Scheme 3). Compounds 16 were
5′-O-dimethoxytritylated in 57-82% yield and further converted
into 5′-O-DMTr-3′-O-phosphoramidites 17a-d using 2-cyano-
ethyl-N,N,N′,N′-tetraisopropylphosphordiamidite and dicyano-
imidazole, which in our hands is a stronger activator than
tetrazole (58-73% yield; Scheme 3). Neither during the
introduction of the DMTr group nor the phosphitylation reaction
did a side reaction at the N8-atom take place.
(21) Riehl, H.; Meier, C. 2000, unpublished results.
(22) Wolfe, J. P.; Wagaw, S.; Marcoux, J.-F.; Buchwald, S. L. Acc. Chem.
Res. 1998, 31, 805. Hartwig, J. F. Acc. Chem. Res. 1998, 31, 852.
(23) Lakshman, M. K. Curr. Org. Synth. 2005, 2, 83.
The overall yields for the preparation of the phosphoramidites
17a-d were 15-19%. Thus, a convincingly efficient synthetic
procedure to 3′-phosphoramidites of C8-arylamine-dG adducts
using palladium-catalyzed C-N bond formation in the key step
has been described.
(24) 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.
(25) De Riccardis, F.; Johnson, F. Org. Lett. 2000, 2, 293.
(26) Schoffers, E.; Olsen, P. D.; Means, J. C. Org. Lett. 2001, 3, 4221.
(27) Wang, Z.; Rizzo, C. J. Org. Lett. 2001, 3, 565.
(28) Gannett, P. M.; Sura, T. P. Synth. Commun. 1993, 23, 1611-1615.
9730 J. Org. Chem., Vol. 71, No. 26, 2006

Oligonucleotides Containing C8-Deoxyguanosine Arylamine Adducts
SCHEME 3. Synthesis of the 5′-O-DMTr-3′-O-phosphoramidites 17a-d
In contrast, the same reaction conditions applied to N2-PAc
compound 14 did not result in the expected products. In fact,
when LiHMDS was used as base we found only decomposition
of the products; weaker bases like K3PO4 or Cs2CO3 gave the
desired cross-coupling products (65-75% yield), but also
considerable N2-deprotection. With NaOAc no reaction took
place. Thus, the PAc group is obviously to labile to resist the
reaction condition needed for efficient cross-coupling. However,
for oligonucleotide synthesis a more labile protecting group for
the N2-position compared to the i-Bu group is desirable. To
introduce such a protecting group we tried to achieve the cross-
coupling with N2-unprotected derivative 12 applying the above-
mentioned conditions. Surprisingly, comparable yields of the
C8-arylamine adducts 18a,b were obtained as in the couplings
starting from the N2-i-Bu intermediate 13 (Table 1). However,
a subsequent blocking of the exocyclic N2-amino group of
compounds 18a,b with the phenoxyacetyl group according to
the protocols of McLaughlin,29 Ogilvie,30 Schulhof,31 or Patil32
failed. Interestingly, also attempts to protect compound 18a as
its N2-i-Bu derivative failed. Possible alternatives could be
groups like formamidine or isopropyl-PAC, which has been used
by Scha¨rer and Rizzo.12,14,15
Site-Specific Synthesis of Oligonucleotides Containing a
C8-Deoxyguanosine Adduct of Different Arylamines. Phos-
phoramidites 17a-d were successfully used in automated
oligonucleotide synthesis applying a modified coupling protocol.
Two sequences were prepared: (i) a sequence of the mouse
c-H-ras proto-oncogene (19 and 20a-d) and (ii) a self-
complementary sequence (21 and 22a,b) shown in Figure 2.
The c-H-ras-sequence 19 was chosen because in earlier
studies Marques and Beland identified this sequence as a hotspot
for mutations of amines and amides (Figure 3).33 The same
approach has already been successfully used for the synthesis
of various site-specifically modified 34-mer oligonucleotides
bearing multiple dG in the sequence. These oligonucleotides
were used as templates for primer extension assays with DNA-
polymerases (data not shown).34
60% (based on the commonly used trityl assay). It has been
observed, that C8-arylamine adducts undergo an oxidative
rearrangement in the presence of strong bases and air ana-
logous to that observed for 8-oxo-2′-deoxyguanosine.35,36 This
reaction that occurs during the deprotection of the oligonu-
cleotides can be avoided by addition of mercaptoethanol to
degassed ammonium hydroxide. The deprotection was ac-
complished at 55 °C overnight followed by purification by
reversed-phase HPLC. The oligonucleotides were character-
ized by LC-MS or MALDI-TOF mass spectrometry and, in
the case of the self-complementary oligonucleotides, by enzy-
matic digestion (see the Supporting Information). The enzy-
matic digestion was necessary because we observed a fragmen-
tation in the MALDI-TOF mass spectrometry of the self-
complementary oligonucleotides. HPLC and LC-MS mass
spectrometry indicate that the purity of all oligonucleotides was
at least 95%.
Melting Temperature (Tm) and Circular Dichroism Stud-
ies. All oligonucleotides were hybridized with the complemen-
tary strands and the effect of the incorporated C8-arylamine
adduct on the thermal stability of the DNA duplex was measured
by UV melting curve analysis (Tm). The results of these
measurements are summarized in Table 2. In general, modifica-
tion of DNA destabilizes the duplex because fewer hydrogen
bonds cause a lowering in the Tm value. Indeed, in the case of
the c-H-ras-sequences 20a-d a decrease in the Tm of the
modified oligonucleotides of 5-7 °C compared to the unmodi-
fied oligonucleotide 19 was observed. However, no signifi-
cant difference was observed in the destabilization effect of
the oligonucleotides modified with borderline carcinogens
(20a-c) and the oligonucleotide modified with the strong
carcinogen 4-aminobiphenyl (20d). The Tm values of all
modified oligonucleotides were found within a range of 1.5 °C
which is within the experimental error.
Comparable observations were made with the self-comple-
mentary sequence 21. However, the difference of the Tm value
of unmodified oligonucleotide 21 and modified oligonucleotides
22a (anisidine-modified) and 22b (4-aminobiphenyl-modified)
is higher (∆ ) 14 °C) due to modification in each strand. Again,
there is no difference in Tm between the p-anisidine- and the
4-aminobiphenyl-modified oligonucleotide.
For the incorporation of these adducts, 50 mg of phosphor-
amidites 17a-d were dissolved in acetonitrile (0.1 M solution).
The coupling step of the adduct phosphoramidite was doubled
compared to regular building blocks with an efficiency of about
FIGURE 2. Sequences for C8-arylamine-adducted oligonucleotides.
J. Org. Chem, Vol. 71, No. 26, 2006 9731

Bo¨ge et al.
FIGURE 3. CD spectra of the unmodified c-H-ras oligonucleotide 19 (s) in comparison to the C8-p-anisidine-modified oligonucleotide 20c
(panel A) and the C8-4-aminobiphenyl-modified oligonucleotide 20d (panel B).
TABLE 2. Tm Studies of Adducted Oligonucleotides (van’t
Hoff’sche Calculation for ∆H and ∆S)
TABLE 3. Tm Studies of Adducted Oligonucleotides 25 and 26
Tm value (°C) against
dA5XdA5 24a-d
oligonucleotide
Tm (°C)
∆H (kcal/mol)
∆S (cal/mol/K)
19
51.6
46.3
46.4
46.0
45.0
33.0
19.1/69.0
20.2/65.6
-82.0
-280.8
oligonucleotide
X ) C
X ) G
X ) T
X ) A
20a
20b
20c
20d
21
-163.9
-541.4
TTTTT(dG)TTTTT 23
TTTTT(Tol-dG) TTTTT 25
TTTTT(ABP-dG) TTTTT 26
40
35
33
28
27
26
30
28
26
27
26
26
-144.4
-480.8
-161.5
-534.5
-175.7
-580.2
-245.5
-773.8
22a
22b
-323.1/-186.3
-288.2/-121.7
-1075/-523.7
-855.3/-384.4
oligonucleotides 22a,b are comparable to this observation
because the second Tm is 50 °C higher for oligonucleotide 22a
and 46 °C for oligonucleotide 22b. In both cases, the lower
melting transition is sensitive to oligo concentration while the
higher was not, which is a strong indication for the proposed
hairpin formation. The unmodified oligonucleotide 21 did not
show a second Tm within the temperature range of 10-85 °C.
Two explanations are possible: (i) the second Tm is higher than
85 °C or (ii) there is no hairpin structure for this oligonucleotide.
If the latter is true, the arylamine modification would stabilize
the hairpin formation.
Interestingly, in the cases of the two modified self-
complementary oligonucleotides 22a,b a second Tm was de-
tected. This may be due to a second stable solution conformation
in addition to the DNA duplex, most probably a hairpin
structure. Kukreti, for example, reported a palindromic sequence
(12mer) for which a second Tm value of the hairpin was found
to be 45 °C above the Tm of the duplex.37 Our results with
(29) Benseler, F.; McLaughlin, L.W. Synth. Commun. 1986, 45.
(30) Wu, T.; Ogilvie, K. K. Tetrahedron Lett. 1988, 29, 4249.
(31) Teoule, R.; Molko, D.; Schulhof, J. C. Nucl. Acids Res. 1987, 15,
397.
(35) Johnson, F.; Huang, C. Y.; Yu, P. L. EnViron. Health Perspect.
1994, 102, 143.
(32) Wadia, M. S.; Patil, D. V. Synth. Commun. 2003, 33, 2725.
(33) Marques, M. M.; Beland, F. A. Chem. Res. Toxicol. 1990, 3, 559.
(34) Meier, C.; Gra¨sl, S.; Detmer, I.; Marx, A Nucl. Nucl. Nucleic Acids
2005, 24, 691.
(36) Shibutani, S.; Gentles, R.; Johnson, F.; Grollmann, A. P. Carcino-
genesis 1991, 12, 813.
(37) Kaushik, M.; Kukreti, R.; Grover, D.; Brahmachari, S.; Kukreti, S.
Nucl. Acids Res. 2003, 31, 6904.
9732 J. Org. Chem., Vol. 71, No. 26, 2006

Oligonucleotides Containing C8-Deoxyguanosine Arylamine Adducts
FIGURE 4. CD spectra of the unmodified self-complementary sequence 21 (s) and the C8-p-anisidine-modified oligonucleotide 22a.
Recently, we have been incorporated dG adducts in the middle
of a homo(T)14 sequence and hybridization experiments were
done.34 For comparison, the unmodified (T)7(dG)(T)7 23 was
hybridized to (dA)7(X)(dA)7 24a-d (X ) dC, T, dA, dG). In
the full-matched situation the Tm value was found to be 39 °C
(see Table 3). A mismatch within the hybrid caused a decrease
of the Tm value of about 10 °C (Tm ∼28.5 °C). Incorporation
of a adducted dG* (modified with p-toluidine 25 and 4-ami-
nobiphenyl 26) into the homo(T) sequence led to a reduction
of 5 °C in the case of the matched (dA)7(dC)(dA)7-strand
independently of the arylamine modification. Hybridization of
the mismatched (dA)7(X)(dA)7-strand with the modified strand
led to the same thermal stability as the mismatched duplex (Tm
∼27.5 °C).34 Thus, obviously the incorporated adducted-dG’s
have a significant less reduction of the thermal stability of the
duplex as compared to a mismatch in the sequence. Further
studies will be conducted to investigate the structure of the C8-
arylamine adducted DNA strands.
In summary, the conclusion that can be taken from the Tm
measurements is that there is no major conformational difference
between the oligonucleotides containing a C8-modification of
a borderline or a strong carcinogen, which has already been
postulated by Cho et al. for the C8-aminobiphenyl adduct from
NMR studies.38 To verify this conclusion, the circular dichroism
spectra of the oligonucleotides were recorded. CD spectra of
modified oligonucleotides 20c and 20d and the corresponding
unmodified oligonucleotide 19 are shown in Figure 3. All c-H-
ras-sequence oligonucleotide duplexes 19 showed the same
spectral characteristics, a maximum at 280-290 nm and a
minimum at 240-250 nm with no change in intensity. Both
indicate that all oligonucleotides of the c-H-ras-sequence adopt
a B-type-DNA-conformation. The shifting of the maxima of
the modified oligonucleotides 20c,d to higher wavelength (5
and 8 nm, respectively) is caused by the (partly) conjugated
aromatic systems to the guanine heterocycle, which can also
be observed in the UV spectra of the adducted oligonucleo-
tides as well as on the nucleoside level (data not shown). In
this context it is reasonable that the strongest shift was observed
in the case of C8-(4-aminobiphenyl)-modified oligonucleotide
20d.
Analogous properties were studied using the self-comple-
mentary sequence. The CD spectra of the unmodified 21 and
the modified oligonucleotide 22a are shown in Figure 4. Again,
there is no conformational difference observable between the
modified and the unmodified oligonucleotide. Comparing these
spectra with those of the c-H-ras-sequence two remarkable
changes can be seen. First, the self-complementary sequence
did not exist in only one conformation. In addition to the B-type-
DNA-conformation (minimum at 240 nm) there is a second
minimum at 280 nm (from the Z-DNA-form). This goes along
with our interpretation of the Tm-measurements that a hairpin
formation is responsible for the second Tm value.
In fact, in all structural studies performed so far point to very
similar conformations of oligonucleotides that are modified by
borderline carcinogens 1-3 or by the strong carcinogen
4-aminobiphenyl 4. However, further studies are needed to
confirm this.
Recently, we ran primer-template extension studies using
standing start and standing start +1 conditions. In these studies,
it became apparent that DNA pol â showed a considerable loss
in fidelity and that replication errors did not take place opposite
to the adduct, but one step downstream. But again no clues were
found, that could explain the different carcinogenic potential
of a borderline carcinogen and a strong carcinogen.34
To add more data to the reported results and to gain more
insights into the reason for the different carcinogenic potential
C8-modified phosphoramidites will be incorporated in further
different DNA sequences. Also, including strong, polycyclic
carcinogens as 2-aminofluorene in these studies would be
interesting for comparison. Structural analysis of modified
oligonucleotides like 21 and 22a,b by means of NMR and
further biochemical studies, e.g., repair studies, may give hints
to explain the difference in carcinogenic potential.
Summary
In summary, we have reported on an efficient strategy for
the synthesis of C8-arylamine adducts of 2′-deoxyguanosine
involving the Buchwald-Hartwig catalyzed cross-coupling
starting from protected 8-bromo-2′-deoxyguanosine derivative
13. The C8-arylamine adducts were site-specifically incorporated
into oligonucleotides using N2-i-Bu-protected phosphoramidites
in solid-phase synthesis. We have examined the properties of
(38) Cho, B. P.; Beland, F. A.; Marques, M. M. Biochemistry 1992, 31,
9587.
J. Org. Chem, Vol. 71, No. 26, 2006 9733

Bo¨ge et al.
Hz, 1H), 3.42 (dd, J ) 9.9, 6.0 Hz, 1H), 3.25 (ddd, J ) 13.4, 6.8,
6.2 Hz, 1H), 2.01 (ddd, J ) 13.4, 6.8, 3.0 Hz, 1H), 0.86 (s, 9H),
0.81 (s, 9H), 0.07 (s, 6H), -0.03 (s, 3H), -0.04 (s, 3H). 13C NMR
(DMSO-d6, 101 MHz): δ (ppm) 171.4, 165.2, 159.6, 159.1, 155.0,
138.3, 129.6, 128.6, 128.4, 128.3, 121.1, 114.6, 87.0, 83.4, 81.2,
72.9, 72.8, 67.8, 35.1, 25.9, 25.8, 18.0, 17.8, -4.5, -4.7, -5.3,
-5.3. FABMS: calcd (M) 797.4308, obsd (M + H)+ 798.5021.
General Procedure (I) for the Amination of O6-Benzyl-8-
bromo-3′,5′-bis(tert-butyldimethylsilyl)-2′-deoxyguanosine De-
rivatives. A dried flask was purged with nitrogen and charged with
bromide (12, 13 or 14), 1.5 equiv of K3PO4, 10 mol % of tris-
(dibenzylideneacetone)dipalladium(0) (Pd2dba3), 30 mol % of
racemic 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP), and
2 equiv of amine. Dry 1,2-DME (15 mL) was added, and the
reaction vessel was stirred at 80 °C until the reaction was complete
(TLC analysis). The reaction mixture was allowed to cool to room
temperature, and then 1 mL of saturated sodium bicarbonate solution
was added. After addition of 10 mL of brine, the layers were
separated and the aqueous 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 of
brine and 2 mL of water. Then the organic layer was dried over
sodium sulfate, and the solvent was removed in vacuo. Purification
by chromatography on silica gel, eluting with 10-30% ethyl acetate
in hexanes, gave the desired product.
two sequences containing different arylamines by combination
of UV melting studies and CD spectroscopy. Such C8-arylamine
adducts formed by borderline carcinogens and strong carcino-
gens play an important role in their mutagenicity due to
alteration of the structure of the DNA double helix. However,
no important differences have been observed so far in the
structure of DNA-oligonucleotides modified by borderline or
strong carcinogens. Further biochemical studies with the modi-
fied oligonucleotides with regard to replication or repair are
currently underway in our laboratories.
Experimental Section
O6-Benzyl-8-bromo-3′,5′-bis(tert-butyldimethylsilyl)-2′-deoxy-
guanosine (12). 8-Br-dG was prepared according to refs 28 and
34. The spectral data were in accordance with those reported in ref
14. Compound 12 was prepared from 8-Br-dG according to refs
14 and 34. The spectral data were in accordance with those reported
in ref 14.
N2-Isoutyryl-O6-benzyl-8-bromo-3′,5′-bis(tert-butyldimethyl-
silyl)-2′-deoxyguanosine (13). In a flask, 1.15 g (1.73 mmol) of
O6-Bn-8-Br-3′,5′-TBDMS-2′-dG (12) was twice coevaporated with
4 mL of pyridine and then dissolved in 10 mL of dry pyridine.
Then, 0.37 mL of isobutyryl chloride was added slowly via syringe,
and the solution was stirred for 1 h at room temperature. After
addition of 1 mL of methanol and stirring for 5 min, the solvent
was removed in vacuo followed by coevaporation with toluene.
The residue was redissolved in 20 mL of water and 20 mL of
CH2Cl2, and the layers were separated. The aqueous layer was
extracted twice with 20 mL of CH2Cl2, and the combined organic
layers were washed with 20 mL of water and then dried over sodium
sulfate. The solvent was removed in vacuo, and purification of the
residue by chromatography on silica gel (5% ethyl acetate/hexanes)
gave 1.22 g (96%) of (13) as a colorless foam.1H NMR (DMSO-
d6, 400 MHz): δ (ppm) 10.41 (s, 1H), 7.53-7.33 (m, 5H), 6.26
(dd, J ) 7.4, 6.1 Hz, 1H), 5.60 (d, J ) 12.1 Hz, 1H), 5.57 (d, J )
12.1 Hz, 1H), 4.99-4.95 (m, 1H), 3.84-3.77 (m, 2H), 3.69-3.62
(m, 1H), 3.51 (ddd, J ) 13.1, 6.1, 6.1 Hz, 1H), 2.80 (sept, J ) 6.8
Hz, 1H), 2.26 (ddd, J ) 13.1, 7.4, 4.8 Hz, 1H), 1.10 (d, J ) 6.8
Hz, 3H), 1.09 (d, J ) 6.8 Hz, 3H), 0.88 (s, 9H), 0.75 (s, 9H), 0.12,
(d, J ) 8.5 Hz, 6H), -0.11 (d, J ) 9.4 Hz, 6H). 13C NMR (DMSO-
d6, 101 MHz): δ (ppm) 174.5, 158.8, 153.2, 151.8, 136.1, 129.2,
128.7, 128.6, 128.5, 118.1, 88.1, 85.9, 72.2, 67.9, 63.4, 36.8, 34.8,
25.9, 19.5, 19.4, 18.1, 17.8, -4.7, -4.8, -5.3, -5.4. HRFABMS:
calcd (M + H)+ 734.2769, obsd 734.2794.
N2-Isobutyryl-O6-benzyl-8N-phenylamino-3′,5′-bis(tert-bu-
tyldimethylsilyl)-2′-deoxyguanosine (15a). The general procedure
(I) was conducted with 350 mg (0.48 mmol) of bromide 13 (reaction
time 49 h) and afforded 273 mg (77%) of a slightly pink foam.
1H NMR (DMSO-d6, 400 MHz): δ (ppm) 10.13 (s, 1H), 9.14
(s, 1H), 7.69-6.93 (m, 10H), 6.41 (dd, J ) 7.3, 6.3 Hz, 1H), 5.60
(d, J ) 12.2 Hz, 1H), 5.56 (d, J ) 12.2 Hz, 1H), 4.94 (ddd, J )
4.3, 6.6, 4.3 Hz, 1H), 3.86 (dd, J ) 10.6, 4.7 Hz, 1H), 3.80 (ddd,
J ) 4.7, 4.7, 6.0 Hz, 1H), 3.71 (dd, J ) 10.6, 6.0 Hz, 1H), 3.50
(ddd, J ) 13.1, 6.3, 6.3 Hz, 1H), 2.81 (sept, J ) 6.8 Hz, 1H), 2.17
(ddd, J ) 13.1, 7.3, 4.3 Hz, 1H), 1.10 (d, J ) 6.8 Hz, 3H), 1.09 (d,
J ) 6.8 Hz, 3H), 0.89 (s, 9H), 0.77 (s, 9H), 0.13 (d, J ) 8.1 Hz,
6H), -0.09 (d, J ) 3.2 Hz, 6H). 13C NMR (DMSO-d6, 101 MHz):
δ (ppm) 174.4, 156.8, 152.7, 149.4, 148.7, 140.4, 136.8, 128.8,
128.7, 128.6, 128.3, 121.8, 118.6, 115.7, 88.0, 83.0, 72.5, 67.3,
63.7, 37.3, 34.5, 25.9, 19.6, 19.4, 18.1, 17.8, -4.6, -4.7, -5.3,
-5.4. HRFABMS: calcd (M + H)+ 747.4086, obsd 747.4066.
N2-Isobutyryl-O6-benzyl-8N-(4-methylphenylamino)-3′,5′-bis-
(tert-butyldimethylsilyl)-2′-deoxyguanosine (15b). The general
procedure (I) was conducted with 450 mg (0.61 mmol) of bromide
13 (reaction time 50 h) and afforded 350 mg (75%) of a light yellow
1
N2-Phenoxyacetyl-O6-benzyl-8-bromo-3′,5′-bis(tert-butyldi-
methylsilyl)-2′-deoxyguanosine (14). To 69 mg (0.51 mmol) of
1-hydroxybenzotriazole, suspended in 2 mL of dry CH3CN, was
added 188 mg (0.48 mmol) of phenoxyacetyl chloride. As soon as
the 1-hydroxybenzotriazole had dissolved, dry pyridine (3 mL) was
added to dissolve any precipitated salt. This slightly red solution
was added dropwise to 200 mg (0.3 mmol) of O6-Bn-8-Br-3′,5′-
TBDMS-2′-dG (12) at 0 °C, which was dissolved in 2 mL of dry
pyridine. The mixture was then stirred overnight at ambient
temperature. After TLC analysis indicated complete reaction, the
mixture was cooled again to 0 °C and the reaction stopped by
addition of 1 mL of water. After 5 min of stirring at 0 °C, 4 mL of
saturated sodium bicarbonate solution was added and stirred for
another 15 min. After separation of the layers, the aqueous layer
was extracted three times with ethyl acetate. The combined organic
layers were dried over sodium sulfate, and the solvent was removed
in vacuo. Purification of the residue by chromatography on silica
gel (10-20% ethyl acetate/hexanes) gave 447 mg (35%) of (14)
as a yellow solid. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 10.48
(s, 1H), 7.52-7,50 (m, 2H), 7.38-7.33 (m, 3H), 7.28-7.26 (m,
2H), 6.94-6.90 (m, 3H), 6.10 (dd, J ) 7.1, 7.1 Hz, 1H), 5.53 (s,
2H), 4.89 (d, J ) 16.0 Hz, 2H), 4.62 (ddd, J ) 2.8, 5.6, 3.1 Hz,
1H), 3.74 (ddd, J ) 5.6, 6.0, 3.1 Hz, 1H), 3.58 (dd, J ) 9.9, 3.1
foam. H NMR (DMSO-d6, 400 MHz): δ 10.13 (s, 1H), 9.05 (s,
1H), 7.58-7.49 (m, 4H), 7.40-7.31 (m, 3H), 7.12-7.08 (m, 2H),
6.39 (dd, J ) 7.3, 6.2 Hz, 1H), 5.59 (d, J ) 12.1 Hz, 1H), 5.55 (d,
J ) 12.1 Hz, 1H), 4.93 (ddd, J ) 4.3, 6.6, 4.0 Hz, 1H), 3.86 (dd,
J ) 10.6, 5.1 Hz, 1H), 3.79 (ddd, J ) 5.1, 4.0, 5.8 Hz, 1H), 3.70
(dd, J ) 10.6, 5.8 Hz, 1H), 3.50 (ddd, J ) 13.1, 6.2, 6.6 Hz, 1H),
2.80 (sept, J ) 6.8 Hz, 1H), 2.23 (s, 3H), 2.15 (ddd, J ) 13.1, 7.3,
4.3 Hz, 1H), 1.09 (d, J ) 6.8 Hz, 3H), 1.09 (d, J ) 6.8 Hz, 3H),
0.89 (s, 9H), 0.77 (s, 9H), 0.13 (d, J ) 8.1 Hz, 6H), -0.09 (d, J )
3.1 Hz, 6H). 13C NMR (DMSO-d6, 101 MHz): δ (ppm) 174.3,
156.5, 152.6, 149.1, 148.8, 137.6, 136.7, 130.5, 129.0, 128.5, 128.4,
128.1, 118.7, 115.6, 87.7, 82.8, 72.3, 67.1, 63.5, 37.0, 34.4, 25.7,
20.4, 19.4, 19.3, 18.0, 17.7, -4.8, -4.9, -5.5, -5.5. HRFABMS:
calcd (M + H)+ 761.4242, obsd 761.4216.
N2-Isobutyryl-O6-benzyl-8N-(4-methoxyphenylamino)-3′,5′-
bis(tert-butyldimethylsilyl)-2′-deoxyguanosine (15c). The general
procedure (I) was conducted with 250 mg (0.34 mmol) of bromide
13 (reaction time 51 h) and afforded 200 mg (76%) of a slightly
violet foam: 1H NMR (DMSO-d6, 500 MHz): δ (ppm) 10.20 (s,
1H), 9.06 (s, 1H), 7.69-6.97 (m, 9H), 6.48 (dd, J ) 6.6, 7.3 Hz,
1H), 5.67 (d, J ) 12.1 Hz, 1H), 5.63 (d, J ) 12.1 Hz, 1H), 5.02
(ddd, J ) 6.6, 4.2, 4.2 Hz, 1H), 3.96 (dd, J ) 5.2, 11.0 Hz, 1H),
3.89 (ddd, J ) 5.2, 4.2, 5.9 Hz, 1H), 3.80 (dd, J ) 5.9, 11.0 Hz,
9734 J. Org. Chem., Vol. 71, No. 26, 2006

Oligonucleotides Containing C8-Deoxyguanosine Arylamine Adducts
1H), 3.61 (ddd, J ) 13.0, 6.6, 6.6 Hz, 1H), 2.90 (sept, J ) 6.7 Hz,
1H), 2.25 (ddd, J ) 13.0, 4.2, 7.3 Hz, 1H), 1.20 (d, J ) 6.7 Hz,
3H), 1.19 (d, J ) 6.7 Hz, 3H), 1.00 (s, 9H), 0.87 (s, 9H), 0.23 (d,
J ) 7.6 Hz, 6H), 0.01 (d, J ) 2.7 Hz, 6H). 13C NMR (DMSO-d6,
101 MHz): δ (ppm) 174.4, 156.5, 154.7, 152.9, 149.5, 149.1, 136.9,
133.4, 128.7, 128.6, 128.2, 120.8, 115.8, 114.1, 87.9, 82.9, 72.6,
67.3, 63.7, 55.3, 37.2, 34.5, 25.9, 19.6, 19.4, 18.1, 17.9, -4.6, -4.7,
-5.3, -5.4. HRFABMS: calcd (M + H)+ 777.4191, obsd
777.4204.
butyryl-8N-arylamino-3′,5′-bis(tert-butyldimethylsilyl)-2′-deoxy-
guanosine derivative in THF was treated with 2.5 equiv of a 1.0 M
solution of tetrabutylammonium fluoride in THF at room temper-
ature. The reaction was stirred until the reaction was complete (TLC
analysis), concentrated in vacuo, and purified by chromatography
on silica gel with 20% CH3OH in CH2Cl2.
N2-Isobutyryl-8N-phenylamino-2′-deoxyguanosine (16a). The
general procedure (II) was conducted with 800 mg (1.07 mmol) of
compound 15a and 50 mg of Pd/C in 20 mL of dry CH3OH and
afforded 696 mg (99%) of a colorless solid. 1H NMR (DMSO-d6,
400 MHz): δ (ppm) 11.99 (s, 1H), 11.21 (s, 1H), 8.66 (s, 1H),
7.57-7.54 (m, 2H), 7.29-7.24 (m, 2H), 6.95-6.91 (m, 1H), 6.32
(dd, J ) 8.7, 6.0 Hz, 1H), 4.52-4.48 (m, 1H), 3.88-3.83 (m, 2H),
3.68 (dd, J ) 13.5, 7.9 Hz, 1H), 3.40 (ddd, J ) 13.2, 8.7, 5.9 Hz,
1H), 2.75 (sept, J ) 6.8 Hz, 1H), 2.13 (ddd, J ) 13.2, 6.0, 1.8 Hz,
1H), 1.12 (d, J ) 6.8 Hz, 3H), 0.89 (s, 9H), 0.81 (s, 9H), 0.11 (s,
6H), -0.01 (d, J ) 4.6 Hz, 6H). 13C NMR (DMSO-d6, 101 MHz):
δ (ppm) 179.5, 153.7, 147.3, 146.1, 145.8, 140.8, 128.6, 121.2,
117.8, 117.8, 87.6, 83.8, 73.2, 63.1, 36.5, 34.7, 25.7, 19.0, 18.8,
18.0, 17.8, -4.7, -4.8, -5.4, -5.5. HRFABMS: calcd (M + H)+
657.3616, obsd 657.3577.
O6-Benzyl-8N-(4-methoxyphenylamino)-3′,5′-bis(tert-butyldi-
methylsilyl)-2′-deoxyguanosine (18a). The general procedure (I)
was conducted with 3.02 g (4.54 mmol) of bromide 12 (reaction
1
time 70 h) and afforded 2.00 g (62%) of a yellow foam. H NMR
(DMSO-d6, 500 MHz): δ (ppm) 8.47 (s, 1H), 7.51 (d, J ) 9.0 Hz,
2H), 7.47 (d, J ) 7.2 Hz, 2H), 7.38 (dd, J ) 7.2 Hz, 3H), 6.88 (d,
J ) 9.0 Hz, 2H), 6.29 (dd, J ) 6.9 Hz, 1H), 5.97 (s, 2H), 5.46 (s,
2H), 4.63 (ddd, J ) 6.2, 3.1, 3.1 Hz, 1H), 3.84 (m, J ) 13.2, 9.6,
5.5 Hz, 2 H), 3.70 (s, 3 H), 3.68 (dd, J ) 13.2, J ) 5.5 Hz, 1H),
3.43 (ddd, J ) 13.3, 6.7 Hz, 1H), 2.10 (ddd, J ) 13.3, 6.7, 3.2 Hz,
1H), 0.90 (s, 9H), 0.81 (s, 9H), 0.12 (s, 6H), -0.02 (s, 3H), -0.03
(s, 3H). 13C NMR (DMSO-d6, 101 MHz): δ (ppm) 157.6, 157.4,
147.2, 139.1, 137.3, 133.9, 128.6, 128.5, 128.1, 120.0, 114.0, 87.3,
83.0, 72.9, 66.6, 63.2, 55.3, 36.6, 25.9, 18.0, -5.2. FABMS: calcd
(M) 706.3704, obsd (M + H)+ 707.3211.
The general procedure (III) was conducted with 330 mg (0.50
mmol) of N2-isobutyryl-8N-phenylamino-3′,5′-bis(tert-butyldi-
methylsilyl)-2′-deoxyguanosine in 6 mL of THF (reaction time 2
N2-Isobutyryl-O6-benzyl-8N-(4-biphenylamino)-3′,5′-bis(tert-
butyldimethylsilyl)-2′-deoxyguanosine (15d). The general proce-
dure (I) was conducted with 300 mg (0.41 mmol) of bromide 13
(reaction time 51 h) and afforded 269 mg (80%) of a light-yellow
h). Purification afforded 212 mg (99%) of a colorless solid. H
1
NMR (DMSO-d6, 400 MHz): δ (ppm) 11.98 (s, 1H), 11.56 (s,
1H), 8.87 (s, 1H), 7.78-7.76 (m, 2H), 7.30-7.26 (m, 2H), 6.96-
6.92 (m, 1H), 6.42 (dd, J ) 9.9, 5.6 Hz, 1H), 5.99, (s, 1H), 5.35
(d, J ) 3.2 Hz, 1H), 4.44-4.43 (m, 1H), 3.97 (ddd, J ) 2.0, 2.0,
2.0 Hz, 1H), 3.77, (s, 2H), 2.73 (sept, J ) 6.9 Hz, 1H), 2.54 (ddd,
J ) 12.9, 9.9, 5.9 Hz, 1H), 2.04 (ddd, J ) 12.9, 5.6, 1.3 Hz, 1H),
1.11 (d, J ) 6.9 Hz, 6H). 13C NMR (DMSO-d6, 101 MHz): δ
(ppm) 179.8, 153.7, 146.9, 146.6, 145.2, 140.2, 128.6, 121.2, 117.9,
116.3, 87.4, 83.2, 71.3, 61.3, 38.4, 34.8, 18.9. HRFABMS: calcd
(M + H)+ 429.1886, obsd 429.1934.
1
foam. H NMR (DMSO-d6, 400 MHz): δ (ppm) 10.15 (s, 1H),
9.30 (s, 1H), 7.81-7.27 (m, 14H), 6.44 (dd, J ) 6.2, 7.4 Hz, 1H),
5.61 (d, J ) 12.1 Hz, 1H), 5.57 (d, J ) 12.1 Hz, 1H), 4.96 (ddd,
J ) 6.7, 4.3, 4.3 Hz, 1H), 3.87 (dd, J ) 5.0, 10.6 Hz, 1H), 3.81
(ddd, J ) 5.0, 4.3, 6.0 Hz, 1H), 3.72 (dd, J ) 6.0, 10.6 Hz, 1H),
3.53 (ddd, J ) 13.0, 6.2, 6.7 Hz, 1H), 2.81 (sept, J ) 6.7 Hz, 1H),
2.19 (ddd, J ) 13.0, 4.3, 7.4 Hz, 1H), 1.11 (d, J ) 6.7 Hz, 3H),
1.10 (d, J ) 6.7 Hz, 3H), 0.90 (s, 9H), 0.77 (s, 9H), 0.14 (d, J )
8.0 Hz, 6H), -0.09 (d, J ) 3.3 Hz, 6H). 13C NMR (DMSO-d6,
101 MHz): δ (ppm) 156.9, 152.7, 149.5, 148.6, 140.0, 139.9, 136.8,
133.4, 129.0, 128.7, 128.6, 128.3, 127.0, 126.9, 126.2, 118.9, 115.7,
88.0, 83.0, 72.5, 67.3, 63.7, 37.2, 34.6, 25.9, 19.6, 19.4, 18.1, 17.9,
-4.6, -4.7, -5.3, -5.4. HRFABMS: calcd (M + H)+ 823.4399,
obsd 823.4482.
N2-Isobutyryl-8N-(4-methylphenylamino)-2′-deoxyguano-
sine (16b). The general procedure (II) was conducted with 500 mg
(0.66 mmol) of compound 15b and 50 mg of Pd/C in 15 mL of
1
dry CH3OH and afforded 437 mg (99%) of a colorless solid. H
NMR (DMSO-d6, 500 MHz): δ (ppm) 12.02 (s, 1H), 11.26 (s,
1H), 8.53 (s, 1H), 7.47-7.45 (m, 2H), 7.09-7.07 (m, 2H), 6.30
(dd, J ) 8.5, 6.1 Hz, 1H), 4.52 (d, J ) 5.8 Hz, 1H), 3.87-3.83
(m, 2H), 3.67 (dd, J ) 13.3, 7.6 Hz, 1H), 3.38 (ddd, J ) 13.3, 8.5,
5.8 Hz, 1H), 2.75 (sept, J ) 6.9 Hz, 1H), 2.24 (s, 3H), 2.11 (ddd,
J ) 13.3, 6.1, 1.8 Hz, 1H), 1.12 (d, J ) 6.9 Hz, 3H), 1.12 (d, J )
6.9 Hz, 3H), 0.89 (s, 9H), 0.82 (s, 9H), 0.11 (s, 6H), -0.01 (d, J
) 5.4 Hz, 6H). 13C NMR (DMSO-d6, 101 MHz): δ (ppm) 147.4,
146.3, 138.2, 129.9, 129.0, 118.0, 117.7, 87.6, 83.7, 73.2, 63.1,
36.5, 34.8, 25.8, 25.7, 20.3, 19.0, 18.8, 18.0, 17.8, -4.7, -4.8,
-5.4, -5.5. HRFABMS: calcd (M + H)+ 671.3773, obsd
671.3769.
O6-Benzyl-8N-(4-biphenylamino)-3′,5′-bis(tert-butyldimethyl-
silyl)-2′-deoxyguanosine (18b). The general procedure (I) was
conducted with 1.40 g (1.56 mmol) of bromide 12 (reaction time
55 h) and afforded 1.03 g (65%) of a light-yellow foam. 1H NMR
(DMSO-d6, 400 MHz): 8.05 (s, 1H), 7.52-7.20 (m, 14H), 6.50
(s, 2H), 6.20 (dd, J ) 6.8, 7.4 Hz, 1H), 5.48 (s, 2H), 4.51 (ddd, J
) 6.7, 4.3, 4.3 Hz, 1H), 3.81 (dd, J ) 10.6, 5.0 Hz, 1H), 3.73
(ddd, J ) 5.0, 4.3, 6.0 Hz, 1H), 3.69 (dd, J ) 10.6, 6.0 Hz, 1H),
3.63 (ddd, J ) 13.0, 6.2, 6.8 Hz, 1H), 2.26 (ddd, J ) 13.0, 4.3, 7.4
Hz, 1H), 0.89 (s, 9H), 0.85 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H),
-0.04 (s, 3H), -0.05 (s, 3H). 13C NMR (DMSO-d6, 101 MHz): δ
(ppm) 160.2, 159.9, 154.3, 148.5, 140.9, 137.8, 136.8, 128.9, 128.6,
128.4, 128.2, 127.5, 127.4, 127.2, 125.8, 125.5, 114.4, 82.5, 72.4,
72.3, 67.0, 62.9, 37.2, 26.0, 25.9, 18.1, 17.9, -4.5, -4.7, -5.2,
-5.3. HRFABMS: calcd (M) 752.3967, obsd (M + H)+ 753.4244.
General Procedure (II) for the Debenzylation of N2-Isobu-
tyryl-O6-benzyl-8N-arylamino-3′,5′-bis(tert-butyldimethylsilyl)-
2′-deoxyguanosine Derivatives. A dried flask was purged with
nitrogen and charged with the N2-isobutyryl-O6-benzyl-8N-aryl-
amino-3′,5′-bis(tert-butyldimethylsilyl)-2′-deoxyguanosine deriva-
tive and Pd/C. Dry CH3OH was added, and the reaction was stirred
under hydrogen atmosphere, at room temperature, for 1 h. The
reaction mixture was centrifuged several times with CH3OH,
filtered, and concentrated in vacuo to give the pure product.
General Procedure (III) for the Desilylation of N2-Iso-
butyryl-8N-arylamino-3′,5′-bis(tert-butyldimethylsilyl)-2′-deoxy-
guanosine Derivatives. In a dried flask, a solution of the N2-iso-
The general procedure (III) was conducted with 876 mg (1.31
mmol) of N2-isobutyryl-8N-(4-methylphenylamino)-3′,5′-bis(tert-
butyldimethylsilyl)-2′-deoxyguanosine in 10 mL of THF (reaction
time 2 h). Purification afforded 845 mg (99%) of a colorless solid.
1H NMR (DMSO-d6, 400 MHz): δ (ppm) 11.97 (s, 1H), 11.55 (s,
1H), 8.78 (s, 1H), 7.67-7.65 (m, 2H), 7.10-7.07 (m, 2H), 6.41
(dd, J ) 9.9, 5.5 Hz, 1H), 5.96, (dd, J ) 4.6, 4.6 Hz, 1H), 5.34 (d,
J ) 3.4 Hz, 1H), 3.96 (ddd, J ) 2.8, 1.9, 2.0 Hz, 1H), 3.78 (ddd,
J ) 11.8, 4.6, 1.9 Hz, 1H), 3.74 (ddd, J ) 11.8, 4.6, 2.0 Hz, 1H),
2.73 (sept, J ) 6.8 Hz, 1H), 2.53 (ddd, J ) 13.0, 9.9, 5.8 Hz, 1H),
2.03 (ddd, J ) 13.0, 5.5, 1.2 Hz, 1H), 1.11 (d, J ) 6.8 Hz, 6H).
13C NMR (DMSO-d6, 101 MHz): δ (ppm) 179.9, 153.8, 147.0,
146.6, 145.6, 137.9, 130.2, 129.1, 118.1, 116.6, 87.5, 83.4, 71.5,
61.4, 38.5, 34.9, 20.5, 19.0. HRFABMS: calcd (M + H)+ 443.2043,
obsd 443.2070.
N2-Isobutyryl-8N-(4-methoxyphenylamino)-2′-deoxyguano-
sine (16c). The general procedure (II) was conducted with 626 mg
(0.81 mmol) of compound 15c and 50 mg of Pd/C in 15 mL of dry
J. Org. Chem, Vol. 71, No. 26, 2006 9735

Bo¨ge et al.
CH3OH and afforded 548 mg (99%) of a colorless solid. 1H NMR
(DMSO-d6, 500 MHz): δ (ppm) 11.96 (s, 1H), 11.18 (s, 1H), 8.44
(s, 1H), 7.50-7.48 (m, 2H), 6.88-6.86 (m, 2H), 6.30 (dd, J )
8.7, 6.0 Hz, 1H), 4.50 (d, J ) 5.8 Hz, 1H), 3.88-3.84 (m, 2H),
3.71 (s, 3H), 3.68 (dd, J ) 13.6, 7.7 Hz, 1H), 3.40 (ddd, J )
13.2, 8.7, 5.8 Hz, 1H), 2.75 (sept, J ) 6.9 Hz, 1H), 2.11 (ddd, J )
13.2, 6.0, 1.6 Hz, 1H), 1.12 (d, J ) 6.9 Hz, 6H), 0.90 (s, 9H), 0
.81 (s, 9H), 0.11 (d, J ) 1.4 Hz, 6H), -0.01 (d, J ) 6.9 Hz, 6H).
13C NMR (DMSO-d6, 101 MHz): δ (ppm) 179.4, 154.3, 153.6,
147.3, 147.0, 145.5, 133.7, 120.2, 117.7, 113.8, 87.6, 83.7, 73.2,
63.1, 55.2, 36.5, 34.7, 25.8, 19.0, 18.8, 18.1, 17.8, -4.7, -4.8,
-5.4, -5.5. HRFABMS: calcd (M + H)+ 687.3722, obsd
687.3723.
The general procedure (III) was conducted with 550 mg (0.80
mmol) of N2-isobutyryl-8N-(4-methoxyphenylamino)-3′,5′-bis(tert-
butyldimethylsilyl)-2′-deoxyguanosine in 8 mL of THF (reaction
time 3 h). Purification afforded 352 mg (96%) of a colorless solid.
1H NMR (DMSO-d6, 400 MHz): δ (ppm) 11.96 (s, 1H), 11.55 (s,
1H), 8.74 (s, 1H), 7.68-7.65 (m, 2H), 6.89-6.85 (m, 2H), 6.40
(dd, J ) 9.9, 5.5 Hz, 1H), 5.95 (dd, J ) 4.7, 4.7 Hz, 1H), 5.34 (d,
J ) 3.3 Hz, 1H), 4.43 (ddd, J ) 3.3, 4.5, 1.4 Hz, 1H), 3.96 (ddd,
J ) 2.0, 2.0, 2.0 Hz, 1H), 3.77-3.75 (m, 2H), 3.72 (s, 3H), 2.73
(sept, J ) 6.9 Hz, 1H), 2.55 (ddd, J ) 13.0, 9.9, 4.5 Hz, 1H), 2.03
(ddd, J ) 13.0, 5.5, 1.4 Hz, 1H), 1.11 (d, J ) 6.9 Hz, 6H). 13C
NMR (DMSO-d6, 101 MHz): δ (ppm) 179.7, 154.1, 153.6, 146.9,
146.3, 145.8, 133.5, 119.6, 116.4, 113.8, 87.3, 83.2, 71.4, 61.3,
55.2, 38.3, 34.7, 18.9. HRFABMS: calcd (M + H)+ 459.1992,
obsd 459.2007.
N2-Isobutyryl-8N-(4-biphenylamino)-2′-deoxyguanosine (16d).
The general procedure (II) was conducted with 695 mg (0.84
mmol) of compound 15d and 50 mg of Pd/C in 20 mL of dry
CH3OH and afforded 613 mg (99%) of a colorless solid. 1H
NMR (DMSO-d6, 400 MHz): δ (ppm) 12.01 (s, 1H), 11.21 (s,
1H), 8.85 (s, 1H), 7.69-7.58 (m, 6H), 7.44-7.40 (m, 2H), 7.31-
7.27 (m, 1H), 6.35 (dd, J ) 8.6, 6.0 Hz, 1H), 4.53-4.50 (m, 1H),
3.89-3.83 (m, 2H), 3.68 (dd, J ) 14.0, 8.4 Hz, 1H) 3.46 (ddd, J
) 13.3, 8.6, 5.9 Hz, 1H), 2.76 (sept, J ) 6.7 Hz, 1H), 2.15 (ddd,
J ) 13.3, 6.0, 1.6 Hz, 1H), 1.13 (d, J ) 6.7 Hz, 6H), 0.89 (s, 9H),
0.82 (s, 9H), 0.11 (s, 6H), -0.00 (d, J ) 4.2 Hz, 6H). 13C NMR
(DMSO-d6, 101 MHz): δ (ppm) 179.5, 153.8, 147.4, 146.0, 145.8,
140.4, 139.9, 132.8, 128.8, 126.8, 126.7, 126.0, 118.1, 117.9, 87.7,
83.8, 73.3, 63.1, 36.4, 34.7, 25.8, 19.0, 18.8, 18.1, 17.9, -4.7, -4.8,
-5.4, -5.4. HRFABMS: calcd (M + H)+ 733.3929, obsd
733.3916.
sulfate and the solvent was removed in vacuo. Purification of the
residue by chromatography on silica gel with CH3OH (0-10%) in
CH2Cl2 gave the desired product.
General Procedure (V) for the Phosphitylation of N2-Iso-
butyryl-8N-arylamino-O5′-dimethoxytrityl-2′-deoxyguanosine De-
rivatives. The N2-isobutyryl-8N-arylamino-2′-deoxyguanosine de-
rivative was twice coevaporated with dry acetonitrile, dissolved in
a mixture of dry CH3CN and dry CH2Cl2 (1:1), and 1 equiv of
dicyanoimidazole solution (0.25 M in CH3CN) and 1.5 equiv of
bis-N,N′-diisopropylamino(2-cyanoethyl) phosphite were added. The
solution was stirred at room temperature until no more starting
material was detected (TLC analysis). Then, the reaction was
stopped by adding saturated NaHCO3 solution. The layers were
separated, and the aqueous layer was extracted three times with
CH2Cl2. The combined organic layers were dried over sodium
sulfate, and the solvent was removed in vacuo. Purification of the
residue by chromatography on aluminum oxide delivered the desired
product.
N2-Isobutyryl-8N-phenylamino-O3′-[(2-cyanoethoxy)-(N,N-di-
isopropylamino)phosphinyl]-O5′-dimethoxytrityl-2′-deoxygua-
nosine (17a). The general procedure (IV) was conducted with 290
mg (0.68 mmol) of compound 16a in 15 mL of dry pyridine
(reaction time 3 h). Purification afforded 406 mg (82%) of a slightly
1
yellow solid. H NMR (DMSO-d6, 400 MHz): δ (ppm) 12.00 (s,
1H), 11.06 (s, 1H), 8.87 (s, 1H), 7.68-7.65 (m, 2H), 7.30-7.25
(m, 4H), 7.17-7.10 (m, 7H), 6.95-6.91 (m, 4H), 6.73-6.64 (m,
1H), 6.43 (dd, J ) 7.6, 5.8 Hz, 1H), 5.16 (d, J ) 5.0 Hz, 1H), 4.53
(dddd, J ) 5.0, 7.2, 4.9, 4.8 Hz, 1H), 3.98 (ddd, J ) 7.9, 4.8, 2.7
Hz, 1H), 3.67 (s, 3H), 3.66 (s, 3H), 3.47 (dd, J ) 10.2, 7.9, Hz,
1H), 3.18 (ddd, J ) 13.2, 7.2, 5.8 Hz, 1H), 3.08 (dd, J ) 10.2, 2.7
Hz, 1H), 2.70 (sept, J ) 6.8 Hz, 1H), 2.23 (ddd, J ) 13.2, 7.6, 4.9
Hz, 1H), 1.10 (d, J ) 6.8 Hz, 3H), 1.08 (d, J ) 6.8 Hz, 3H).13C
NMR (DMSO-d6, 101 MHz): δ (ppm) 179.6, 157.9, 157.8, 153.7,
146.9, 145.9, 145.6, 144.9, 140.9, 135.7, 135.6, 129.7, 129.6, 128.6,
127.8, 127.4, 126.4, 121.0, 117.9, 117.5, 112.8, 112.7, 86.5, 85.2,
83.1, 70.8, 64.8, 54.9, 54.8, 37.4, 34.6, 19.0, 18.8. HRFABMS:
calcd (M + H)+ 731.3115, obsd 731.3118.
The general procedure (V) was conducted with 390 mg (0.53
mmol) of N2-isobutyryl-8N-phenylamino-O5′-dimethoxytrityl-2′-
deoxyguanosine in 20 mL of dry CH3CN/CH2Cl2, 2.12 mL (0.53
mmol) of dicyanoimidazole solution in CH3CN, and 241 mg (0.80
mmol) of (2-cyanoethoxy)bis(diisopropylamino)phosphine (reaction
time 1 h). Purification with CH2Cl2/CH3OH and lyophilization with
benzene afforded 332 mg (67%) of a colorless solid. 1H NMR (500
MHz, C6D6): δ (ppm) 11.11 (s, 2H), 9.61 (s, 2H), 8.07 (s, 2H),
7.85-6.70 (m, 36H), 6.42 (dd, J ) 6.6, 6.6 Hz, 1H), 6.36 (dd, J )
6.3, 6.3 Hz, 1H), 4.88-4.80 (m, 2H), 4.49-4.45 (m, 2H), 3.67-
3.26 (m, 26H), 2.92-2.86 (m, 1H), 2.65-2.59 (m, 1H), 2.50-
2.39 (m, 3H), 2.17 (ddd, J ) 17.2, 5.8, 1H), 2.05 (ddd, J ) 16.7,
5.6, 5.6 Hz, 1H), 1.93 (ddd, J ) 16.7, 6.9, 5.3 Hz, 1H), 1.17-1.01
(m, 36H). 13C NMR (126 MHz, C6D6): δ (ppm) 178.7, 159.3,
159.2, 147.0, 146.8, 145.4, 145.3, 140.7, 140.6, 136.1, 136.1, 136.0,
136.0, 130.7, 130.6, 130.6, 130.5, 129.3, 128.8, 128.7, 128.6, 128.3,
128.2, 128.0, 127.2, 127.1, 121.9, 121.9, 118.9, 118.7, 118.4, 118.2,
113.6, 87.1, 86.7, 86.6, 86.4, 86.4, 85.9, 75.1, 74.9, 74.1, 73.9, 64.1,
58.8, 58.6, 58.5, 58.4, 54.9, 54.9, 43.7, 43.6, 43.5, 43.5, 38.7, 38.5,
36.1, 24.8, 24.7, 24.6, 24.6, 20.6, 20.5, 20.3, 20.3, 19.3, 19.2, 19.1,
19.1, 1.4. 31P NMR (202 MHz, C6D6): δ (ppm) 149.76, 147.90.
MALDI-MS: calcd 930.4282, obsd 953.4237 (M + Na+)
The general procedure (III) was conducted with 421 mg (0.57
mmol) of N2-isobutyryl-8N-(4-biphenylamino)-3′,5′-bis(tert-bu-
tyldimethylsilyl)-2′-deoxyguanosine in 8 mL of dry THF (reaction
time 2 h). Purification afforded 288 mg (99%) of a slightly yellow
1
solid. H NMR (DMSO-d6, 400 MHz): δ (ppm) 12.00 (s, 1H),
11.59 (s, 1H), 9.00 (s, 1H), 7.89-7.87 (m, 2H), 7.64-7.60 (m,
4H), 7.45-7.41 (m, 2H), 7.32-7.29 (m, 1H), 6.44 (dd, J ) 9.9,
5.6 Hz, 1H), 6.05 (dd, J ) 4.6, 4.6 Hz, 1H), 5.36 (d, J ) 3.2 Hz,
1H), 4.48-4.43 (m, 1H), 4.00-3.97 (m, 1H), 3.83-3.75 (m, 2H),
2.74 (sept, J ) 6.8 Hz, 1H), 2.56 (ddd, J ) 13.0, 9.9, 5.8 Hz, 1H),
2.07 (dd, J ) 13.0, 5.6 Hz, 1H), 1.12 (d, J ) 6.8 Hz, 6H). 13C
NMR (DMSO-d6, 101 MHz): δ (ppm) 179.7, 153.6, 146.9, 146.6,
145.0, 139.9, 139.7, 132.9, 128.8, 126.8, 126.7, 126.1, 118.2, 116.4,
87.4, 83.3, 71.3, 61.3, 52.0, 38.5, 34.7, 18.9, 7.1. HRFABMS: calcd
(M + H)+ 505.2199, obsd 505.2183.
General Procedure (IV) for the Dimethoxytritylation of N2-
Isobutyryl-8N-arylamino-2′-deoxyguanosine Derivatives. The N2-
isobutyryl-8N-arylamino-2′-deoxyguanosine derivative was twice
coevaporated with pyridine and then dissolved in dry pyridine. After
that, 2 equiv dimethoxytrityl chloride were added and the solution
was stirred at room temperature until the reaction was complete
(TLC analysis). The reaction was stopped by adding a saturated
NaHCO3 solution. After addition of CH2Cl2 the layers were
separated and the aqueous layer was extracted three times with
CH2Cl2. The combined organic layers were dried over sodium
N2-Isobutyryl-8N-(4-methylphenylamino)-O3′-[(2-cyanoethoxy)-
(N,N′-diisopropylamino)phosphinyl]-O5′-dimethoxytrityl-2′-de-
oxyguanosine (17b). The general procedure (IV) was conducted
with 500 mg (1.13 mmol) of compound 16b in 25 mL of dry
pyridine (reaction time 3 h). Purification afforded 605 mg (72%)
of a slightly yellow foam. 1H NMR (DMSO-d6, 500 MHz): δ (ppm)
11.99 (s, 1H), 11.04 (s, 1H), 8.73 (s, 1H), 7.57-7.56 (m, 2H), 7.28-
7.26 (m, 2H), 7.16-7.08 (m, 9H), 6.72-6.65 (m, 4H), 6.41 (dd, J
) 7.4, 6.1 Hz, 1H), 5.15 (d, J ) 5.2 Hz, 1H), 4.52 (dddd, J ) 5.2,
7.0, 4.9, 4.7 Hz, 1H), 3.98 (ddd, J ) 8.0, 4.7, 2.7 Hz, 1H), 3.68 (s,
9736 J. Org. Chem., Vol. 71, No. 26, 2006

Oligonucleotides Containing C8-Deoxyguanosine Arylamine Adducts
3H), 3.67 (s, 3H), 3.46 (dd, J ) 10.1, 8.0, Hz, 1H), 3.17 (ddd, J )
13.1, 7.0, 6.1 Hz, 1H), 3.08 (dd, J ) 10.1, 2.7 Hz, 1H), 2.70 (sept,
J ) 6.8 Hz, 1H), 2.25 (s, 3H), 2.22 (ddd, J ) 13.1, 7.4, 4.9 Hz,
1H), 1.10 (d, J ) 6.8 Hz, 3H), 1.08 (d, J ) 6.8 Hz, 3H).13C NMR
(DMSO-d6, 101 MHz): δ (ppm) 179.8, 158.1, 158.0, 153.8, 147.1,
146.3, 145.7, 145.1, 138.4, 135.8, 135.7, 129.9, 129.9, 129.8, 129.2,
127.9, 127.6, 126.6, 118.0, 117.9, 112.9, 112.9, 86.6, 85.3, 83.2,
71.0, 64.9, 55.0, 55.0, 37.6, 34.7, 20.5, 19.2, 18.9. HRFABMS:
calcd (M + H)+ 745.3350, obsd 745.3363.
127.2, 127.2, 120.4, 120.3, 118.9, 118.8, 118.2, 114.6, 113.6, 113.6,
87.1, 86.6, 86.5, 86.4, 86.3, 86.2, 85.6, 75.0, 74.8, 74.0, 73.8, 64.1,
64.0, 58.8, 58.6, 58.6, 58.5, 55.2, 54.9, 54.9, 43.6, 43.6, 43.5, 43.5,
38.6, 38.4, 36.1, 30.2, 24.8, 24.7, 24.6, 20.6, 20.5, 20.3, 20.3, 19.1,
1.4. 31P NMR (202 MHz, C6D6): δ (ppm) 149.36, 148.08. ESI-
MS: calcd 960.4310, obsd. 983.4671 (M + Na+).
N2-Isobutyryl-8N-(4-biphenylamino)-O3′-[(2-cyanoethoxy)-
(N,N-diisopropyl-amino)phosphinyl]-O5′-dimethoxytrityl-2′-
deoxyguanosine (17d). The general procedure (IV) was conducted
with 695 mg (1.38 mmol) of compound 16d in 30 mL of dry
pyridine (reaction time 3 h). Purification afforded 800 mg (72%)
of a colorless solid. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 12.02
(s, 1H), 11.07 (s, 1H), 9.00 (s, 1H), 7.78-7.76 (m, 2H), 7.65-
7.60 (m, 4H), 7.45-7.42 (m, 2H), 7.31-7.26 (m, 3H), 7.16-7.11
(m, 7H), 6.72-6.65 (m, 4H), 6.45 (dd, J ) 6.4, 6.0 Hz, 1H), 5.17
(d, J ) 5.0 Hz, 1H), 4.56-4.52 (m, 1H), 3.99 (ddd, J ) 8.0, 4.7,
2.7 Hz, 1H), 3.67 (s, 3H), 3.66 (s, 3H), 3.47 (dd, J ) 9.9, 8.0 Hz,
1H), 3.20 (ddd, J ) 13.1, 7.1, 6.4 Hz, 1H), 3.09 (dd, J ) 9.9, 2.7
Hz, 1H), 2.71 (sept, J ) 6.7 Hz, 1H), 2.25 (ddd, J ) 13.1, 7.6, 6.0
Hz, 1H), 1.11 (d, J ) 6.7 Hz, 3H), 1.09 (d, J ) 6.7 Hz, 3H). 13C
NMR (DMSO-d6, 101 MHz): δ (ppm) 179.6, 157.9, 157.8, 153.7,
146.9, 145.7, 144.9, 140.4, 139.9, 135.7, 135.6, 132.6, 129.7, 129.6,
128.9, 127.8, 127.4, 126.8, 126.6, 126.4, 126.0, 117.9, 117.9, 112.8,
112.7, 86.5, 85.2, 83.2, 70.8, 64.8, 54.9, 54.8, 37.4, 34.6, 19.0, 18.8.
HRFABMS: calcd (M + H)+ 807.3506, obsd 807.3600.
The general procedure (V) was conducted with 400 mg (0.50
mmol) of N2-isobutyryl-8N-(4-biphenylamino)-O5′-dimethoxytrityl-
2′-deoxyguanosine in 20 mL of dry CH3CN/CH2Cl2, 2.0 mL (0.50
mmol) of dicyanoimidazole solution in acetonitrile, and 224 mg
(0.74 mmol) of (2-cyanoethoxy)bis(diisopropylamino)phosphine
(reaction time 1 h). Purification with CH2Cl2/CH3OH and lyo-
philization with benzene afforded 291 mg (58%) of a slightly yellow
solid. 1H NMR (500 MHz, C6D6): δ (ppm) 11.18 (s, 2H), 9.58 (s,
2H), 7.83 (s, 2H), 7.76-6.68 (m, 44H), 6.27 (dd, J ) 6.4, 6.0 Hz,
1H), 6.21 (dd, J ) 6.3, 6.3 Hz, 1H), 4.85-4.75 (m, 2H), 4.50-
4.42 (m, 2H), 3.60-3.28 (m, 25H), 3.25-3.17 (m, 1H), 2.88-
2.80 (m, 1H), 2.55 (ddd, J ) 13.6, 6.0, 3.3 Hz, 1H), 2.51-2.42
(m, 2H), 2.37 (ddd, J ) 16.8, 7.6, 4.7 Hz, 1H), 2.12 (ddd, J )
16.8, 6.2, 4.5 Hz, 1H), 2.00 (ddd, J ) 16.8, 5.7, 5.7 Hz, 1H), 1.91
(ddd, J ) 16.8, 6.9, 5.1 Hz, 1H), 1.19-1.02 (m, 36H). 13C NMR
(126 MHz, C6D6): δ (ppm) 178.8, 159.3, 159.2, 146.8, 146.6, 145.4,
145.4, 141.4, 140.1, 140.0, 136.1, 136.1, 136.0, 136.0, 134.6, 134.6,
130.7, 130.7, 130.6, 130.6, 129.1, 128.8, 128.7, 128.6, 128.3, 128.2,
128.0, 127.5, 127.2, 127.2, 127.0, 126.9, 119.0, 118.8, 118.8, 118.3,
113.6, 113.6, 87.1, 86.7, 86.7, 86.4, 86.4, 85.9, 75.0, 74.8, 74.0,
73.9, 64.1, 58.8, 58.7, 58.6, 58.5, 54.9, 54.9, 45.7, 43.7, 43.6, 43.5,
38.8, 38.6, 36.1, 24.8, 24.7, 24.6, 20.6, 20.6, 20.4, 20.3, 19.1, 19.1,
1.4. 31P NMR (202 MHz, C6D6): δ (ppm) 149.71, 147.95. ESI-
MS: calcd 1006.4650, obsd 1029.4918 (M + Na+).
Synthesis of the Oligonucleotides. The oligonucleotides were
synthesized using isobutyryl-protected dA, isobutyryl-protected dG,
benzyl-protected dC, and T phosphoramidites on a 300+ DNA
synthesizer (Eppendorf) on a 1 µmol scale using phosphoramidites
and solid supports from ChemGenes. The manufacturer’s standard
synthesis protocol was followed except at the incorporation of the
modified phosphoramidites (17a-d), which were coupled twice
and for a longer time.
The oligonucleotide was purified by HPLC using triethylam-
moniumacetate buffer (pH 6.9) (solvent 1) and acetonitrile (solvent
2) on a C-18 reversed-phase column with UV detection. The solvent
gradient was as follows: initially 99% solvent 1, then a 50 min
linear gradient to 23% solvent 2; 10 min 100% solvent 2; 10 min
100% of solvent 1.
LC-MS for 19: m/z calcd for (M + H+) 4469.2, found 4469.0
LC-MS for 20a: m/z calcd for (M - H+) 4562.5, found 4562.3
LC-MS for 20b: m/z calcd for (M - H+) 4571.8, found 4572.0
LC-MS for 20c: m/z calcd for (M) 4590.1, found 4590.5
LC-MS for 20d: m/z calcd for (M - H+) 4634.2, found 4634.0
The general procedure (V) was conducted with 500 mg (0.69
mmol) of N2-isobutyryl-8N-(4-methylphenylamino)-O5′-dimeth-
oxytrityl-2′-deoxyguanosine in 25 mL of dry CH3CN/CH2Cl2, 2.76
mL (0.69 mmol) of dicyanoimidazole solution in CH3CN, and 304
mg (1.01 mmol) of (2-cyanoethoxy)bis(diisopropylamino)phosphine
(reaction time 1 h). Purification with CH2Cl2/CH3OH and lyo-
philization with benzene afforded 463 mg (73%) of a slightly yellow
foam. 1H NMR (500 MHz, C6D6): δ (ppm) 12.01 (s, 2H), 9.19 (s,
2H), 8.92 (s, 2H), 7.74-6.69 (m, 34H), 6.18 (dd, J ) 6.4, 6.0 Hz,
1H), 6.13 (dd, J ) 6.3, 6.0 Hz, 1H), 4.85-4.77 (m, 2H), 4.50-
4.45 (m, 2H), 3.59-3.23 (m, 25H), 3.21-3.15 (m, 1H), 2.80 (ddd,
J ) 14.0, 6.0, 3.0 Hz, 1H), 2.48 (ddd, J ) 13.3, 6.0, 2.9 Hz), 2.38
(sept, J ) 6.9 Hz, 1H), 2.31-2.21 (m, 2H), 2.15 (s, 6H), 2.04-
1.92 (m, 2H), 1.87 (ddd, J ) 16.9, 7.0, 5.6 Hz, 1H), 1.14-0.95
(m, 36H). 13C NMR (126 MHz, C6D6): δ (ppm) 178.8, 159.3,
159.2, 155.3, 155.2, 147.6, 147.3, 147.1, 146.4, 146.2, 145.4, 145.4,
138.3, 138.1, 136.1, 136.1, 136.0, 136.0, 131.1, 131.0, 130.7, 130.6,
130.6, 130.5, 129.8, 128.8, 128.7, 128.6, 128.3, 128.2, 128.0, 127.2,
127.1, 118.9, 118.9, 118.8, 118.6, 118.6, 118.3, 113.6, 113.6, 87.1,
86.7, 86.6, 86.4, 86.4, 85.8, 75.1, 74.9, 74.0, 73.9, 64.1, 58.8, 58.6,
58.5, 58.4, 54.9, 54.9, 43.6, 43.6, 43.5, 43.5, 38.7, 38.4, 36.2, 36.1,
24.8, 24.7, 24.6, 24.6, 20.9, 20.6, 20.6, 20.4, 20.3, 19.2, 19.2, 19.1,
1.4. 31P NMR (202 MHz, C6D6): δ (ppm) 149.97, 147.79 MALDI-
MS: calcd 944.4365, obsd. 967.3248 (M + Na+).
N2-Isobutyryl-8N-(4-methoxyphenylamino)-O3′-[(2-cyano-
ethoxy)-(N,N-diisopropylamino)phosphinyl]-O5′-dimethoxytri-
tyl-2′-deoxyguanosine (17c). The general procedure (IV) was
conducted with 340 mg (0.74 mmol) of compound 16c in 20 mL
of dry pyridine (reaction time 2 h). Purification afforded 320 mg
1
(57%) of a colorless foam. H NMR (DMSO-d6, 400 MHz): δ
(ppm) 11.97 (s, 1H), 11.04 (s, 1H), 8.65 (s, 1H), 7.61-7.58 (m,
2H), 7.28-7.26 (m, 2H), 7.17-7.11 (m, 7H), 6.89-6.86 (m, 2H),
6.73-6.70 (m, 2H), 6.68-6.65 (m, 2H), 6.40 (dd, J ) 7.4, 6.3 Hz,
1H), 5.15 (d, J ) 4.9 Hz, 1H), 4.52 (dddd, J ) 4.9, 7.0, 4.8, 4.6
Hz, 1H), 3.98 (ddd, J ) 7.9, 4.6, 2.8 Hz, 1H), 3.72 (s, 3H), 3.68
(s, 3H), 3.67 (s, 3H), 3.45 (dd, J ) 10.1, 7.9 Hz, 1H), 3.18 (ddd,
J ) 13.0, 7.0, 6.3 Hz, 1H), 3.08 (dd, J ) 10.1, 2.8 Hz, 1H), 2.70
(sept, J ) 6.8 Hz, 1H), 2.22 (ddd, J ) 13.0, 7.4, 4.8 Hz, 1H), 1.10
(d, J ) 6.8 Hz, 3H), 1.08 (d, J ) 6.8 Hz, 3H). 13C NMR (DMSO-
d6, 101 MHz): δ (ppm) 179.7, 158.1, 158.0, 154.2, 153.7, 147.1,
146.8, 145.6, 145.1, 135.8, 135.7, 134.1, 129.9, 129.8, 127.9, 127.6,
126.6, 119.7, 118.0, 114.0, 112.9, 112.9, 86.6, 85.3, 83.2, 71.0,
64.9, 55.4, 55.1, 55.0, 52.2, 37.5, 34.7, 19.2, 18.9. HRFABMS:
calcd (M + H)+ 761.3299, obsd 761.3324.
The general procedure (V) was conducted with 395 mg (0.52
mmol) of N2-isobutyryl-8N-(4-methoxyphenylamino)-O5′-dimethoxy-
trityl-2′-deoxyguanosine in 20 mL of dry CH3OH/CH2Cl2, 2.08 mL
(0.52 mmol) of dicyanoimidazole solution in acetonitrile, and 235
mg (0.78 mmol) of bis-N,N′-diisopropylamino(2-cyanoethyl) phos-
phite (reaction time 1 h). Purification with CH2Cl2/CH3OH and
lyophilisation with benzene afforded 278 mg (56%) of a slightly
yellow foam. 1H NMR (500 MHz, C6D6): δ (ppm) 10.99 (s, 2H),
9.63 (s, 2H), 7.70 (s, 2H), 7.67-6.70 (m, 34H), 6.34 (dd, J ) 6.6,
6.6 Hz), 6.27 (dd, J ) 6.5, 6.5 Hz, 1H), 4.86-4.78 (m, 2H), 3.64-
3.16 (m, 32H), 2.79-2.72 (m, 1H), 2.54-2.35 (m, 4H), 2.18 (ddd,
J ) 17.0, 5.4, 5.4 Hz, 1H), 2.01 (ddd, J ) 16.8, 5.8, 5.8 Hz, 1H),
1.94 (ddd, J ) 16.8, 6.1, 6.1 Hz, 1H), 1.18-1.02 (m, 36H). 13C
NMR (126 MHz, C6D6): δ (ppm) 178.7, 159.3, 159.2, 155.5, 155.4,
147.6, 147.5, 145.3, 145.3, 136.1, 136.0, 136.0, 135.9, 133.9, 133.8,
130.7, 130.6, 130.6, 130.5, 128.8, 128.7, 128.6, 128.3, 128.2, 128.0,
J. Org. Chem, Vol. 71, No. 26, 2006 9737

Bo¨ge et al.
MALDI-TOF for 21: m/z calcd 3643.465, found 1183.1,
1147.59, 1486.17, 1833.28
MALDI-TOF for 22a: m/z calcd 3766.625, found 1485.64,
1565.67, 1765.7, 1845.63
mM phosphate buffer, 140 mM NaCl, 1 mM EDTA, pH 6.8). The
UV absorption at 260 nm was monitored as a function of
temperature. The temperature was increased at a rate of 0.5
°C/min from 5 to 80 °C.
MALDI-TOF for 22b: m/z calcd 3810.531, found 1173.2,
1486.2, 1563.9
Circular Dichroism Measurements. CD measurements were
carried out with the same solutions as the Tm studies and at 25 °C.
Samples were scanned from 350 to 220 nm at 0.5 nm intervals
averaged over 1 s.
Enzymatic Digestion of Oligonucleotides 21 and 22a,b.
Enzymatic digestion of the oligonucleotides was performed by
treatment of purified oligonucleotides (2 nmol) in a reaction buffer
(pH 5.3, 100 µL, 0.03 M sodium acetate) with 5 µL of ZnSO4
solution (20 mM) and 5 units of Nuclease P1 stirred at 37 °C
overnight. This was followed by the addition of 20 µL of a buffer
(pH 9, 50 mM, Tris/HCl) and 5 units of alkaline phosphatase, and
the reaction was stirred at 37 °C overnight. The reaction mixture
was concentrated, solved in methanol and filtered. After concentra-
tion of the sample, the pellet was solved in 50 µL water and injected
directly on HPLC using water (solvent 1) and CH3CN (solvent 2).
The gradient was as follows: initially 100% solvent 1, then a 20
min linear gradient to 25% solvent 2; 5 min 100% solvent 2; 10
min 100% of solvent 1.
Acknowledgment. We thank the Deutsche Forschungsge-
meinschaft (Bonn, Germany) for financial support and WACK-
ER Fine Chemicals (Burghausen, Germany) for reagents. F.
Seela, University of Osnabru¨ck is acknowledged for recording
ESI-MS spectra of the oligonucleotides.
Supporting Information Available: 1H, 13C, and 31P NMR
spectra for all compounds. MALDI-TOF and LC-MS spectra,
HPLC traces for the enzyme digest, and CD spectra of the adducted
oligonucleotides. This material is available free of charge via the
Internet at http://pubs.acs.org.
Thermal Melting Studies. Equal amounts of the two comple-
mentary strands (2 nmol) were dissolved in 1 mL of buffer (10
JO061803T
9738 J. Org. Chem., Vol. 71, No. 26, 2006