A facile incorporation of the aldehyde function into DNA: 3-Formylindole nucleoside as an aldehyde-containing universal nucleoside

Article abstract of DOI:10.1016/S0040-4039(02)00888-2

The first facile incorporation of an aldehyde function into DNA without any protection/deprotection of the aldehyde was achieved by the use of 3-formylindole 2′-deoxynucleoside (1). This building block is very easily prepared and efficiently incorporated into oligonucleotides. In addition, 1 acted as a universal nucleoside in duplex. The post-synthetic modification of oligonucleotides bearing 1 with functionalized molecules was also effective.

Full text of DOI:10.1016/S0040-4039(02)00888-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 4581–4583
A facile incorporation of the aldehyde function into DNA:
3-formylindole nucleoside as an aldehyde-containing universal
nucleoside
Akimitsu Okamoto, Kazuki Tainaka and Isao Saito*
Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, CREST,
Japan Science and Technology Corporation, Kyoto 606-8501, Japan
Received 25 March 2002; accepted 10 May 2002
Abstract—The first facile incorporation of an aldehyde function into DNA without any protection/deprotection of the aldehyde
was achieved by the use of 3-formylindole 2%-deoxynucleoside (1). This building block is very easily prepared and efficiently
incorporated into oligonucleotides. In addition, 1 acted as a universal nucleoside in duplex. The post-synthetic modification of
oligonucleotides bearing 1 with functionalized molecules was also effective. © 2002 Published by Elsevier Science Ltd.
The assembling of functionalized oligonucleotides
(ODNs) into complementary nucleic acids with target
sequences has led to a rich variety of technologies
exploiting new functionalities of ODNs.1–5 The develop-
ment of a new synthetic method for ODNs possessing
diverse functionalities is of great interest. The aldehyde
function is often used to couple biopolymers to other
molecules by the process of reductive amination or the
formation of adducts with hydroxylamines, hydrazines,
and semicarbazides as nucleophiles.6–11 However, alde-
hyde functions have rarely been applied for post-syn-
thetic modification of ODNs, because suitable aldehyde
Scheme 1. Synthesis of 3-formylindole deoxynucleoside 1 and phosphoramidite 6. (a) Sodium hydride, acetonitrile, 50°C, 1 h, and
then 2, acetonitrile, rt, 3 h (74%); (b) lithium hydroxide, THF–water (1:1), rt, 3.5 h (90%); (c) 4,4%-dimethoxytrityl chloride,
N,N-dimethylaminopyridine, pyridine, rt, 5 h (73%); (d) (iPr2N)2PO(CH2)2CN, tetrazole, acetonitrile, rt, 2 h (quant.).
Keywords: oligonucleotides; post-synthetic modification; aldehydes; universal nucleosides.
* Corresponding author. Tel.: +81-75-753-5656; fax: +81-75-753-5676; e-mail: saito@sbchem.kyoto-u.ac.jp
0040-4039/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0040-4039(02)00888-2

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A. Okamoto et al. / Tetrahedron Letters 43 (2002) 4581–4583
building blocks for automatic ODN synthesis have not
been available and such complicated and unwieldy pro-
cesses as protection/deprotection and oxidative cleav-
age of diols were unavoidable during ODN
synthesis.10–14 It would be very useful to have an alde-
hyde building block mimicking nucleotides, especially if
ODNs bearing multiple aldehyde functions are
like nitroindole17,18 and methylbenzimidazole19–21
nucleosides known as ‘universal’ nucleosides. Thus, the
aldehyde function can be easily incorporated into any
desired site of DNA by the use of 1.
We next examined the modification of the formylindole
1 of ODN 5%-d(GCGATG1GTAGCG)-3% with func-
tionalized hydrazines (Fig. 2). The fully protected ODN
after solid-phase synthesis on an automated DNA syn-
thesizer was treated with 10 mM N,N-diphenylhy-
drazine in the presence of 10 mM sodium acetate in
ethanol at 60°C for 24 h.22 After incubation, the
oligomer was deprotected by aqueous ammonia and
then analyzed by HPLC. It was shown in an HPLC
profile that a formyl group in the oligomer was con-
verted to the corresponding diphenylhydrazone in 86%
yield. The composition of the oligomer after HPLC
required. In addition, as
a compatible aldehyde
modifier, it is desirable that the aldehyde building block
can be applied in the desired sequence, regardless of the
type of complementary bases in duplex DNA.
We herein describe a convenient method for incorporat-
ing an aldehyde function into the desired position of an
ODN. The aldehyde building block 3-formylindole 2%-
deoxynucleoside (1) was easily prepared in a few steps,
and its incorporation into ODN was achieved without
any protection/deprotection of the formyl group during
the synthesis of 1-containing ODN. The synthesized
ODNs formed stable duplexes regardless of the type of
base opposite 1. In addition, post-synthetic modifica-
tion of ODN bearing an aldehyde function with func-
tionalized hydrazides efficiently furnished the
corresponding modified ODNs.
The synthetic route for 1 and its phosphoramidite is
shown in Scheme 1. Commercially available 3-formyl-
indole (2) was coupled with deoxyribosyl chloride (3)15
to give 4 (74%). The hydrolytic deprotection of 4
afforded a free deoxynucleoside 1 (90%).16 The primary
alcohol of 1 was protected by the 4,4%-dimethoxytrityl
group (73%) and quantitatively converted to cyanoethyl
phosphoramidite 6.
Figure 1. HPLC profiles for ODN containing 3-formylindole
deoxynucleoside
1.
(a)
crude
oligomer
5%-d(GC-
GATG1GTAGCG)-3%; (b) nucleosides after enzymatic diges-
tion (snake venom phosphodiesterase, nuclease P1, and
alkaline phosphatase). Elution with a solvent mixture of 0.1
M triethylamine acetate (TEAA), pH 7.0, linear gradient over
20 min from 0 to 10% acetonitrile at a flow rate 1.0 mL/min.
Amidite 6 was employed in the conventional solid-
phase synthesis of ODNs. After ODN synthesis, depro-
tection was conducted with aqueous ammonia at 55°C
for 16 h, and the resulting ODNs were purified by
HPLC. The composition of the oligomer was deter-
mined by MALDI-TOF mass spectrometry (calcd
4048.67 for [M−H]−, found 4048.14), and the product
analysis obtained from enzymatic digestion with snake
venom phosphodiesterase, nuclease P1, and alkaline
phosphatase. An HPLC profile after enzymatic diges-
tion, as shown in Fig. 1, exhibited five peaks corre-
sponding to four natural bases and 1.
Table 1. Tm measurement of duplex containing 1a
5%-d(GCGATG1GTAGCG)-3%
3%-d(CGCTACNCATCGC)-5%
Entry
N
Tm (°C)
1
2
3
4
C
G
T
52.8
50.0
49.7
52.7
A
a 3 mM strand concentration, 10 mM PIPES buffer, 10 mM magne-
sium chloride, 100 mM sodium chloride (pH 7.0).
The stability of the duplex ODN containing 1 was
investigated by monitoring the melting temperatures
(Tm) of the duplex. The duplexes 5%-d(GC-
GATG1GTAGCG) - 3%/5% - d(CGCTACNCATCGC) - 3%
(N=C, G, T, and A) were prepared for Tm measure-
ments, and the effects of the complementary bases on
duplex stability were compared. In all Tm measure-
ments, sigmoidal curves on the change of A254 were
obtained, and the Tm values were calculated from the
first derivative of the curve. The results of the Tm
measurements are shown in Table 1. Selectivity of
base-pairing with
1 was not observed for these
duplexes. Their Tm values were less than the duplex
containing the natural A–T base pair (59.2°C). Thus,
the stability of a duplex containing 1 was not influenced
by the base opposite 1, and 1 in duplex ODN behaved
Figure 2. Products given by post-synthetic modification of
5%-d(GCGATG1GTAGCG)-3%.

A. Okamoto et al. / Tetrahedron Letters 43 (2002) 4581–4583
4583
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Figure 3. HPLC profiles after enzymatic digestion (snake
venom phosphodiesterase, nuclease P1 and alkaline phos-
phatase) of 1-containing ODN modified by diphenylhy-
drazine. Elution with a solvent mixture of 0.1 M triethylamine
acetate (TEAA), pH 7.0, linear gradient over 20 min from 0
to 10% acetonitrile at a flow rate 1.0 mL/min.
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16. Spectral data of 1: H NMR (DMSO-d6) l 9.92 (s, 1H),
purification was determined by MALDI-TOF mass
spectrometry (calcd 4214.89 for [M−H]−, found
4215.15) and the product analysis obtained from enzy-
matic digestion. An example of an HPLC profile after
enzymatic digestion is shown in Fig. 3. The peaks
shown in Fig. 3 suggest that N,N-diphenylhydrazine
was effectively incorporated into 1 in ODN without any
side reaction.23 Similarly, ferrocenecarbohydrazide was
1
8.55 (s, 1H), 8.09 (d, 1H, J=7.6 Hz), 7.70 (d, 1H, J=8.4
Hz), 7.29 (m, 2H), 6.42 (t, 1H, J=6.4 Hz), 5.36 (d, 1H,
J=4.0 Hz), 4.97 (t, 1H, J=5.2 Hz), 4.37 (m, 1H), 3.87 (q,
1H, J=4.0 Hz), 3.55 (m, 2H), 2.49 (m, 1H), 2.35 (m, 1H);
MS (FAB, NBA/CH2Cl2) m/e 261 [M+H]+; HRMS
(FAB) calcd for C14H16O4N [M+H]+ 262.1080, found
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also incorporated into
1
of ODN 5%-d(GC-
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[M−H]−, found 4275.23). Ferrocene-modified ODNs
have been noted as excellent electrochemical probes in
recent years.24
20. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org.
In conclusion, the facile incorporation of an aldehyde
function into DNA without any protection/deprotec-
tion of the aldehyde was achieved by the use of 3-
formylindole 2%-deoxynucleoside (1). This building
block is very easily prepared and efficiently incorpo-
rated into oligonucleotides. In addition, 1 can be used
as a universal nucleoside in duplex oligonucleotides.
The post-synthetic modification of 1-containing ODN
with a variety of functionalized molecules is also possi-
ble. This post-synthetic modification method will be
applicable to site-selective DNA labeling and bioconju-
gation with various functionalized groups.
Chem. 1998, 63, 9652–9656.
21. Morales, J. C.; Kool, E. T. Biochemistry 2000, 39, 12979–
12988.
22. Post-synthetic modification via on-column conjugation:
A solution of the coupling reagents (10 mM diphenylhy-
drazine hydrochloride, and 10 mM sodium acetate in 1.5
mL ethanol) was prepared. To the solution was added the
resin-bound DNA (containing ꢀ0.5 mmol); it was then
shaken at 60°C for 24 h, and then the solvent was
evaporated in vacuo. The resin was treated with 28%
aqueous ammonia (1 mL) for 16 h at 55°C and concen-
trated under vacuum, then filtered. Oligonucleotides were
purified by reverse phase HPLC on a 5-ODS-H column
(10×150 mm, elution with a solvent mixture of 0.1 M
triethylamine acetate (TEAA), pH 7.0, linear gradient
over 40 min from 5 to 100% acetonitrile at a flow rate 3.0
mL/min).
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