Enhanced reactivity of amino-modified oligonucleotides by insertion of aromatic residue

Article abstract of DOI:10.1016/j.bmcl.2006.07.027

We developed novel amino-modifying reagents, of which an amino group was connected with an aromatic residue by aliphatic linker. It was proved that the insertion of the aromatic residue could increase the reactivity of the amino group on oligonucleotides

Full text of DOI:10.1016/j.bmcl.2006.07.027

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5118–5121
Enhanced reactivity of amino-modified oligonucleotides
by insertion of aromatic residue
Naoshi Kojima,a Maiko Sugino,a Akiko Mikami,a Ken Nonaka,b Yumi Fujinawa,b
Isamu Muto,c Kenichi Matsubara,b Eiko Ohtsukaa and Yasuo Komatsua,*
aResearch Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST),
2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
bDNA Chip Research Inc., 1-1-43, Suehirocho, Tsurumi-ku, Yokohama 230-0045, Japan
cHitachi Software Engineering Co. Ltd., 1-1-43, Suehirocho, Tsurumi-ku, Yokohama 230-0045, Japan
Received 16 May 2006; revised 28 June 2006; accepted 11 July 2006
Available online 28 July 2006
Abstract—We developed novel amino-modifying reagents, of which an amino group was connected with an aromatic residue by
aliphatic linker. It was proved that the insertion of the aromatic residue could increase the reactivity of the amino group on oligo-
nucleotides in comparison with conventional amino-modification.
Ó 2006 Elsevier Ltd. All rights reserved.
Terminal modification of oligonucleotides is necessary
for immobilization of oligonucleotide probes on solid
surface. There are many kinds of terminal modifications
such as sulfhydryl group,1 hydrazide,2 anthraquinone3
and acrylamide4 for attachment on the solid surface;
among them, the aliphatic amine is most cost effective
and produces less side products under standard depro-
tecting condition after oligonucleotide synthesis.5 Thus,
oligonucleotides modified with aliphatic amine have
been frequently used for a gene analysis system requir-
ing diverse oligonucleotide probes.6 Besides aliphatic
amino groups, there are nucleosidic-type amino modifi-
ers to date.7 However, these nucleosidic amino modifiers
are able to form base pairs with opposite nucleotide in
binding with target strand. Therefore, these nucleosidic
amino modifiers are not suitable for universal termi-
nal-modification for preparation of diverse oligonucleo-
tide probes. On the other hand, polycyclic aromatic
residues inserted into oligonucleotides have been well
studied.8 Although, these aromatic groups are known
to increase the hybridization affinity for a complementary
double strand, the effects of the aromatic residues on
the reactivity of primary amino group have not been
examined. We expected that the aromatic groups would
improve the reactivity of primary amine and assist the
purification steps of amino-modified oligonucleotides
by their hydrophobic properties.
We designed and synthesized a series of amidite units,
which have a primary amino group and an aromatic res-
idue, by connecting with a short (S) or a long (L) linker
of various lengths (see Supplementary data). Naphtha-
lene (N) and anthracene (A) were selected as the aromat-
ic groups (Fig. 1). The synthesized amidite units are
referred to as SN, SNO, ssN, LNO, and LAO. The large
letter ‘O’ indicates the carbamate structure in the linker
between the amino and the aromatic residue. Although
ssN consists of a hexamethylene linker and carbamate
group, similarly to the structure of SNO, the naphtha-
lene residue of ssN is very proximal to the primary
amino group.
Amino-modified oligodeoxynucleotides of 25 bases
(ODNs, X-25) were chemically synthesized by introduc-
ing each amino modifier at the 50-ends (Fig. 2b). As con-
trol molecules, conventional amino-modified ODN
(Con-25) and PL-25 were prepared. In PL-25, the amin-
ohexyl group is tethered to the ODN by a propanediol
phosphoric acid (Fig. 2a). PL-25 was synthesized to esti-
mate the effect of linker length on the reactivity of amino
group. Photocleavable (PC) amino-modifying reagent,
which is commercially available, has an o-nitro-benzyl
Keywords: Amino-modified oligonucleotide; Hydrophobic interaction;
Aromatic group.
*
Corresponding author. Tel.: +81 11 857 8437; Fax +81 11 857
8954; e-mail: komatsu-yasuo@aist.go.jp
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.027

N. Kojima et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5118–5121
5119
Figure 2. (a) 50-Terminal structures of Con-, PL-, and PC-modified
ODNs. Con is referred to as conventional amino modification. (b)
Sequence of the amino-modified ODN X-25, which has a primary
amino group at the 50-terminus (X = Con, PL, SN, SNO, ssN, LNO,
LAO, and PC). (c) Percentage of products in coupling reactions of
X-25 with FITC. The ratios were calculated from HPLC analyses of
30-min reaction. Black and white bars represent the reactions in 10%
and 40% dimethylformamide solutions, respectively.
Figure 1. Structures of amino-modifying amidite units. The large
letters, N, A, S, and L, indicate naphthalene, anthracene, and short
and long linkers, respectively.
group as an aromatic residue. Since the reactivity of the
amino group has not been studied, PC-25 was also syn-
thesized to evaluate the hydrophobic effect of the phenyl
group.
We carried out coupling reactions of the amino-
modified ODNs with fluorescein isothiocyanate (FITC)
in aqueous solution containing 10% dimethylformam-
ide. All amino-modified ODNs with the aromatic
groups reacted with FITC more efficiently than
Con-25 (black bars, Fig. 2c). Especially, ssN-25 showed
the highest reactivity among all ODNs. LAO-25 also
showed higher reactivity than LNO-25. The reactivity
of PC-25 was slightly higher than that of Con-25, but
less than that of other modifications. PL-25 without
an aromatic residue showed the lowest reactivity among
all probes. These results suggest that the presence of
hydrophobic residue increased the reactivity of the
amino-group in coupling reactions. It is thought that
the hydrophobic residue of the amino-modified ODNs
facilitates the association with FITC by a hydrophobic
interaction between the aromatic group and FITC.
The amino-reactivity is also related to the distance
In chemical synthesis of amino-modified ODNs, some
amount of incomplete ODNs, which fail to combine
with the amino modifier at the final step of the synthesis,
are generated. It is generally difficult to remove these
impurities from the conventional amino-modified ODNs
by a reverse-phase column chromatography because
there is little difference between the hydrophobic proper-
ties of these ODNs. Analysis of reverse-phase HPLC of
the synthesized amino-modified ODNs revealed that all
ODNs with the aromatic residue could be easily separat-
ed from the impurities due to the hydrophobicity of the
aromatic moiety (see Supplementary data). The struc-
tures of ODNs were confirmed by MALDI-TOF/MS
spectrometry analyses.9

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N. Kojima et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5118–5121
between the amino group and the aromatic residue, as
suggested from the effective coupling reaction of ssN-
25. The labeling reactions were also checked in the solu-
tion containing 40% dimethylformamide (white bars,
Fig. 2c). In this solution, all amino-modified ODNs
reacted quite efficiently, and the effect of the aromatic
residue seemed to be diminished. This result indicates
that the ‘more hydrophobic’ condition suppressed the
hydrophobic interaction, which becomes more impor-
tant factor for the efficient coupling reactions in the
hydrophilic condition. In practical labeling experiments,
reactions are generally carried out in the solution con-
taining dimethylformamide or dimethylsulfoxide to dis-
solve reporter groups, and as low concentration of
organic solvent as possible makes the purification of
labeled oligonucleotides successful in gel filtration or
reverse-phase column. Therefore, the ssN is effective
modification for labeling reactions.
Fluorescence spectra of FITC in conjugation with the
ODNs are shown in Figure 3. Significant fluorescent
quenching was observed only in LAO-25, which was
presumably caused by some kind of interaction of fluores-
cein with an anthracene residue on the identical ODN. In
ssN-25, the fluorescent quenching was not observed.
We also tested coupling reactions with other reporter
groups, such as Cy5-succinimidyl ester or FAM-succin-
imidyl ester, and also with biotin-succinimidyl ester. In
all cases, amino-modified ODNs with the aromatic resi-
due reacted more efficiently than the conventional ami-
no group, and ssN-modified ODN showed the most
effective coupling reactions with these reporter groups
similarly to the reaction with FITC (data not shown).10
Figure 4. Crosslinking reactions between complementary double-
stranded oligonucleotides. (a) Sequences of donor (X-25) and acceptor
(F-AS16rG, F-AS15rG) ODNs. 50-End of the acceptors was labeled
with fluorescein (F), and each acceptor had a guanosine (rG) at the 30-
end to produce dialdehyde groups by periodate oxidation. (b) Analysis
of the crosslinking reactions of X-25/F-AS16rG by denaturing
polyacrylamide gel electrophoresis. The donor X-25 was added after
periodate oxidation for 60 min, and each lane indicates the analyses of
10- and 30-min reactions from the additions of donor strand. CL and
F-AS16rG (ox) indicate the crosslinked products and F-AS16rG
oxidized by periodate ions, respectively. (c) Observed rate constants
(kobs) of the crosslinking reactions. White and black bars represent the
observed rate constants for F-AS15rG and F-AS16rG, respectively.
The primary amino group is able to react with an
aldehyde group to form a transient Schiff base, which
is stabilized under reducing conditions.11 We next eval-
uated the crosslinking rates of these amino-modified
ODNs in covalent bond formation between complemen-
tary double-stranded ODNs. 50-Fluorescein-labeled
ODNs of 15 or 16 bases (F-AS15rG or F-AS16rG,
Fig. 4a) were prepared as the 50-side fragment (acceptor
strand) of the ligated hairpin structure. Both strands had
a riboguanosine at the 30-end to generate 20,30-dialde-
hyde group by periodate oxidation.
As the results of crosslinking reactions, all amino-
modified ODNs (donor strands) provided crosslinked
products, and the oxidized acceptor strands were simul-
taneously decreased as shown in Figure 4b. Observed
rate constants for the crosslinking reactions were calcu-
lated from the percentages of the products (Fig. 4c).
Interestingly, ssN-25 exhibited the fastest crosslinking
rate. The crosslinking rate was about threefold higher
than that of Con-25. This result is consistent with that
observed in the coupling reaction with FITC under
‘hydrophilic’ condition. In contrast, other ODNs con-
taining an aromatic residue showed almost the same
reaction rate as Con-25. The reason for the significant
reactivity of ssN is not clear, but the characteristic struc-
ture of ssN might contribute to the effective coupling
Figure 3. Fluorescence emission spectra of ODNs coupled with FITC.
Solid line, Con-25; broken line, ssN-25; dotted line, LAO-25.

N. Kojima et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5118–5121
Table 1. Melting temperatures (°C) of crosslinked products
5121
Supplementary data
Con-25
ssN-25
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.07.027.
AS15rG
AS16rG
66.6 (35.6)a
64.1 (34.5)
64.0 (35.8)
64.0 (37.5)
a Melting temperatures of double-stranded DNAs before crosslinking
reactions are listed in parentheses.
References and notes
reaction by facilitating the hydrophobic interaction of
naphthalene residue with nucleobase at the 30-end of
the oxidized acceptor strand.
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mal stabilities were examined (see Supplementary data).
All crosslinked products showed Tm values of about
30 °C higher than those of the double-stranded ODNs
without the covalent linkage (Table 1). The results of
the thermal stabilities and the mass spectroscopic analy-
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We showed that the reactivity of the amino group on the
oligonucleotides was increased by the introduction of an
aromatic residue. Especially, it was found that the amino
group close to the aromatic residue could react very effi-
ciently with FITC and aldehyde groups in comparison
with the conventional amino modification. Although
anthracene is thought to be more effective than naphtha-
lene residue in respect of hydrophobicity, anthracene
yields its own fluorescence, and this character is not
appropriate for fluorescence assay using other dyes.
Therefore, ssN, which has a naphthalene residue, can be
a profitable amino-modification for oligonucleotides
and is applicable for various biological studies. For in-
stance, it will be useful for efficient immobilization of
probes onto oligonucleotide array because the ssN-
modified oligonucleotides are thought to react efficiently
with the reactive groups on the array surface. This effect
will improve the sensitivity of the oligonucleotide array.
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9. MALDI-TOF/MS spectra; Con-25: calcd. 7770.39, found
7769.38; PL-25: calcd. 7908.40, found 7908.16; SN-25:
calcd. 7997.49, found 7997.52; SNO-25: calcd. 8027.50,
found 8027.47; ssN-25: calcd. 8027.50, found 8027.69;
LNO-25: calcd. 8131.54, found 8131.90; LAO-25: calcd.
8182.56, found 8182.56.
Acknowledgments
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We thank Dr. Lim Chun Ren (DNA Chip Research
Inc.), Dr. Junya Hashida, and Youji Ueda (Hitachi
Software Engineering Co. Ltd.) for their helpful discus-
sions. We also thank Naonori Inoue (Hokkaido Univer-
sity) for technical assistance in MALDI-TOF/MS
measurement. This work was carried out by financial
support from National Institute of Advanced Industrial
Science and Technology (AIST).