Efficient synthesis of oligonucleotide conjugates on solid-support using an (aminoethoxycarbonyl)aminohexyl group for 5′-terminal modification

Article abstract of DOI:10.1016/j.bmcl.2009.02.121

Solid-support conjugation at the 5′-terminal primary amine of oligonucleotides is a convenient and powerful method for introducing various functional groups. However, conventional aliphatic amines do not necessarily provide conjugates with sufficient yiel

Full text of DOI:10.1016/j.bmcl.2009.02.121

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2144–2147
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of oligonucleotide conjugates on solid-support using
an (aminoethoxycarbonyl)aminohexyl group for 50-terminal modification
*
Naoshi Kojima, Toshie Takebayashi, Akiko Mikami, Eiko Ohtsuka, Yasuo Komatsu
Research 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
a r t i c l e i n f o
a b s t r a c t
Solid-support conjugation at the 50-terminal primary amine of oligonucleotides is a convenient and pow-
erful method for introducing various functional groups. However, conventional aliphatic amines do not
necessarily provide conjugates with sufficient yields. To improve the modification efficacy, we used
the amino-linker (aminoethoxycarbonyl)aminohexyl group (ssH-linker), for solid-support conjugation.
In the ssH-linker terminal modification, reactive free amino group could be easily presented onto a
solid-support due to rapid removal of the amino-protecting group, and activated amino acids or choles-
terol molecules could be covalently connected more efficiently than to typical 6-aminohexyl-linkers.
Based on these results, the ssH-linker can be a useful terminal modification for the solid-support conju-
gation of functional molecules.
Article history:
Received 28 January 2009
Revised 17 February 2009
Accepted 27 February 2009
Available online 4 March 2009
Keywords:
Amino-linker
Oligonucleotide
Conjugation
Ó 2009 Elsevier Ltd. All rights reserved.
Solid-phase synthesis
Oligonucleotides (ONTs) are functionalized by attaching fluoro-
phores, hydrophobic molecules1 or peptides2 to achieve gene
detection or gene delivery. These functional molecules can be
covalently connected to a primary amine on the ONTs. An aliphatic
amine as typified by the 6-aminohexyl group (C6-linker, Fig. 1a) at
the 50-terminus of ONTs has been used for these functionalizations.
The conjugations with the amino terminus of the ONTs are carried
out in solution- or as a solid-phase reaction.3 The reaction method
is selected as the functionalization demands. When an anhydrous
condition in organic solvent is required for the conjugation reac-
tion, then a solid-support reaction is suitable.
In the solid-phase reactions, the protecting groups are first re-
moved from the 50-terminal amines on the solid-support after
the ONTs are synthesized, and free amino groups are presented
onto the solid-support, followed by coupling with target mole-
cules. The monomethoxytrityl (MMT) group has generally been
used as the protection for a 50-terminal aliphatic amine. However,
the MMT group is not rapidly removed from the aliphatic amine,
even with stringent acidic treatment, which sometimes causes
depurination of the ONTs. Furthermore, the reactivity of the pri-
mary amine is not sufficient for obtaining conjugates at high yields.
Thus, excess amounts of exogenous molecules and longer reaction
times are required for conjugations with the conventional amino
group of ONTs.4
reported that ssH-linker modified ONTs (ssH-ONTs) reacted more
efficiently with active esters in aqueous solution as compared with
C6-linker modified ONTs (C6-ONTs). In addition, the MMT group of
the ssH-linker could be removed much faster than that of the C6-
linker under mild acidic conditions. However, the potential use of
the ssH-linker for solid-phase modification has not been investi-
gated. We describe here the utility of the ssH-linker for solid-sup-
port reactions.
We first examined the acid labilities of the protecting groups for
the C6- and ssH-linkers since 4,40-dimethoxy-400-methanesulfinyl-
trityl (DMS(O)MT, Fig. 1a) was recently reported as a novel protect-
ing group for the C6-linker.6 C6- and ssH-modified 25-base ONTs
synthesized with ‘trityl on mode’ (X-25; X = C6- or ssH-linker,
Fig. 1b) were treated with a mild acidic solution (1% acetic acid),
and the observed rate constants (kobs) were determined from the
percentage of ‘trityl off’ ONTs (see Supplementary data for details).
The MMT group of ssH-25 was almost completely cleaved from the
amine in 20 min (kobs = 0.25 minꢀ1) as reported in a previous
study,5a whereas C6-25 protected with DMS(O)MT group gave a
60% ‘trityl off’ product yield (kobs = 0.048 minꢀ1, also see Fig. S1
in Supplementary data). MMT protected C6-25 yielded only a small
amount of ‘trityl off’ ONTs after 20 min. These results show that the
ssH-linker is superior to the C6-linker in the removal of the pro-
tecting group. The fast deprotection of the MMT group from the
ssH-linker is responsible for the aminoethyl carbamate structure,
as described in a previous report.5a This MMT deprotection was
also checked on a DNA synthesizer, and the MMT group was found
to be completely removed from ssH-25 synthesized on a controlled
pore glass (CPG)-support by a single ‘detritylate procedure’ step
Recently, we developed a new amino-linker with an (amino-
ethoxycarbonyl)aminohexyl structure (ssH-linker, Fig. 1a).5 We
* Corresponding author. Tel.: +81 11 857 8437; fax: +81 11 857 8954.
E-mail address: komatsu-yasuo@aist.go.jp (Y. Komatsu).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.121

N. Kojima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2144–2147
2145
gates depends on the coupling efficiency between a carbonyl group
of the amino acid and a terminal amine of the ONTs.7 Initially, C6-
or ssH-modified penta-thymidylic acids (X-T5; X = C6- or ssH-lin-
ker, Fig. 1b) were used for the coupling reactions on the CPG-sup-
ports. After the MMT groups were removed on a DNA synthesizer,
the CPG-supports were washed with diisopropylethylamine (DIEA)
in DMF in order to neutralize the 50-terminal amino groups.8 Next,
N-protected phenylalanine (N-Fmoc-Phe) or its pentafluorophenyl
ester (N-Fmoc-Phe-OPfp) were added to the support by combining
(benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluoro-
phosphate (PyBOPÒ),8 O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyl-
uronium hexafluorophosphate (HBTU)9 or 1-ethyl-3-(30-
dimethylaminopropyl)carbodiimide hydrochloride (EDC)–1H-ben-
zotriazole (HOBt) in the presence of DIEA.10 The reactions were
then analyzed by HPLC after deprotection of the Phe-X-T5
(X = C6- or ssH-linker) with aqueous ammonia. When PyBOP was
used as a coupling reagent for N-Fmoc-Phe conjugation, ssH-T5
gave a 74.1% yield of Phe-conjugates after 2 min of reaction,
whereas C6-T5 gave a 54.1% yield (Fig. 2a; also see Fig. S2 and Ta-
ble S1 in the Supplementary data).11 Similarly, ssH-T5 conjugated
efficiently with N-Fmoc-Phe in the presence of HBTU. Although
the reactions using EDC–HOBt or N-Fmoc-Phe-OPfp gave products
with lower efficiencies in comparison with PyBOP or HBTU, ssH-T5
showed about 10% higher coupling yields than C6-T5. These results
indicate that the ssH-linker is able to react with a target molecule
on a solid-support as well as in solution.5a
We then carried out the same coupling reactions (N-Fmoc-Phe/
PyBOP) with amino-modified ONTs containing all four nucleobases
(X-25; X = C6- or ssH-linker). However, reactions using ssH-25 and
C6-25 resulted in the predominant formation of 50-N-acetylated
ONTs, and their molecular weights were confirmed by MALDI-
TOF/MS measurements (Fig. 2b). Strömberg and co-workers re-
ported a similar 50-N-acetylation as a serious side reaction during
amino acid coupling with amino-modified ONTs on a CPG-sup-
port.9 They explained that the acetyl groups were derived from
acetylated nucleobases formed by the capping reagent for ONT
synthesis, and eliminated these reactive acetyl species by washing
Figure 1. Amino-modified ONTs. (a) 50-terminal structures of the amino-modified
ONTs. The protecting group of the ssH-linker is MMT, and the C6-linker is MMT or
DMS(O)MT. (b) Sequences of the oligonucleotides. X indicates the ssH-linker or C6-
linker.
(3% trichloroacetic acid in dichloromethane for 60 s for a 0.2 lmol
scale synthesis), and could be detected by the trityl-monitor of a
DNA synthesizer. On the other hand, repetitive treatments (usually
3–4 times) were necessary to remove the MMT group from C6-25.
Next, we investigated the coupling reactions with ssH- and C6-
modified ONTs on a CPG-support. First, an amino acid conjugation
was performed, because the total synthesis of peptide-ONT conju-
Figure 2. Conjugations of the amino-modified ONTs with N-Fmoc-Phe. (a) Reactions of X-T5 with N-Fmoc-Phe or N-Fmoc-Phe-OPfp.10 The gray and black bars indicate C6-T5
and ssH-T5, respectively. The coupling reagents used in each reaction are listed in the horizontal bar, and the reaction times are shown below the graph. HPLC analyses of the
conjugation reactions of ssH-25 with N-Fmoc-Phe after the DIEA wash (b), and after omitting the DIEA wash (c). Ac-ssH-25 indicates the 50-N-acetylated ONT. See the
Supplementary data for the HPLC conditions.

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N. Kojima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2144–2147
Figure 4. HPLC analyses of the conjugation reactions of ssH-25 with cholesteryl
chloroformate. (a) Reaction in the absence of DMAP,13 and (b) reaction in the
presence of DMAP.14 Ac-ssH-25 indicates the 50-N-acetylated ONT. See the
Supplementary data for the HPLC conditions.
tion within the solid-support. In each reaction, chol-X-25 was pro-
duced to some extent, but the 50-N-acetylated form was the major
product (Fig. 4a). Although the trans-acetylation within the solid-
support was suppressed by this method, we thought that acetyla-
tion to the 50-terminal amine competed with the coupling reaction
due to the low reactivity of cholesteryl chloroformate. Thus, to
activate the cholesteryl chloroformate, dimethylaminopyridine
(DMAP) was added to the reaction mixture. Under these reaction
Figure 3. HPLC analyses of the conjugation reactions with cholesteryl chlorofor-
mate.13 (a) Structure of cholesteryl chloroformate. (b) Reaction of C6-T15, and (c)
reaction of ssH-T15. These reactions were performed without DMAP. Ac-C6-T15
and Ac-ssH-T15 indicate the 50-N-acetylated ONTs. See the Supplementary data for
HPLC conditions.
conditions,14 the chol conjugates increased successfully to
a
the CPGs with 2% morpholine solution prior to the MMT removal.
We simply omitted the neutralization process after the acid treat-
ment for MMT removal. Interestingly, the formation of 50-N-acety-
lated ONTs was drastically suppressed, and the yields of the Phe-
conjugates increased to 73.7% for ssH-25 and 68.6% for C6-25
(Fig. 2c). These results suggested that 50-N-acetylated ONTs are
probably produced by intermolecular or intramolecular transfer
of the acetyl groups from nucleobases (trans-acetylation), via the
neutralization of the 50-terminal amino-groups on the CPG-sup-
port. In addition, this acetylation was strongly dependent on the
sequence of the amino-modified ONTs (data not shown), and this
is consistent with a previous report.9
The conjugation of lipophilic groups to synthetic ONTs, such as
antisense or siRNA molecules, is shown to mediate the cellular up-
take of the conjugates.1a,12 We next tried to conjugate ssH-ONTs
with cholesterol (chol) on a CPG support. Although chol-ONT con-
jugates are generally synthesized using chol-phosphoramidite
units, cholesteryl chloroformate (Fig. 3a) is inexpensive and thus
a solid-phase reaction using cholesteryl chloroformate is cost-
effective. Amino-modified pentadeca-thymidylic acids (X-T15;
X = C6- or ssH-linker, Fig. 1b) were treated with cholesteryl chloro-
formate in the presence of DIEA.13 As a result of these reactions,
ssH-T15 could provide about 1.5-fold more chol conjugate than
C6-T15, as shown in Fig. 3 (chol-ssH-T15, 76.0%; chol-C6-T15,
51.7%, also see Table S2 in the Supplementary data). Subsequently,
we carried out these conjugation reactions to X-25 (X = C6 or ssH-
linker) without the neutralization process to prevent trans-acetyla-
73.5% yield for ssH-25, whereas C6-25 gave a 54.6% conjugate yield
(Fig. 4b). We also used the ssH-linker for the conjugation reaction
of RNA (X-r21; X = ssH-linker, Fig. 1b) with cholesteryl chlorofor-
mate in the presence of DMAP on a CPG-support.15 Expectedly,
the ssH-linker gave chol-RNA conjugates at a 65.9% yield (Fig. S3
in the Supplementary data). These results showed that the ssH-lin-
ker is a promising terminal modification for the easy and cost-
effective preparation of chol-RNA conjugates.
In summary, we have demonstrated efficient solid-phase conju-
gations with the 50-terminus of ONTs using the ssH-linker. The
ssH-linker is superior to the C6-linker not only for the removal of
the protecting group from the 50-terminal amine, but also for the
conjugation efficiency on a solid-support. Although the 50-terminal
amine of ONTs with a hetero-sequence was acetylated during the
conjugation reactions, we found that the acetylation could be sup-
pressed by keeping the terminal amino groups protonated before
the conjugation. Furthermore, it was found that the activation of
exogenous molecules could reduce the acetylation in the case of
cholesterol couplings. From these results, we conclude that the
ssH-linker can be a very useful terminal modification for the func-
tionalization of oligonucleotides.
Acknowledgements
We thank Dr. Noriaki Minakawa and Dr. Kousuke Sato (Hokka-
ido University) for technical assistance with the MALDI-TOF/MS

N. Kojima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2144–2147
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measurements. This work was supported financially by the
National Institute of Advanced Industrial Science and Technology
(AIST).
8. Ocampo, S. M.; Albericio, F.; Fernández, I.; Vilaseca, I.; Eritja, R. Org. Lett. 2005,
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Supplementary data
9. Zaramella, S.; Yeheskiely, E.; Strömberg, R. J. Am. Chem. Soc. 2004, 126, 14029.
10. The 50-amino-modified ONTs on the CPG-support (0.1
lmol) were treated with
Detailed experimental protocols of the solid-phase conjuga-
tion reactions, list of results, and HPLC chromatograms of the
reactions. Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2009.
02.121.
N-Fmoc-Phe (10 equiv), the coupling reagent (10 equiv) and DIEA (20 equiv) in
DMF (0.5 mL), or N-Fmoc-Phe-OPfp ester (10 equiv) and DIEA (20 equiv) in
DMF (0.5 mL). See the Supplementary data for more details.
11. The products were confirmed by MALDI-TOF/MS measurements. See the
Supplementary data.
12. (a) Wolfrum, C.; Shi, S.; Jayaprakash, K. N.; Jayaraman, M.; Wang, G.; Pandey, R.
K.; Rajeev, K. G.; Nakayama, T.; Charrise, K.; Ndungo, E. M.; Zimmermann, T.;
Koteliansky, V.; Manoharan, M.; Stoffel, M. Nat. Biotechnol. 2007, 25, 1149; (b)
Lorenz, C.; Hadwiger, P.; John, M.; Vornlocher, H.-P.; Unverzagt, C. Bioorg. Med.
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References and notes
13. The 50-amino-modified ONTs on the CPG-support (0.1
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cholesteryl chloroformate (40 equiv) and DIEA (80 equiv for X-T15, 20 equiv
for X-25) in dichloromethane (0.5 mL) at room temperature for 15 min. See the
Supplementary data for more details. The longer ONTs were used in the
cholesterol coupling reactions for the purpose of increasing the solubility of the
chol-ONT conjugates in water.
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14. The 50-amino-modified ONTs on the CPG-support (0.1
lmol) were treated with
cholesteryl chloroformate (40 equiv), DMAP (20 equiv) and DIEA (20 equiv) in
dichloromethane (0.5 mL) at room temperature for 15 min. See the
Supplementary data for details.
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Sugimoto, T.; Sato, K.; Matsubara, K.; Ohtsuka, E. Bioorg. Med. Chem. 2008, 16,
15. The 50-amino-modified ONT on the CPG-support (0.1
lmol) was treated with
cholesteryl chloroformate (150 equiv), DMAP (20 equiv) and DIEA (20 equiv) in
dichloromethane (0.5 mL) at room temperature for 5 min. See the
Supplementary data for details.